WO2007002830A2 - Diagnostic d'un hyperinsulinisme et d'un diabete de type ii et protection contre ceux-ci a partir de proteines exprimees de façon differentielle dans la peau et identifiees par proteomique - Google Patents

Diagnostic d'un hyperinsulinisme et d'un diabete de type ii et protection contre ceux-ci a partir de proteines exprimees de façon differentielle dans la peau et identifiees par proteomique Download PDF

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WO2007002830A2
WO2007002830A2 PCT/US2006/025373 US2006025373W WO2007002830A2 WO 2007002830 A2 WO2007002830 A2 WO 2007002830A2 US 2006025373 W US2006025373 W US 2006025373W WO 2007002830 A2 WO2007002830 A2 WO 2007002830A2
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protein
human
mouse
proteins
diabetes
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WO2007002830A3 (fr
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John J. Kopchick
Edward List
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Ohio University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • IA-PCT relating to diabetes-related differential expression in liver, PCT/US04/17322, filed June 2, 2004 (atty docket Kopchick7 A-PCT ) and PCT/US 04/21944, filed July 8, 2004 (atty docket Kopchick8A-PCT), related to age-related differential expression; PCT/US04/3760, filed Nov. 5, 2004 (atty docket Kopchickl2.
  • IA-PCT relating to diabetes-related differential expression in pancreas; US Prov. Appl. Ser. No.60/591,077, filed July 27, 2005 (atty docket Kopchickl3-USA), relating to diabetes-related differential expression in white adipose tissue; US Prov. Appl. Ser. No.
  • the invention relates to various nucleic acid molecules and proteins, and their use in (1) diagnosing hyperinsulinemia and type II diabetes, or conditions associated with their development, and (2) protecting mammals (including humans) against them. Description of the Background Art
  • Obesity is a major cause of premature morbidity and mortality, especially in the United States, where caloric intake often exceeds energy expenditure.
  • obesity predisposes individuals to type 2 diabetes mellitus (non-insulin dependent diabetes mellitus, NIDDM), which is characterized by insulin resistance, impaired glucose- stimulated insulin secretion, and pancreatic ⁇ -cell dysfunction.
  • NIDDM non-insulin dependent diabetes mellitus
  • obesity-induced type 2 diabetes often involves a progression from a normal phenotype through an insulin, resistant/hyperinsulinemic state to overt diabetes. These stages are replicated in C57BL/6J mice fed a diet composed of 58% kcal from fat but not those fed a diet with only 14% kcal fat. Mice exposed to the high-fat diet become relatively obese and often develop hyperinsuliner ⁇ ia and diabetes.
  • Type 1 diabetes insulin dependent diabetes mellitus
  • pancreatic ⁇ -cells (1) Type 1 diabetes is relatively rare and beyond the scope of this discussion.
  • type 2 diabetes is extremely prevalent. According to recent estimates, type 2 diabetes affects about 5.9% of the population in the United States or 17 million individuals, and is predicted to affect 300 million people worldwide by 2025 (2). The pathogenesis of type 2 diabetes is not completely understood, however, it is closely associated with increased body fat (9).
  • Type 2 diabetes is characterized by insulin resistance, impaired glucose-stimulated insulin secretion, and ⁇ -cell dysfunction.
  • insulin resistance induces insulin hypersecretion and ultimately pancreatic ⁇ -cell failure (3,4).
  • pancreatic ⁇ -cell failure 3,4
  • peripheral insulin resistance stimulates the pancreatic islet cells to hyper-secrete insulin in order to maintain glucose homeostasis.
  • the islet cells eventually fail and the symptoms of clinical diabetes are manifested (5,6). It is important to recognize that hyperinsulinemia may result from a combination of increased insulin production and decreased utilization by hepatic, muscle and adipose tissue, and that once established, hyperinsulinemia leads to global insulin resistance in all insulin-sensitive tissues.
  • Obesity is clearly a global epidemic and growing medical problem in the United States (7,9). In 1980, approximately 14.5% of the U.S. population was considered clinically obese whereas currently more than 22.5% of the Americans are clinically obese; and that number continues to rise (7,9). Obesity is defined as an excess of body fat relative to lean body mass (8,9) or by a body-mass-index (BMI; weight divided by the square of height) of 30 kg m-2 or greater (8). By these criteria, about 60 million individuals in the U.S. are obese and related medical spending is ⁇ $90 billion/year (10). Obesity contributes to premature morbidity and mortality and is associated with the development of type 2 diabetes mellitus (11,12). Obesity-related health risks also include hypertension, dyslipidemia, peripheral vascular disease, and cardiovascular disease, collectively referred to as metabolic syndrome (9).
  • Adipocytes have depot-specific properties. For example, visceral adipocytes are larger than subcutaneous adipocytes and are more efficient at breaking down stored lipids (7,9). Visceral adipocytes release free fatty acids and other adipocyte-secreted products directly into the liver through the portal vein (18). Consequently, there is a flood of fatty acids into the blood and liver that can either a) inhibit insulin-stimulated glucose uptake into muscle; b) decrease the efficiency of insulin clearance by the liver (7,9); c) increase gluconeogenesis (19); and/or d) potentiate glucose-stimulated insulin secretion (19). Visceral adipocytes were recently shown to express more plasminogen activator inhibitor than subcutaneous adipocytes, providing a possible link between visceral obesity and vascular disease (20,21).
  • Chronic hyperglycemia invariably produces macro- and microvascular pathology that in many ways resemble the consequences of aging (22,23).
  • Increased intracellular glucose causes impaired blood flow, increased vascular permeability and excess production of extracellular matrix (ECM) molecules, ultimately resulting in edema, ischemia and hypoxia-induced neovascularization (24).
  • Macrovascular damage results from arterial endothelial cell dysfunction and vascular smooth muscle cell proliferation and increases susceptibility to myocardial infarct (MI), cerebral vascular accident (CVA) and peripheral vascular disease (PVD).
  • MI myocardial infarct
  • CVA cerebral vascular accident
  • PVD peripheral vascular disease
  • Microvascular damage primarily affects the retina, renal glomeruli and peripheral nerves and often causes blindness, end-stage renal disease and neuropathies (24).
  • peripheral neuropathies characterized by multi-focal demyelination and axonal loss similar to that seen with micro-vascular ischemia, are present in half of the patients with type 2 diabetes, especially those with poor glycemic control, and are the most common cause of non-traumatic amputations (9) Because the risk of developing complications precedes diabetes, it is important to distinguish between the consequences of insulin-resistance and those of hyperglycemia, hi addition, identification of individuals who are vulnerable to diabetes-related complications is a critical component of any comprehensive diabetes intervention program.
  • the hexosamine pathway is normally used in the biosynthesis of proteoglycans and O-liriked glycoproteins. However, excess intracellular glucose can be diverted to this pathway resulting in inappropriately modified proteins or aberrant transcriptional activation of glucose-responsive genes (24). Overexpression of the rate-limiting enzyme for hexosamine synthesis in cells and transgenic mice leads to insulin resistance, presumably due to diminished translocation of the glucose transporter GLUT4 to the plasma membrane (25). The polyol pathway can be activated at higher concentrations of intracellular glucose.
  • Glucose can be reduced to sorbitol by the NADPH-dependent enzyme, aldose reductase, and sorbitol can be oxidized to fructose by the NAD+-dependent enzyme sorbitol dehydrogenase. Consequently, the cell's redox potential is disrupted making it more vulnerable to oxidative stress (24,26).
  • Intracellular hyperglycemia can also increase the levels of diacylglycerol (DAG), a lipid second messenger that activates most protein kinase C (PKC) isoforms.
  • DAG diacylglycerol
  • PKC protein kinase C
  • PKC protein kinase
  • AGE advanced glycation end-products
  • AGEs damage cells by interfering with protein function and through inappropriate interactions with cell-surface or nuclear receptors, leading to diverse cellular responses such as the secretion of inflammatory cytokines and generation of reactive oxygen species, altered cell migration and adhesion, or transcription of stress-related genes.
  • the receptor for AGEs (RAGE) is normally expressed at low levels but is upregulated by high concentrations of ligand, such as those observed in the vasculature of diabetics (24,29).
  • each of these hyperglycemia-induced pathways appears to involve excessive production of superoxide (O2-) by fee mitochondrial electron-transport chain (24,30).
  • hyperglycemia increases the production of reactive oxygen species in cultured bovine aortic endothelial cells. This increase was prevented when the cell's ability to utilize electron donors produced by tricarboxylic acid (TCA) cycle, but not glycolysis, was disrupted (30). Abolition of oxidative phosphorylation with pharmacological agents or overexpression of uncoupling protein-1 (UCPl) also prevented the production of free radicals in the presence of excess glucose, as well as the accumulation of AGEs and sorbitol and the activation of PKC (30). Similar experiments indicated that hyperglycemia-induced flux through the hexosamine pathway requires superoxide production and activation of this pathway increases the transcriptional activity of the SpI transcription factor by O-glycosylation (31).
  • DNA microarray analysis was used to compare gene expression (mRNA levels) in white adipose tissue (epididymal fat pads) from different strains of lean and obese (ob-/ob- ) mice with varying degrees of hyperglycemia.
  • Obese mice downregulated genes involved in adipocyte differentiation, lipid metabolism and the mitrochondrial electron-transport chain and upregulated genes associated with the cytoskeleton and extracellular matrix and involved hi immune system function (32).
  • hi addition, 88 genes were identified whose expression correlated with the level of hyperglycemia.
  • the genes encoding the non-receptor protein tyrosine phosphatase PTPKl and the transcription factor Disheveled decreased as hyperglycemia increased, whereas phosphatase inhibitor-2-like protein and fructose- 1,6 bis-phosphatase increased with elevated plasma glucose (32).
  • Feeding behavior is influenced by the subjective experience of appetite and the physiological signals that control hunger and satiety (36).
  • a key component of this system is the adipocyte-derived hormone leptin, its receptor in the hypothalamus (37), and the janus-kinase/STAT-3 signal transduction cascade (38). Circulating leptin modulates the release of neuropeptides from the specific neurons in the hypothalamus that either stimulate ⁇ e.g.
  • mice lacking MCH are hypophagic and lean (39), whereas mice lacking the receptor for a- MSH, the melanocortin-4 receptor (MC4-R), are hyperphagic and obese (40). Loss-of- function mutations in the MC4-R gene in humans result in morbid obesity and episodic binge eating (41).
  • mice lacking the gene encoding leptin (ob-/ob-) (43) or its receptor (db-/db-) (44) are hyperphagic, obese and diabetic, presumably because they are in a state of perceived starvation.
  • Abnormalities in ob-/ob- mice also include decreases in body temperature, activity, immune function and fertility (37), illustrating the complex relationship between nutritional status, energy homeostasis and reproduction.
  • leptin deficiency in humans produces obesity and other metabolic anomalies such as hypogonadism or insulin-resistance, but not diabetes (45,46).
  • caloric restriction results in loss of lean and fat mass whereas administration of leptin selectively reduces fat mass.
  • Mice with diet-induced obesity are less sensitive to chronic infusions of exogenous leptin and consequently tend to lose less weight than their lean counterparts (47).
  • mice with a complete absence of the insulin receptor appear normal at birth but die shortly after in a state of severe hyperinsulinemia, hyperglycemia and ketoacidosis (49).
  • mice with targeted disruption of the insulin receptor in skeletal muscle have increased adiposity and elevated serum free fatty acids and triglycerides, but are not hyperglycemic, hyperinsulinemic or glucose intolerant. These data suggest that impaired fat metabolism is a consequence of insulin resistance and other tissues are important for glucose disposal (49). Mice with selective deletion of GLUT4 in white and brown adipose tissue have impaired insulin-stimulated glucose uptake in adipose tissue and develop insulin resistance and glucose intolerance (50). It is important to recognize that differential glucose transport may contribute to the differences observed in tissue susceptibility to hyperglycemia-induced tissue damage.
  • adipocytes can function as endocrine cells, producing not only fatty acids, but also several bioactive peptides (51,52).
  • a comprehensive discussion about the synthesis, secretion, and regulation of "adipokines" is beyond the scope of this discussion, but several relevant examples will be highlighted.
  • resistin Two recently discovered hormones, resistin (53) and adiponectin (also called Acrp30, adipocyte complement related protein 30IdDa) are synthesized and secreted by adipocytes and are intimately involved in glucose and lipid metabolism (54).
  • resistin "resists" insulin-stimulated glucose uptake and impairs glucose tolerance.
  • Resistin gene expression is induced during adipocyte differentiation and serum levels are elevated in genetic models of obesity and diabetes (ob-/ob- and db/db) and high-fat diet- induced obesity (53).
  • Adiponectin gene expression is induced during adipocyte ' differentiation and its secretion is stimulated by insulin. Adiponectin appears to increase tissue sensitivity to insulin.
  • adiponectin Several missense mutations in the adiponectin gene have been identified in individuals with type 2 diabetes (55). Serum levels of adiponectin are reduced in human and animal models of obesity and insulin resistance. For example, spontaneously occurring obesity and diabetes in rhesus monkeys correspond with a decrease in circulating adiponectin (56). Intraperitoneal administration of recombinant adiponectin in mice inhibits gluconeogenesis and glucose secretion in mouse hepatocytes (57). Intravenous injection of recombinant adiponectin transiently decreased hyperglycemia in ob-/ob- mice and streptozotocin-treated mice without altering serum insulin levels (58).
  • mice lacking adiponectin have been reported to display moderate insulin resistance, impaired glucose tolerance (59) and increased ⁇ -oxidation without insulin resistance and glucose intolerance, despite being fed a high-fat diet (60). The reasons for this discrepancy are unclear, but may involve genetic differences in the strains of mice used in the studies.
  • Adipsin is a serine protease secreted by adipocytes following differentiation and it may play a role in stimulating triglyceride acylation (61).
  • the expression of adipsin is greatly reduced in many rodent models of diabetes (52).
  • Adipocytes also secrete the inflammatory cytokine tumor necrosis factor (TNF-a)
  • TNF-a tumor necrosis factor
  • It has recently been shown that TNF-a is expressed at high levels in the adipocytes of obese animals and humans (63,64) and may possibly play a role in insulin resistance (62).
  • Genetically obese mice (ob-/ob-) lacking TNF- ⁇ are protected from obesity-induced insulin resistance (65).
  • the Zucker diabetic fatty (ZDF) rat has defective leptin receptors and develops type 2 diabetes where compensatory insulin hypersecretion is accompanied by an increase in ⁇ -cell mass and subsequent ⁇ -cell failure is attributed to apoptosis rather than lack of proliferation (66).
  • the ⁇ -cells in these animals display altered gene expression of key metabolic enzymes such as glucokinase and ion channels involved in Ca2+-dependent exocytosis (67), supporting the relationship between diabetes, impaired glucose sensitivity and insulin secretion.
  • Mice lacking the insulin receptor subsrate IRS-2 are insulin resistant and diabetic but fail to display an increase in ⁇ -cell mass, suggesting this molecule is necessary for compensation (68).
  • Partial pancreatectomy (Px) in rats is another model of type 2 diabetes that leads to a period of ⁇ -cell hypertrophy and neogenesis followed by diminished insulin secretion and hyperglycemia, without confounding factors like specific gene mutations or toxin- induced ⁇ -cell degeneration.
  • Weir and colleagues (69-71) have shown that altered ⁇ -cell islet gene expression depends on the magnitude and duration of hyperglycemia. For example, hyperglycemia increased expression of the mRNA encoding the mitochondrial uncoupling protein-2 (UCP-2) and decreased those encoding insulin and the glucose transporter GLUT2 (70).
  • PPAR peroxisome proliferator-activated receptor
  • UCPs mitochondrial uncoupling proteins
  • BAT brown adipose tissue
  • UCP2 is widely distributed
  • UCP3 is expressed primarily in skeletal muscle and BAT (35).
  • the ⁇ -cells of UCP2- deficient mice have increased glucose-stimulated insulin secretion and activation of UCP- 2 in leptin-deficient mice correlates with ⁇ -cell dysfunction (73).
  • body weight is regulated by genetic and environmental factors. Nonetheless, in the absence of predisposing genetic influences, obesity results when energy consumption exceeds energy expenditure. Obesity contributes to premature morbidity and mortality and is associated with the development of type 2 diabetes mellitus. We believe that the physiological consequences of obesity and type 2 diabetes correspond to distinct protein profiles indicative of stage and severity of disease progression.
  • proteomics-based (98); it directly identifies differentially expressed proteins with the aid of two-dimensional gel-electrophoresis and mass spectrometry. Unlike genomics-based methods, it can detect differential expression of post- or co-translationally modified ⁇ roteins(99,100). Proteomic analysis has been used to detect disease associated polymorphisms in mouse brain (101).
  • mice reared on a high-fat diet are relatively obese compared to age-matched controls fed a normal diet, and display progressive deterioration in glucose homeostasis. Consequently, proteins which are expressed at higher or lower levels in such mice, as compared to those on a normal (low fat) diet, are likely to be involved in the disease progression.
  • mice reared on each diet were monitored at regular intervals for evidence of obesity and diabetes (i.e. weight; glucose and insulin levels).
  • the physiological parameters were correlated with the relative abundance of proteins that are differentially expressed or modified as a consequence of obesity and diabetes.
  • the insulin-sensitive tissues i.e. liver, skeletal muscle, white adipose, pancreas
  • tissues susceptible to diabetes-related complications i.e. kidney, heart, brain
  • proteins whose timing and pattern of expression are believed to correlate with the stage and severity of obesity and diabetes. We believe that there are significant differences in the way each tissue responds to diet-induced obesity and diabetes.
  • Serum and skin are also believed to contain such proteins. Serum in any event is clinically relevant, has established age-and diabetes-related biomarkers, and is readily accessible. Skin is considered worth studying because it is readily available and can be obtained using a minimally invasive punch biopsy that might also extract the associated endothelial-rich vascular tissue.
  • tissue may refer to tissues which are part of an organ (e.g., heart tissue) or tissues which aren't (e.g., muscle tissues, subcutaneous tissue, etc.).
  • tissue as used herein, is intended to include serum and skin. Should it be desirable to refer to tissues other than serum, the term “solid tissue” will be used.
  • the tissue of interest was skin. That is, the skin samples were compared to identify mouse proteins which were expressed at different levels in skin from normal, hyperinsulinemic and/or diabetic mice of a particular age, and the identification of differentially expressed mouse proteins in Master Tables 101-103 is strictly with respect to differential expression in skin. However, the findings with respect to skin may be compared with the differential expression findings vis-a-vis other tissues.
  • Insulin-sensitive tissues tissues susceptible to hyperglycemia-related damage, and serum, were harvested from the experimental animals at different stages of disease severity. In parallel, tissue were prepared for proteomic analysis.
  • Proteins can be isolated from distinct subcellular fractions by differential gradient ultracentrifugation or homogenized as total protein lysates and then resolved by two- dimensional gel-electrophoresis. The relative abundance of each protein were determined by densitometry and differentially expressed or modified proteins were excised from the gels and prepared for mass spectrometry. Peak intensity spectra were used to predict the peptide fragments found in each sample. When necessary, a protein's identity was confirmed by western blot analysis and its pattern of expression was determined by immunocytochemistry.
  • each protein "spot” was assigned an intensity corresponding to its relative pixel density and a grid location based on its location in the gel. Proteins were selected for further analysis if their relative abundance is altered as a consequence of obesity and diabetes. Protein "spots” were manually excised from the gels and prepared for automated mass spectrometry analysis. The peptide mass fingerprint data were thoroughly analyzed to determine the confidence of the predictions.
  • mice proteins usually tissue-specific
  • the corresponding mouse protein profiles provide insight about the control of functional mouse protein networks and reveal novel targets for the diagnosis and treatment of type 2 diabetes.
  • a “profile” we mean the state of the proteome at a particular stage of the disease progression (normal to hyperinsulinemic to diabetic; or normal to overweight to obese; these two progressions are related but not necessarily synchronized) and, more particularly, the elements of the proteome which have changed relative to the other stage(s).
  • Corresponding human proteins can be identified by searching human protein sequence databases for homologous proteins.
  • the sequences in the protein databases are determined either by directly sequencing the protein or, more commonly, by sequencing a DNA, and then determining the translated amino acid sequence in accordance with the Genetic Code.
  • AU of the mouse sequences in the mouse polypeptide database are referred to herein as "mouse proteins” regardless of whether they are in fact full length sequences (i.e., encoded by a full-length DNA).
  • human sequences in the human polypeptide database are referred to as "human proteins”.
  • mammalian subjects Since the progression is from normal to hyperinsulinemic, and thence from hyperinsulinemic to type II diabetic, one may define mammalian subjects as being more favored or less favored, with normal subjects being more favored than hyperinsulinemic subjects, and hyperinsulinemic subjects being more favored than type II diabetic subjects. The subjects' state may then be correlated with their gene expression activity.
  • normal and "control” are used interchangeably in this specification, unless expressly stated otherwise.
  • the control or normal subject is a mouse which is normal vis-a-vis fasting insulin and fasting glucose levels.
  • normal means normal relative to those parameters, and does not necessitate that the mouse be normal in every respect.
  • a mouse protein is said to have exhibited a favorable behavior if, for a particular mouse age of observation, its average level of expression in mice which are in a more favored state is higher than that in mice which are in a less favored state.
  • a mouse protein is said to have exhibited an unfavorable behavior if, for a particular mouse age of observation, its average level of expression in mice which are in a more favored state is lower than that in mice which are in a less favored state.
  • mice When we observe the mice at several different ages, it is possible for their expression behavior to vary from time point to time point.
  • the mouse protein would be classified as an unfavorable protein with respect to the subject comparison in question.
  • mice protein were observed at an age other than one of the ages noted in the Examples, we would have observed a still stronger differential expression behavior. Nonetheless, we must classify the mouse proteins on the basis of the behavior which we actually observed, not the behavior which might have been observed at some other age.
  • a behavior is considered strong if the ratio of the higher level to the lower level is at least two-fold.
  • a mouse protein may still be identified as significantly favorable or unfavorable even if none of its observed behaviors are strong as defined above.
  • a behavior which is not strong i.e., is less than a two-fold change
  • the mean level of expression in one group of mice e.g., diabetic mice at week 8 of the diabetes-inducing diet
  • a second group of age-matched mice e.g., control mice at week 8 of the control diet
  • the differential behavior observed is both strong and consistent.
  • a mouse protein which was more strongly expressed in hyperinsulinemic tissue than in either normal or type II diabetic tissue will be deemed both "unfavorable”, by virtue of the controlrhyperinsulinemic comparison, and "favorable”, by virtue of the hyperinsulinemic: diabetic comparison. This is one of several possible “mixed” expression patterns.
  • mixed proteins are those exhibiting a combination of favorable and unfavorable behavior.
  • a mixed protein can be used as would a favorable protein if its favorable behavior outweighs the unfavorable. It can be used as would an unfavorable protein if its unfavorable behavior outweighs the favorable. Preferably, they are used in conjunction with other agents that affect their balance of favorable and unfavorable behavior.
  • Use of mixed proteins is, in general, less desirable than use of purely favorable or purely unfavorable proteins, but it is not excluded.
  • a mouse protein is classified on the basis of the strongest C- HI behavior among the ages tested, the strongest HI-D behavior among the ages tested, and the strongest C-D behavior among the ages tested. If at least one of these three behaviors is significantly favorable, and none of the others of these three behaviors is significantly unfavorable, the mouse protein will be classified as wholly favorable and listed in subtable IA of Master Table 1. However, that does not mean that it may not have exhibited a weaker but unfavorable expression behavior at some tested age.
  • the “favorable”, “unfavorable” and “mixed” mouse proteins of the present invention include the mouse database proteins listed in the Master Table.
  • mouse proteins of interest also include mouse proteins which, while not listed in the table, correspond to (i.e., homologous to, i.e., which could be aligned in a statistically significant manner to) such mouse proteins or genes, and mouse proteins which are at least substantially identical or conservatively identical to the listed mouse proteins.
  • Related proteins were identified by searching a database comprising human proteins for sequences corresponding to (i.e., homologous to, i.e., which could be aligned in a statistically significant manner to) the mouse protein. More than one human protein may be identified as corresponding to a particular mouse protein.
  • human proteins are used in a manner analogous to that already discussed in the case of “mouse proteins”.
  • corresponding does not mean identical, but rather implies the existence of a statistically significant sequence similarity, such as one sufficient to qualify the human protein as a homologous proteinas defined below.
  • the greater the degree of relationship as thus defined i.e., by the statistical significance of the alignment, measured by an E value), the more close the correspondence.
  • the human proteins which most closely correspond, directly or indirectly, to the mouse proteins are preferred, such as the one(s) with the highest, top two highest, top three highest, top four highest, top five highest, and top ten highest homologies (lowest E values) for the BlastP alignment to a particular mouse protein.
  • the human proteins deemed to correspond to our mouse proteins are identified in the Master Tables.
  • a partial protein may still have biological activity, and a molecule which binds the partial protein may also bind the full-length protein so as to antagonize a biological activity of the full-length protein.
  • the protein sequences may of course also be used in the design of probes intended to identify the full length gene or protein sequence.
  • a human protein For the sake of convenience, we refer to a human protein as favorable if (1) it is listed in Master Table 1 as corresponding to a favorable mouse protein, or (2) it is at least substantially identical or conservatively identical to a listed protein per (1).
  • a human protein We define a human protein as unfavorable in an analogous manner.
  • agents useful in screening humans at risk for progression toward hyperinsulinemia or toward type II diabetes or protecting humans at risk thereof from progression from a normoinsulinemic state to a hyperinsulinemic state, or from either to a type II diabetic state.
  • Agents which bind the "favorable" and "unfavorable” human proteins e.g., an antibody vs. a human protein identified as corresponding to a favorable or unfavorable mouse protein
  • a subject with one or more elevated “unfavorable” and/or one or more depressed “favorable” proteins is at increased risk, and one with one or more elevated “favorable” and/or one or more depressed “unfavorable” proteins is at decreased risk.
  • the assay may be used as a preliminary screening assay to select subjects for further analysis, or as a formal diagnostic assay.
  • the identification of the related proteins may also be useful in protecting humans against these disorders.
  • Human proteins corresponding to favorable mouse proteins (and of the mouse proteins, or other corresponding nonhuman proteins, if biologically active in humans), to protect against the disorder(s);
  • Such proteins in diagnostic agents, in assays to measure progression toward hyperinsulinemia or type II diabetes, or protection against the disorder(s), or to estimate related end organ damage such as kidney damage;
  • the agents of the present invention may be used in conjunction with known anti-aging or anti-age-related disease agents. It is of particular interest to use the agents of the present invention in conjunction with an agent disclosed in one of the related applications cited above, in particular, an antagonist to CIDE-A, the latter having been taught in USSN 60/474,606, filed June 2, 2003 (arty docket Ko ⁇ chick7), and PCT/US04/17322, filed June 2, 2004 (atty docket Kopchick7A- PCT ), hereby incorporated by reference in their entirety. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
  • a “full length” gene is here defined as (1) a naturally occurring DNA sequence which begins with an initiation codon (almost always the Met codon, ATG), and ends with a stop codon in phase with said initiation codon (when introns, if any, are ignored), and thereby encodes a naturally occurring polypeptide with biological activity, or a naturally occurring precursor thereof, or (2) a synthetic DNA sequence which encodes the same polypeptide as that which is encoded by (1).
  • the gene may, but need not, include introns.
  • a "full-length” protein is here defined as a naturally occurring protein encoded by a full-length gene, or a protein derived naturally by post-translational modification of such a protein. Thus, it includes mature proteins, proproteins, preproteins and preproproteins. It also includes substitution and extension mutants of such naturally occurring proteins.
  • Some protein "spots” will represent post-translational modifications of the same protein while others may represent heterogeneity due to genetic polymorphisms. For example, 2D gels often reveal a "charge" train representing a difference in phosphorylation states of the same protein.
  • a mouse is considered to be a diabetic subject if, regardless of its fasting plasma insulin level, it has a fasting plasma glucose level of at least 190 mg/dL.
  • a mouse is considered to be a hyperinsulinemic subject if its fasting plasma insulin level is at least 0.67 ng/mL and it does not qualify as a diabetic subject.
  • a mouse is considered to be "normal” if it is neither diabetic nor hyperinsulinemic. Thus, normality is defined in a very limited manner.
  • a mouse is considered “obese” if its weight is at least 15% in excess of the mean weight for mice of its age and sex.
  • a mouse which does not satisfy this standard may be characterized as "non-obese", the term “normal” being reserved for use in reference to glucose and insulin levels as previously described.
  • a human is considered a diabetic subject if, regardless of his or her fasting plasma insulin level, the fasting plasma glucose level is at least 126 mg/dL.
  • a human is considered a hyperinsulinemic subject if the fasting plasma insulin level is more than 26 micro International Units/mL
  • a human is considered to be “normal” if it is neither diabetic nor hyperinsulinemic. Thus, normality is defined in a very limited manner.
  • a human is considered “obese” if the body mass index (BMI) (weight divided by height squared) is at least 30 kg/m 2 .
  • BMI body mass index
  • a human who does not satisfy this standard may be characterized as "non-obese", the term "normal” being reserved for use in reference to glucose and insulin levels as previously described.
  • a human is considered overweight if the BMI is at least 25 kg/m 2 .
  • we define overweight to include obese individuals consistent with the recommendations of the National Institute of Diabetes and Digestive and Kidney Diseases(TSHDDK).
  • THDDK National Institute of Diabetes and Digestive and Kidney Diseases
  • the following are risk factors for diabetes type II: older (e.g., at least 45; see below); excessive weight (see below); first-degree relative with diabetes mellitus; member of high risk ethnic group (black, Hispanic, Native American, Asian); history of gestational diabetes mellitus or delivering a baby weighing more than 9 pounds (4.032 kg); hypertensive (>140/90 mm Hg); HDL cholesterol level >35 mg/dL (0.90 mmol/L); and triglyceride level > 250 mg/dL (2.S3 mmol/L).
  • the diagnostic and protective methods of the present invention are applied to human subjects exhibiting one or more of the aforementioned risk factors. Likewise, in a preferred embodiment, they are applied to human subjects who, while not diabetic, exhibit impaired glucose homeostasis (110 to ⁇ 126 mg/dL).
  • the age of the subjects is at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, and at least 75.
  • the BMIs of the human subjects is at least 23, at least 24, at least 25 (i.e., overweight by our criterion), at least 26, at least 27, at least 28, at least 29, at least 30 (i.e., obese), at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, or over 40.
  • an antagonist of a protein or other molecule may be obtained by preparing a combinatorial library, as described below, of potential antagonists, and screening the library members for binding to the protein or other molecule in question. The binding members may then be further screened for the ability to antagonize the biological activity of the target.
  • the antagonists may be used therapeutically, or, in suitably labeled or immobilized form, diagnostically.
  • Substances known to interact with an identified mouse or human protein e.g., agonists, antagonists, substrates, receptors, second messengers, regulators, and so forth
  • binding molecules which bind them, are also of utility.
  • binding molecules can likewise be identified by screening a combinatorial library.
  • MS mass spectrum
  • the mass spectrum (MS) of the peptide mixture resulting from the digestion of a protein by an enzyme provides a "fingerprint" by which the protein can be identified, provided that the protein has a sequence which is published in a sequence database.
  • the identification software determines what fragments would be generated from that database protein if it were subjected to the same treatment as was the recovered protein, and calculates their masses.
  • the program also determines how good a fit there is between the set of mass peaks observed for the actual protein, and the set of mass peaks generated in silico for each database protein.
  • Tandem mass spectrometry deliberately induces fragmentation of a precursor ion and then analyzes the resulting fragments. Since the precursor ion is itself derived from one of the peptide fragments of the original protein, the analysis is called MS/MS.
  • the recovered protein was identified, based on the mass spectrogram of its digest, using one or more of the following analytical tools: Mascot MS, Mascot MS/MS (for up to four fragments of the protein), and MS-FIT.
  • Mascot MS for up to four fragments of the protein
  • MS-FIT MS-FIT.
  • Each of these tools generates a match score which is a measure (although not the only conceivable one) of the degree of fit.
  • the score can take into account, e.g., the apparent molecular weight of the peak, the mass difference between the observed and predicted peaks, and whether the matching predicted fragment has any missed cleavages.
  • the match score is given in the form of the Probability-Based MOWSE score.
  • the human proteins of Master Table 1 are those which are homologous to the mouse proteins with the better match scores. The higher the score, the higher the number of masses matched, and/or the higher the quality of the peak match.
  • the human protein of interest is preferably homologous to a mouse protein for which the Mascot-MS match score is at least 64, more preferably at least 75, even more preferably at least 100.
  • the human protein of Master Table 1 is preferably homologous to a mouse protein for which the Mascot MS/MS score for at least one fragment is at least 24, more preferably at least 27, even more preferably at least 50. This is especially desirable if the mouse protein does not satisfy the Mascot MS match score desideratum stated above.
  • the E value of the top scoring mouse database protein will depend on whether the recovered mouse protein is actually in the database, the accuracy of the database sequence (inaccuracies will reduce the score and hence the E value for that score), and on the specified mass tolerance (the higher the tolerance, the more likely it is that a database protein will match some masses by chance alone).
  • the E value is the number of times that an alignment scoring at least as good as the one observed would occur in the course of the database search (given the number of database sequences) through chance alone. Consequently, the lower the E value, the more significant the result.
  • MOWSE score is based on the scoring system described in Pappin et al., Current Biology, 3(6): 327 (1993). The higher the MOWSE score, the better the fit.
  • each database mouse protein of Master Table 1 Preferably, for each database mouse protein of Master Table 1, at least one of the following desiderata applies:
  • the Mascot MS E value is not more than 0.05
  • the Mascot MS E value is more preferably less than e-3, even more preferably less than e-4, still more preferably less than e-5, most preferably less than e-6.
  • the Mascot MS/MS E value is more preferably less than e-3, even more preferably less than e-4, still more preferably less than e-5, most preferably less than e-6.
  • MS-FIT MOWSE score is more preferably more than 100, even more preferably more than 1000, still more preferably more than 10,000, most preferably more than 100,000.
  • the analyzed protein may be a cleavage product of the database protein
  • the ratio of the number of matched peaks to the number of total peaks (preferably at least 1:10, more preferably at least 1:5);
  • the percentage of the database protein which is covered by the matched peaks (preferably at leas 10%, more preferably at least 20%, even more preferably at least 30%);
  • the apparent molecular weight of the protein is smaller than the calculated molecular weight of the database protein, this may be because the isolated protein corresponds to a fragment of the database protein. If the matched peptide fragments (actual vs. predicted) can be localized to one region of the database protein, e.g., the C-terminal, and that region is similar in molecular weight to the observed molecular weight, then this would support the hypothesis that the isolated protein was a fragment of the database protein.
  • a human protein can be said to be identifiable as corresponding (homologous) to a mouse protein if it can be aligned by BlastP to the mouse protein, where any alignment by BlastP is in accordance with the default parameters set forth below, and the expected value (E) of each alignment (the probability that such an alignment would have occurred by chance alone) is less than e-10. (Note that because this is a negative exponent, a value such as e-50 is less than e-10.).
  • the E value is less than e-50, more preferably less than e-60, still more preferably less than e-70, even more preferably less than e-80, considerably more preferably less than e-90, and most preferably less than e-100. Desirably, it is true for two or even all three of these conditions.
  • a longer (possibly full length) mouse gene may be identified by a BlastN search of the mouse DNA database, using the mouse DNA exactly corresponding to the identified mouse protein as a query sequence.
  • the mouse protein encoded by the longer mouse database DNA may then be deduced using the genetic code, and itself used in a BlastP search of human proteins.
  • Master table 1 assembles a list of human protein corresponding to each of the mouse proteins identified herein. These human proteins form a set and can be given a percentile rank, with respect to E value, within that set.
  • the human proteins of the present invention preferably are those scorers with a percentile rank of at least 50%, more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%.
  • human protein For each mouse protein in Master Table 1, there is a particular human protein which provides the best alignment match as measured by BlastP, i.e., the human protein with the best score (lowest e-value). These human proteins form a subset of the set above and can be given a percentile rank within that subset, e.g., the human proteins with scores in the top 10% of that subset have a percentile rank of 90% or higher.
  • the human proteins of the present invention preferably are those best scorer subset proteins with a percentile rank within the subset of at least 50%, more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90%.
  • BlastP can report a very low expected value as AO.0". This does not truly mean that the expected value is exactly zero (since any alignment could occur by chance), but merely that it is so infinitesimal that it is not reported.
  • the documentation does not state the cutoff value, but alignments with explicit E values as low as e-178 (624 bits) have been reported as nonzero values, while a score of 636 bits was reported as A0.0".
  • a human protein may be said to be functionally homologous to the mouse protein if the human protein has ' at least one biological activity in common with the mouse protein encoded by said mouse protein.
  • the human proteins of interest also include those that are substantially and/or conservatively identical (as defined below) to the homologous and/or functionally homologous human proteins defined above.
  • Searches may also take cognizance, intermediately, of known proteins other than mouse or human ones, e.g., use the mouse sequence to identify a known rat sequence and then the rat sequence to identify a human one.
  • mouse protein which appears similar to a human protein, then that human protein may be used (especially in humans) for purposes analogous to the proposed use of the mouse protein in mice.
  • the disclosed protein In determining whether the disclosed proteins have significant similarities to known proteins, one would generally use the disclosed protein as a query sequence in a search of a sequence database.
  • the results of several such searches are set forth in the Examples. Such results are dependent, to some degree, on the search parameters. Preferred parameters are set forth in Example 1.
  • the results are also dependent on the content of the database. While the raw similarity score of a particular target (database) sequence will not vary with content (as long as it remains in the database), its informational value (in bits), expected value, and relative ranking can change. Generally speaking, the changes are small.
  • cognate proteins include not only those set forth in the examples, but those which would have been highly ranked (top ten, more preferably top three, even more preferably top two, most preferably the top one) in a search run with the same parameters on the date of filing of this application.
  • the degree of differential expression may be expressed as the ratio of the higher expression level to the lower expression level. Preferably, this is at least 2-fold, and more preferably, it is higher, such as at least 3 -fold, at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold.
  • the human protein of interest corresponds to a mouse protein for which the degree of differential expression places it among the top 10% of the mouse proteins in the appropriate subtable.
  • a protein is down-regulated in more favored mammals, or up-regulated in less favored mammals, (i.e., an "unfavorable protein") then several therapeutic utilities are apparent.
  • an agent which is an antagonist of the unfavorable protein, or of a downstream product through which its activity is manifested e.g., a signaling intermediate
  • a downstream product through which its activity is manifested e.g., a signaling intermediate
  • This antagonist could be an antibody, a peptide, apeptoid, a nucleic acid, a peptide nucleic acid (PNA) oligomer, a small organic molecule of a kind for which a combinatorial library exists (e.g., a benzodiazepine), etc.
  • An antagonist is simply a binding molecule which, by binding, reduces or abolishes the undesired activity of its target.
  • the antagonist if not an oligomeric molecule, is preferably less than 1000 daltons, more preferably less than 500 daltons.
  • an agent which degrades, or abets the degradation of the protein or of a downstream product which mediates its activity may be used to curb the effective period of activity of the protein.
  • an agent which down-regulates expression of the gene may be used to reduce levels of the corresponding protein and thereby inhibit further damage.
  • This agent could inhibit transcription of the gene in the subject, or translation of the corresponding messenger RNA.
  • Possible inhibitors of transcription and translation include antisense molecules and repressor molecules.
  • the agent could also inhibit a post-translational modification (e.g., glycosylation, phosphorylation, cleavage, GPI attachment) required for activity, or post-translationally modify the protein so as to inactivate it.
  • a post-translational modification e.g., glycosylation, phosphorylation, cleavage, GPI attachment
  • it could be an agent which down- or up-regulated a positive or negative regulatory gene, respectively.
  • an antisense molecule would require knowledge of the sequence of the gene (to inhibit transcription) or of the mRNA (to inhibit translation), it is possible to identify a repressor molecule without knowing the identity of the sequence to which it binds.
  • an assay can be used to determine the level of the unfavorable protein or the corresponding mRNA in a sample. Such an assay could be for quality control purposes, if the sample were from in vitro production of the protein. If the sample is from a subject, this can, if desired, be correlated with prognostic information and used to diagnose the present or future state of the subject, making the assay a diagnostic assay. Elevated levels are indicative of progression, or propensity to progression, to a less favored state, and clinicians may take appropriate preventative, curative or ameliorative action.
  • the unfavorable protein, or a suitable fragment thereof may be used in labeled or immobilized form as an assay reagent, in the assaying of a sample to determine the level of the protein. (It would compete with the sample protein.)
  • a substance which binds the unfavorable protein may be used in labeled or immobilized form as an assay reagent to label or capture the sample protein.
  • the complementary strand of the gene, or the corresponding cDNA, or a specifically hybridizing fragment of the gene or cDNA may be used in labeled form as a hybridization probe to detect messenger KNA (or its cDNA) and thereby monitor the level of expression of the gene in a subject.
  • the protein may be administered for therapeutic purposes.
  • an agent which is an agonist of the protein, or of a downstream product through which its activity (of inhibition of progression to a less favored state) is manifested, or of a signaling intermediate maybe used to foster its activity.
  • an agent which inhibits the degradation of that protein or of a downstream product or of a signaling intermediate maybe used to increase the effective period of activity of the protein.
  • an expression vector comprising an expressible DNA encoding the favorable protein maybe administered to the subject ("gene therapy") to increase the level of expression of the protein in vivo. It could be a vector which carries a copy of the gene, but which expresses the gene at higher levels than does the endogenous expression system.
  • an agent which up-regulates expression of the gene encoding the favorable protein may be used to increase levels of that protein and thereby inhibit further progression to a less favored state. It could be an agent which up- or down-regulates a positive or negative regulatory gene. Or it could be an agent which modifies in situ the regulatory sequence of the endogenous gene by homologous recombination.
  • assay including diagnostic) utilities for the favorable protein, and related nucleic acids, exist.
  • the protein, or a binding molecule therefor may be used, preferably in labeled or immobilized form, as an assay reagent in an assay for said protein product or downstream product.
  • Depressed levels of the favorable protein are indicative of damage, or possibly of a propensity to damage, and clinicians may take appropriate preventative, curative or ameliorative action.
  • the complementary strand of the corresponding gene, or its cDNA, or a specifically hybridizing fragment of the gene or cDNA maybe used in labeled form as a hybridization probe to detect messenger RNA and thereby monitor the level of expression of the gene in a subject.
  • mutant proteins which are substantially identical (as defined below) to the parental protein (peptide).
  • the fewer the mutations the more likely the mutant protein is to retain the activity of the parental protein.
  • the effect of mutations is usually (but not always) additive. Certain individual mutations are more likely to be tolerated than others.
  • a protein is more likely to tolerate a mutation which:
  • (a) is an amino acid substitution rather than an insertion or deletion of one or more amino acids
  • (b) is an insertion or deletion of one or more amino acids at either terminus, rather than internally, or, if internal, at a domain boundary, or a loop or turn, rather than in an alpha helix or beta strand;
  • (e) is a substitution of one amino acid for another of similar size, charge, and/or hydrophobicity, and does not destroy a disulfide bond or other crosslink;
  • (f) is at a site which is subject to substantial variation among a family of homologous proteins to which the protein of interest belongs.
  • Surface residues may be identified experimentally by various labeling techniques, or by 3-D structure mapping techniques like X-ray diffraction and NMR. A 3-D model of a homologous protein can be helpful.
  • Residues forming the binding site maybe identified by (1) comparing the effects of labeling the surface residues before and after complexing the protein to its target, (2) labeling the binding site directly with affinity ligands, (3) fragmenting the protein and testing the fragments for binding activity, and (4) systematic mutagenesis (e.g., alanine- scanning mutagenesis) to determine which mutants destroy binding. If the binding site of a homologous protein is known, the binding site may be postulated by analogy.
  • Protein libraries may be constructed and screened that a large family (e.g., 10 8 ) of related mutants may be evaluated simultaneously. Hence, the mutations are preferably conservative modifications as defined below.
  • a mutant protein (peptide) is substantially identical to a reference protein (peptide) if (a) it has at least 10% of a specific binding activity or a non-nutritional biological activity of the reference protein, and (b) is at least 50% identical in amino acid sequence to the reference protein (peptide). It is "substantially structurally identical” if condition (b) applies, regardless of (a).
  • Percentage amino acid identity is determined by aligning the mutant and reference sequences according to a rigorous dynamic programming algorithm which globally aligns their sequences to maximize their similarity, the similarity being scored as the sum of scores for each aligned pair according to an unbiased PAM250 matrix, and a penalty for each internal gap of -12 for the first null of the gap and -4 for each additional null of the • same gap.
  • the percentage identity is the number of matches expressed as a percentage of the adjusted (i.e., counting inserted nulls) length of the reference sequence.
  • sequence is not merely substantially identical but rather is at least 51%, at least 66%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical in sequence to the reference sequence.
  • “Semi-Conservative Modifications” are modifications which are not conservative, but which are (a) semi-conservative substitutions as hereafter defined; or (b) single or multiple insertions or deletions internally, but at interdomain boundaries, in loops or in other segments of relatively high mobility. Semi-conservative modifications are preferred to nonconservative modifications. Semi-conservative substitutions are preferred to other semi-conservative modifications.
  • Non-conservative substitutions are preferred to other non-conservative modifications.
  • no more than about five amino acids are inserted or deleted at a particular locus, and the modifications are outside regions known to contain binding sites important to activity.
  • insertions or deletions are limited to the termini.
  • a conservative substitution is a substitution of one amino acid for another of the same exchange group, the exchange groups being defined as follows
  • Residues Pro, GIy and Cys have special conformational roles. Cys participates in formation of disulfide bonds. GIy imparts flexibility to the chain. Pro imparts rigidity to the chain and disrupts ⁇ helices. These residues may be essential in certain regions of the polypeptide, but s ⁇ bstitutable elsewhere.
  • “Semi-conservative substitutions” are defined herein as being substitutions within supergroup I/H/TH or within supergroup IVYV, but not within a single one of groups I- V. They also include replacement of any other amino acid with alanine. If a substitution is not conservative, it preferably is semi-conservative.
  • Non-conservative substitutions are substitutions which are not “conservative” or “semi-conservative”.
  • “Highly conservative substitutions” are a subset of conservative substitutions, and are exchanges of amino acids within the groups Phe/Tyr/Trp, Met/Leu/Ile/Val, His/Arg/Lys, Asp/Glu and Ser/Thr/Ala. They are more likely to be tolerated than other conservative substitutions. Again, the smaller the number of substitutions, the more likely they are to be tolerated.
  • a protein (peptide) is conservatively identical to a reference protein (peptide) it differs from the latter, if at all, solely by conservative modifications, the protein (peptide remaining at least seven amino acids long if the reference protein (peptide) was at least seven amino acids long.
  • a protein is at least semi-conservatively identical to a reference protein (peptide) if it differs from the latter, if at all, solely by semi-conservative or conservative modifications.
  • a protein (peptide) is nearly conservatively identical to a reference protein
  • the core sequence of a reference protein is the largest single fragment which retains at least 10% of a particular specific binding activity, if one is specified, or otherwise of at least one specific binding activity of the referent. If the referent has more than one specific binding activity, it may have more than one core sequence, and these may overlap or not.
  • a peptide of the present invention may have a particular similarity relationship (e.g., markedly identical) to a reference protein (peptide)
  • preferred peptides are those which comprise a sequence having that relationship to a core sequence of the reference protein (peptide), but with internal insertions or deletions in either sequence excluded. Even more preferred peptides are those whose entire sequence has that relationship, with the same exclusion, to a core sequence of that reference protein (peptide).
  • a mutant DNA sequence is substantially identical to a reference DNA sequence if they are structural sequences, and encoding mutant and reference proteins which are substantially identical as described above.
  • mutant sequences are substantially identical if they are regulatory sequences, they are substantially identical if the mutant sequence has at least 10% of the regulatory activity of the reference sequence, and is at least 50% identical in nucleotide sequence to the reference sequence. Percentage identity is determined as for proteins except that matches are scored +5, mismatches -4, the gap open penalty is -12, and the gap extension penalty (per additional null) is -4.
  • DNA sequences may also be considered "substantially identical" if they hybridize to each other under stringent conditions, i.e., conditions at which the Tm of the heteroduplex of the one strand of the mutant DNA and the more complementary strand of the reference DNA is not in excess of 1O 0 C. less than the Tm of the reference DNA homoduplex. Typically this will correspond to a percentage identity of 85-90%.
  • a DNA which encodes a favorable protein may be used in the production of that protein in vitro or in vivo (gene therapy).
  • a DNA which encodes an unfavorable protein may be used in the production of that protein in vitro, and hence to facilitate the use of that protein as a diagnostic agent or as a target in screening for binding and neutralizing substances (antagonists).
  • any coding sequence but preferably one with coding preferences matching those of the intended host.
  • the gene endogenously encoding a favorable human protein of interest is not known, we can teach using any sequence encoding the human protein, but preferably one with human coding preferences. See, e.g., Desai, et al., "Intragenic codon bias in a set of mouse and human genes, Biol., 230(2): 215-25 (Sept. 21, 2004).
  • the DNAs of interest also include DNA sequences which encode peptide (including antibody) antagonists of the proteins of Master Table 1, subtables IB or 1C.
  • a nucleic acid which specifically hybridizes to the human mRNA encoding a favorable human protein may be labeled or immobilized, and then used as a diagnostic agent in assays for that mRNA (or the corresponding cDNA).
  • a nucleic acid which specifically hybridizes to the human mRNA encoding an unfavorable human protein may be used in a like manner, or it may be used therapeutically to inhibit the expression of that human protein.
  • a part of the endogenous human gene encoding the unfavorable human protein (not necessarily the coding sequence). If it isn't, we can isolate the human gene using a probe designed on the basis of the known protein sequence. This could be a mixed probe, a probe with inosine in the degenerate positions, a guessed probe based on human codon preferences, or a combination of the above.
  • One form of therapy is anti-sense therapy.
  • a single stranded nucleic acid molecule which is complementary to the sense strand of the target sequence, is used as a therapeutic agent.
  • the nucleic acid molecule may be DNA, RNA, or an analogue which is resistant to degradation.
  • RNAi therapy uses a double-stranded RNA molecule. Long (>200 nt) double stranded RNAs are known to silence the expression of target genes by their participation in the RNA interference (RNAi) pathway.
  • RNAi RNA interference
  • the dsRNAs are processed into 20-25 nt small interfering RNAs (siRNAs) by the Dicer enzyme.
  • siRNAs assemble into RNA-induced silencing complexes (RISCs), and unwind.
  • RISCs RNA-induced silencing complexes
  • the siRNAs guide the RISCs to complementary messenger RNAs, which are subsequently degraded.
  • siRNAs can be prepared by direct chemical synthesis of the two strands, by in vitro transcription, or , in situ and in vivo, by siRNA expression vectors.
  • the in vitro siRNAs may be delivered by any suitable means, including lipid-mediated transfection and electroporation.
  • target sequences begin with AA dinucleotide and are 21 nt in length. More preferably, the target sequences have a 30-50% GC content, and are of high specificity to the target gene (e.g., not more than 16-17 contiguous pairs of homology to other genes). If the siRNA will be expressed from the RNA pol IE promoter, it is preferable that the target sequence not contain stretches of four successive T's or four successive A's.
  • the siRNA can be designed.
  • it comprises a hairpin structure, i.e., two inverted repeats (one binds the target sequence) which together form the stem of the hairpin structure, and a loop.
  • the loop size is preferably 3-23 nt, and the published loop sequences include AUG, CCC ⁇ UUCG, CCACC, CTCGAG, AAGCUU, CCACACC, and UUCAAGAGA.
  • the hairpin may optionally have a 5' overhang.
  • siRNAs designed according to proprietary and supposedly more accurate algorithms from Atnbion One may also purchase siRNAs designed according to proprietary and supposedly more accurate algorithms from Atnbion.
  • the database DNA appears to be a full-length cDNA or gDNA, that is, it encodes an entire, functional, naturally occurring protein, then it may be used in the expression of that protein.
  • the corresponding human gene is known in full-length, it maybe used to express the human protein.
  • Such expression may be in cell culture, with the protein subsequently isolated and administered exogenously to subjects who would benefit therefrom, or in vivo, i.e., administration by gene therapy.
  • any DNA encoding the same protein may be used for the same purpose, and a DNA encoding a protein which a fragment or a mutant of that naturally occurring protein which retains the desired activity, may be used for the purpose of producing the active fragment or mutant.
  • the encoded protein of course has utility therapeutically and, in labeled or immobilized form, diagnostically.
  • the database DNA may be used as a hybridization probe to isolate the full-length DNA from a suitable DNA (cDNA or gDNA) library.
  • cDNA or gDNA DNA
  • Stringent hybridization conditions are appropriate, that is, conditions in which the hybridization temperature is 5-10 deg. C. below the Tm of the DNA as a perfect duplex.
  • the partial DNA encodes a biologically functional fragment of the cognate protein, it maybe used in a manner similar to the full length DNA, i.e., to produce the functional fragment.
  • mouse gene which encodes that mouse protein is known (which is almost always going to be the case)
  • mouse gene or its cDNA, or a fragment of the gene or cDNA
  • the mouse gene may be used experimentally to isolate the homologous human gene, and the human protein then deduced from the human gene. For particulars, see "genomics cases”.
  • Molecules which bind favorable and unfavorable proteins, or the corresponding nucleic acids may be identified by screening libraries, especially combinatorial libraries, as described below. If the binding target is an unfavorable protein, or the corresponding nucleic acid, the binding molecules may further be screened for antagonist activity. The antagonism may be, e.g., at the receptor level or at the gene expression level. .
  • Combinatorial libraries of special interest are protein/peptide libraries (including antibody, antibody fragment and single chain antibody libraries), nucleic acid libraries, peptoid libraries, peptoid nucleic acid (PNA) libraries, and small organic molecule libraries.
  • library generally refers to a collection of chemical or biological entities which are related in origin, structure, and/or function, and which can be screened simultaneously for a property of interest. Libraries may be classified by how they are constructed (natural vs. artificial diversity; combinatorial vs. noncombinatorial), how they are screened (hybridization, expression, display), or by the nature of the screened library members (peptides, nucleic acids, etc.). For definitions of different types of libraries, see “genomics cases”.
  • combinatorial library refers to a library in which the individual members are either systematic or random combinations of a limited set of basic elements, the properties of each member being dependent on the choice and location of the elements incorporated into it.
  • the members of the library are at least capable of being screened simultaneously. Randomization may be complete or partial; some positions may be randomized and others predetermined, and at random positions, the choices maybe limited in a predetermined manner.
  • the members of a combinatorial library may be oligomers or polymers of some kind, in which the variation occurs through the choice of monomelic building block at one or more positions of the oligomer or polymer, and possibly in terms of the connecting linkage, or the length of the oligomer or polymer, too.
  • the members may be nonoligomeric molecules with a standard core structure, like the 1,4-benzodiazepine structure, with the variation being introduced by the choice of substituents at particular variable sites on the core structure.
  • the members may be nonoligomeric molecules assembled like a jigsaw puzzle, but wherein each piece has both one or more variable moieties (contributing to library diversity) and one or more constant moieties (providing the functionalities for coupling the piece in question to other pieces).
  • chemical building blocks are at least partially randomly combined into a large number (as high as 10 15 ) of different compounds, which are then simultaneously screened for binding (or other) activity against one or more targets.
  • a “composite combinatorial library” is a mixture of two or more simple libraries, e.g., DNAs and peptides, or peptides, peptoids, and PNAs, or benzodiazepines and carbamates.
  • the number of component simple libraries in a composite library will, of course, normally be smaller than the average number of members in each simple library, as otherwise the advantage of a library over individual synthesis is small.
  • Libraries may be characterized by such parameters as size and diversity, see “genomics cases”.
  • the library members maybe presented as solutes in solution, or immobilized on some form of support.
  • the support may be living (cell, virus) or nonliving (bead, plate, etc.).
  • the supports may be separable (cells, virus particles, beads) so that binding and nonbinding members can be separated, or nonseparable (plate).
  • the members will normally be placed on addressable positions on the support.
  • the advantage of a soluble library is that there is no carrier moiety that could interfere with the binding of the members to the support.
  • the advantage of an immobilized library is that it is easier to identify the structure of the members which were positive.
  • the target When screening a soluble library, or one with a separable support, the target is usually immobilized.
  • the target When screening a library on a nonseparable support, the target will usually be labeled.
  • the library may be an library of oligonucleotides (linear, cyclic or branched), and these may include nucleotides modified to increase nuclease resistance and/or chemical stability. Libraries of potential anti-sense or RNAi molecules are of particular interest, but oligonucleotides can also be receptor antagonists.
  • the library may be a library of peptides, linear, cyclic or branched, and may or may not be limited in composition to the 20 genetically encoded amino acids.
  • a peptide library may be an oligopeptide library or a protein library.
  • the oligopeptides are at least five, six, seven or eight amino acids in length. Preferably, they are composed of less than 50, more preferably less than 20 amino acids. In the case of an oligopeptide library, all or just some of the residues may be variable.
  • the oligopeptide maybe unconstrained, or constrained to a particular conformation by, e.g., the participation of constant cysteine residues in the formation of a constraining disulfide bond.
  • Proteins like oligopeptides, are composed of a plurality of amino acids, but the term protein is usually reserved for longer peptides, which are able to fold into a stable conformation.
  • a protein maybe composed of two or more polypeptide chains, held together by covalent or noncovalent crosslinks. These may occur in a homooligomeric or a heterooligomeric state.
  • a peptide is considered a protein if it (1) is at least 50 amino acids long, or (2) has at least two stabilizing covalent crosslinks (e.g., disulfide bonds). Thus, cono toxins are considered proteins.
  • the proteins of a protein library will be characterizable as having both constant residues (the same for all proteins in the library) and variable residues (which vary from member to member). This is simply because, for a given range of variation at each position, the sequence space (simple diversity) grows exponentially with the number of residue positions, so at some point it becomes inconvenient for all residues of a peptide to be variable positions. Since proteins are usually larger than oligopeptides, it is more common for protein libraries than oligopeptide libraries to feature variable positions. In the case of a protein library, it is desirable to focus the mutations at those sites which are tolerant of mutation.
  • protein libraries can be considered a special case of the biased peptide library.
  • oligopeptide libraries there are several reasons that one might screen a protein library instead of an oligopeptide library, including (1) a particular protein, mutated in the library, has the desired activity to some degree already, and (2) the oligopeptides are not expected to have a sufficiently high affinity or specificity since they do not have a stable conformation.
  • variable domains of an antibody possess hypervariable regions and hence, in some embodiments, the protein library comprises members which comprise a mutant of VH or VL chain, or a mutant of an antigen-specific binding fragment of such a chain.
  • VH and VL chains are usually each about 110 amino acid residues, and are held in proximity by a disulfide bond between the adjoing CL and CHl regions to form a variable domain. Together, the VH, VL, CL and CHl form an Fab fragment.
  • the hypervariable regions are at 31- 35, 49-65, 98-111 and 84-88, but only the first three are involved in antigen binding.
  • VH and VL chains there is variation among VH and VL chains at residues outside the hypervariable regions, but to a much lesser degree.
  • a sequence is considered a mutant of a VH or VL chain if it is at least 80% identical to a naturally occurring VH or VL chain at all residues outside the hypervariable region.
  • such antibody library members comprise both at least one VH chain and at least one VL chain, at least one of which is a mutant chain, and which chains may be derived from the same or different antibodies.
  • VH and VL chains may be covalently joined by a suitable linker moiety, as in a "single chain antibody", or they may be noncovalently joined, as in a naturally occurring variable domain, if the joining is noncovalent, and the library is displayed on cells or virus, then either the VH or the VL chain may be fused to the carrier surface/coat protein.
  • the complementary chain may be co-expressed, or added exogenously to the library.
  • the members may further comprise some or all of an antibody constant heavy and/or constant light chain, or a mutant thereof.
  • a peptoid is an analogue of a peptide in which one or more of the peptide bonds (- NH-CO-) are replaced by pseudopeptide bonds, which may be the same or different. It is not necessary that all of the peptide bonds be replaced, i.e., a peptoid may include one or more conventional amino acid residues, e.g., proline.
  • a peptide bond has two small divalent linker elements, -NH- and -CO-.
  • a preferred class of psuedopeptide bonds are those which consist of two small divalent linker elements. Each may be chosen independently from the group consisting of amine (- NH-), substituted amine (-NR-), carbonyl (-CO-), thiocarbonyl (-CS-),methylene (-CH2-), monosubstituted methylene (-CHR-), disubstituted methylene (-CR1R2-), ether (-O-) and thioether (-S-).
  • the more preferred pseudopeptide bonds include:
  • a single peptoid molecule may include more than one kind of pseudopeptide bond.
  • one may vary (1) the side chains attached to the core main chain atoms of the monomers linked by the pseudopeptide bonds, and/or (2) the side chains (e.g., the -R of an -NRCO-) of the pseudopeptide bonds.
  • the monomeric units which are not amino acid residues are of the structure -NR1-CR2-C0-, where at least one of Rl and R2 are not hydrogen. If there is variability in the pseudopeptide bond, this is most conveniently done by using an - NRCO- or other pseudopeptide bond with an R group, and varying the R group. In this event, the R group will usually be any of the side chains characterizing the amino acids of peptides, as previously discussed.
  • R group of the pseudopeptide bond is not variable, it will usually be small, e.g., not more than 10 atoms (e.g., hydroxyl, amino, carboxyl, methyl, ethyl, propyl). If the conjugation chemistries are compatible, a simple combinatorial library may include both peptides and peptoids.
  • PNA oligomer libraries have been made; see e.g. Cook, 6,204,326.
  • a PNA oligomer is here defined as one comprising a plurality of units, at least one of which is a PNA monomer which comprises a side chain comprising a nucleobase.
  • the classic PNA oligomer is composed of (2-aminoethyl)glycine units, with nucleobases attached by methylene carbonyl linkers. That is, it has the structure H- (-HN-CH 2 -CH r N(-CO-CH 2 -B)-CH 2 -CO-) n -OH
  • outer parenthesized substructure is the PNA monomer.
  • the nucleobase B is separated from the backbone N by three bonds, and the points of attachment of the side chains are separated by six bonds.
  • the nucleobase maybe any of the bases included in the nucleotides discussed in connection with oligonucleotide libraries.
  • the bases of nucleotides A, G, T, C and U are preferred.
  • a PNA oligomer may further comprise one or more amino acid residues, especially glycine and proline.
  • the small organic compound library (“compound library”, for short) is a combinatorial library whose members are suitable for use as drugs if, indeed, they have the ability to mediate a biological activity of the target protein.
  • Bunin, et al. generated a I, 4- benzodiazepine library of 11,200 different 2-aminobenzophenone derivatives prepared from 20 acid chlorides, 35 amino acids, and 16 alkylating agents. See Bunin, et al., Proc. Nat. Acad. Sci. USA, 91 :4708 (1994).
  • 2-aminobenzophenone derivatives Since only a few 2-aminobenzophenone derivatives are commercially available, it was later disjoined into 2-aminoarylstannane, an acid chloride, an amino acid, and an alkylating agent. Bunin, et al., Meth. EnzymoL, 267:448 (1996).
  • the arylstannane may be considered the core structure upon which the other moieties are substituted, or all four may be considered equals which are conjoined to make each library member.
  • Heterocylic combinatorial libraries are reviewed generally in Nefzi, et al., Chem. Rev., 97:449-472 (1997).
  • Examples of candidate simple libraries which might be evaluated include derivatives of the following:
  • Cyclic Compounds Containing One Hetero Atom Heteronitrogen pyrroles pentasubstituted pyrroles pyrrolidines pyrrolines prolines indoles beta-carbolines pyridines dihydropyridines
  • the preferred animal subject of the present invention is a mammal.
  • mammal an individual belonging to the class Mammalia.
  • the invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well.
  • Preferred nonhuman subjects are of the orders Primata (e.g., apes and monkeys), Artiodactyla or Perissodactyla (e.g., cows, pigs, sheep, horses, goats), Carnivora (e.g., cats, dogs), Rodenta (e.g., rats, mice, guinea pigs, hamsters), Lagomorpha (e.g., rabbits) or other pet, farm or laboratory mammals.
  • Primata e.g., apes and monkeys
  • Artiodactyla or Perissodactyla e.g., cows, pigs, sheep, horses, goats
  • Carnivora e.g., cats, dogs
  • prevention is intended to include “prevention,” “suppression” and “treatment.” Unless qualified, the term “prevention” will be understood to refer to both prevention of the induction of the disease, and to suppression of the disease before it manifests itself clinically.
  • the preventative or prophylactic use of a pharmaceutical usually involves identifying subjects who are at higher risk than the general population of contracting the disease, and administering the pharmaceutical to them in advance of the clinical appearance of the disease. The effectiveness of such use is measured by comparing the subsequent incidence or severity of the disease, or of particular symptoms of the disease, in the treated subjects against that in untreated subjects of the same high risk group.
  • a particular group e.g., a particular age, sex, race, ethnic group, etc.
  • prophylaxis for the general population, and not just a high risk group. This is most likely to be the case when essentially all are at risk of contracting the disease, the effects of the disease are serious, the therapeutic index of the prophylactic agent is high, and the cost of the agent is low.
  • a prophylaxis or treatment may be curative, that is, directed at the underlying cause of a disease, or ameliorative, that is, directed at the symptoms of the disease, especially those which reduce the quality of life.
  • the protection provided need not be absolute, provided that it is sufficient to carry clinical value.
  • An agent which provides protection to a lesser degree than do competitive agents may still be of value if the other agents are ineffective for a particular individual, if it can be used in combination with other agents to enhance the level of protection, or if it is safer than competitive agents.
  • It is desirable that there be a statistically significant (p 0.05 or less) improvement in the treated subject relative to an appropriate untreated control, and it is desirable that this improvement be at least 10%, more preferably at least 25%, still more preferably at least 50%, even more preferably at least 100%, in some indicia of the incidence or severity of the disease or of at least one symptom of the disease.
  • At least one of the drugs of the present invention may be administered, by any means that achieve their intended purpose, to protect a subject against a disease or other adverse condition.
  • the form of administration may be systemic or topical.
  • administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, transdermal, or buccal routes.
  • parenteral administration can be by bolus injection or by gradual perfusion over time.
  • a typical regimen comprises administration of an effective amount of the drug, administered over a period ranging from a single dose, to dosing over a period of hours, days, weeks, months, or years.
  • suitable dosage of a drag of the present invention will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment;, and the nature of the effect desired.
  • the most preferred dosage can be tailored to the individual subject, as is understood and determinable by one of skill in the art, without undue experimentation. This will typically involve adjustment of a standard dose, e.g., reduction of the dose if the patient has a low body weight.
  • a drag Prior to use in humans, a drag will first be evaluated for safety and efficacy in laboratory animals. In human clinical studies, one would begin with a dose expected to be safe in humans, based on the preclinical data for the drag in question, and on customary doses for analogous drugs (if any). If this dose is effective, the dosage may be decreased, to determine the minimum effective dose, if desired. If this dose is ineffective, it will be cautiously increased, with the patients monitored for signs of side effects.
  • the total dose required for each treatment may be administered by multiple doses or in a single dose.
  • the protein may be administered alone or in conjunction with other therapeutics directed to the disease or directed to other symptoms thereof.
  • Typical pharmaceutical doses, for adult humans, are in the range of 1 ng to 1Og per day, more often 1 mg to Ig per day.
  • the appropriate dosage form will depend on the disease, the pharmaceutical, and the mode of administration; possibilities include tablets, capsules, lozenges, dental pastes, suppositories, inhalants, solutions, ointments and parenteral depots.
  • the drug may be administered in the form of an expression vector comprising a nucleic acid encoding the peptide; such a vector, after incorporation into the genetic complement of a cell of the patient, directs synthesis of the peptide.
  • Suitable vectors include genetically engineered poxviruses (vaccinia), adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses which are or have been rendered nonpathogenic.
  • the compounds of the present invention may be used, in labeled or immobilized form, as assay reagents.
  • assay formats for assay formats, signal producing systems, labels and supports, please see “genomics cases”, hereby incorporated by reference in their entirety.
  • mice were fed either the high fat (HF) diet or a standard lab chow diet. Body weight was monitored bi-weekly. Fasting glucose and insulin levels were measured after various periods of time after commencement of the high fat diet. Consumption of the HF diet resulted in significant, progressive increases in body weight and fasting insulin levels in comparison to consumption of the Std diet. Fasting glucose levels of mice on the HF diet were dramatically increased at the first time point assayed (2 weeks) and remained high through the duration of the experiment. At each time point, several diabetic and control mice were sacrificed and a number of tissues collected.
  • Tissues were harvested at regular intervals during the onset and progression of obesity and type 2 diabetes. Each tissue sample were divided for concurrent histology and proteomic studies. IQ the proteomics studies, separation and visualization of the proteins at a specific time in a specific tissue or "tissue specific protein profile" were established by two-dimensional gel electrophoresis and the relative abundance of each protein were determined by densitometry. Proteins that are differentially expressed or modified as a consequence of obesity and diabetes were excised from the gels and analyzed by mass spectrometry. (Predictions based on the peptide mass fingerprints and deductive reasoning were can be confirmed by western blot analysis and/or irnmunohistochernistry.)
  • Obesity and subsequent hyperinsulinemia and hyperglycemia were induced by feeding a group of 3 week old mice (50 C57BL/6 males) a high-fat diet (Bio-Serve, Frenchtown, NJ, #F1850 High Carbohydrate-High Fat; 56% of calories from fat, 16% from protein and 27% from carbohydrates).
  • Another group of 3 week old mice (20 C57B1/6 males) were fed the normal control diet (PMI Nutrition International Inc., Brentwood, MO, Prolab RMH3000; 14% of calories from fat, 16% from protein and 60% from carbohydrates). The mice were placed onto the respective diets immediately following weaning. Animal weights were determined weekly.
  • Plasma Blood is collected from the tail vein of fasted mice, between 2 p.m. and 4 p.m., using a heparinized capillary tube and stored on ice. Plasma was separated from the cellular components by centrifugation for 10 minutes at 7000 x g and then stored at -80 ° C. Insulin concentrations were determined using the Ultra-Sensitive Rat Rnsulin ELISA kit and rat insulin standards (both from ALPCO: Windham, NH), essentially as instructed by the manufacturer. Values were adjusted by a factor of 1.23 (as determined by the manufacturer) to correct for species differences in the antibody.
  • Normal weight, normal fasting blood glucose and normal fasting plasma insulin levels are defined as the respective mean values of the animals fed the control diet.
  • mice were sacrificed at the appropriate times and the 16 different tissues (Liver, Gastrocnemius, Pancreas, Epididymal Fat, Subcutaneous Fat, Kidney, Stomach Brain, Tongue, Heart, Skin, Small Intestine, Testes, Spleen, Bone & Serum) are harvested. All tissues were harvested at regular intervals for up to 14 time-points during the onset and progression of obesity and type 2 diabetes. Mice were sacrificed by cervical dislocation in the absence of anesthesia. (Euthanasia will be by CO 2 inhalation for animals that are deemed to be suffering.) Each organ is quickly removed and weighed and then maintained on ice during the dissection. This is desirable for the simultaneous preservation of multiple tissues for three distinct applications: proteomics, histology, and KNA analysis. The tissue was placed in 10% formalin for histology or frozen in cryogenic vials with liquid nitrogen for proteomics and RNA analysis.
  • Tissues were dissected in a manner which struck a balance between speed and specificity.
  • the brain for example, is divided into two hemisphere and each hemisphere is divided into cortex, cerebellum, and midbrain, but the liver is not separated into lobes and the heart is not separated into individual chambers.
  • Muscle, skin, WAT, and heart were homogenized in IEF buffer containing non- ionic chaotropes (7 M urea and 2 M thiourea) and zwitterionic detergent (2-4% CHAPS), whereas kidney, liver, pancreas, and brain were lysed by dounce homogenization with a tight-fitting pestle in ice cold sterile lysis buffer containing 0.25 M Sucrose, 50 mM Tris- HCl pH 7.6, 25 mM KCl, 5 mM MgCl 2 , 2 mM DTT and protease inhibitor cocktail (94).
  • the homogenate was placed in tubes and centrifuged at 25,000 rpm (Beckman LE 30) to remove nuclei and other organelles.
  • the supernatant was layered over a 1.5 ml cushion of lysis buffer containing 30% (w/v) sucrose and centrifuged in Beckman LE 80 at 36,000 rpm (130,000 g) for 2.5 hr at 4°C using SW60 rotor.
  • the supernatant (S130) was aliquoted, and stored at -80°C. After removal of the sucrose interface, the polysomal pellet was rinsed twice and then resuspended in -250 ⁇ l of lysis buffer. Samples were maintained on ice until aliquoted, frozen on dry ice and stored at -80°C for subsequent use.
  • Serum was collected by decapitation following cervical dislocation. After removal of cellular component by centrifugation at 7000Xg for 10 min, serum was stored at -80C.
  • IEF isoelectric focusing
  • the protein concentration of each preparation was determined by spectrophotometry (Beckman DU-640) using the Bradford method (BioRad) or the Lowry method. Typically, these fractions yield 100 -3000 ⁇ l samples containing 7-12 ⁇ g protein/ ⁇ l.
  • the yield for crude tissue homogenates ranges from 500 ⁇ l at a concentration of ⁇ 22 ⁇ g/ ⁇ l for white adipose tissue to about -50 ⁇ g/ ⁇ l in 2 ml for liver and skeletal muscle.
  • Serum protein samples were diluted with sample buffer (5M urea, 2M thiourea, 2% CHAPS, 2% SB3-10, 0.1% Bio-lytes, 5OmM Tris/HCl pH 8.8) at final concentration of up to 4mg/ml. Protein was reduced by tributyl phosphine (TBP) for 2hours at room temperature to break disulfide bonds. Alkylating agent, iodoacetamide (IAA; 3.2mg/ml), was added to prevent spontaneous re-oxidation of disulfide bonds.
  • sample buffer 5M urea, 2M thiourea, 2% CHAPS, 2% SB3-10, 0.1% Bio-lytes, 5OmM Tris/HCl pH 8.8
  • TBP tributyl phosphine
  • Alkylating agent iodoacetamide (IAA; 3.2mg/ml) was added to prevent spontaneous re-oxidation of disulfide bonds.
  • the alkylated samples were added to immobilized pH gradient (IPG) strips (Bio- rad) and focused at 4000V for 20,000-30,000 V hrs.
  • the second dimension separation was performed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins based on their masses.
  • Gel images were captured with a high-resolution CCD camera (e.g. Versa-Doc 3000, Bio-Rad) or a laser-scanning device (Fuji FLA-3000G).
  • PDQuest image analysis software package from Bio-Rad was used to interpret and quantify 2-D gel patterns. Before comparing spot quantities between gels, each gel image was optimized and adjusted for image background, spot intensity, streaking, etc., and then normalized to compensate for any variation in spot intensity that is not due to differential protein expression, i.e., variation caused by loading, staining, and imaging between gels.
  • the spot detection wizard function of the PDQuest software helped to optimize the conditions needed to detect all the spots in the gel.
  • the end result of spot detection was three separate images of the same gel: the original gel scan, which is unchanged; the filtered image, with noise and background removed; and a synthetic image, containing ideal Gaussian representations of the spots in the original scan. These Gaussian spots were used for matching and quantization.
  • MALDI-TOF Matrix-Assisted Laser Desorbtion/ Ionization-Time of Flight
  • MALDI-TOF Matrix-Assisted Laser Desorbtion/ Ionization-Time of Flight
  • Each gel spot was assigned an identifier, listed in our tables as the "Orig Well ID”.
  • Protein samples (individual gel spots) were digested with trypsin and subjected to mass spectrometric analysis by MALDI-TOF (Voyager-DE Pro, The Applied Biosystems). At this point, each gel spot was assigned a second identifier, listed in our table as the "Final Well ID”.
  • a peak list was extracted from each mass spectrum obtained by MALDI-TOF and submitted to Matrix Science's Mascot (http://www.matrixscience.corn) for a preliminary database search.
  • the gel spot location data is formatted to facilitate comparisons between gels and with the proteomic databases such as those maintained by the Danish Centre for Human Genome Research at the University of Aarhus (105) (http://biobase.dk/cgi-bm/celis') and the ExPASy (Expert Protein Analysis System) proteomics server fhttp://www.expasy.org/) maintained by the Swiss Institute of Bioinformatics (SIB) at the University of Geneva (106).
  • These databases have tools designed to overcome the enormous computational challenges associated with proteomic analysis. For example, it is possible to search databases (e.g. SWISS-PROT, TrEMBL) for proteins whose theoretical isoelectric point (Pi) 5 molecular weight (Mw), amino acid composition or peptide mass fingerprint match experimentally derived data. Additional tools predict post-translational modifications and protein structure.
  • the quality of each mass spectrum was assessed in terms of resolution and noise. When the mass spectrum was of sufficient quality to use for an analysis, it was assessed for common additional peaks corresponding to peptides from the auto-digestion of trypsin or matrix molecules and keratins contamination. Once a spectrum of acceptable quality was obtained, a peak list was generated for database search.
  • the NCBI database was searched using the specified software.
  • database search the following parameters were specified for MASCOT MS analysis:
  • Mass tolerance instrument dependent, typically 50 ppm or better.
  • the appropriate parameters for an MS-Fit search are same as those listed above for the Mascot MS search. If an MS-Fit option is not listed above, then the default setting is appropriate.
  • the top hit is not necessarily a good hit, nor is it necessarily the correct hit. A number of factors need to be considered. Species. If the search was performed on the entire database, then a hit could be on a non- mouse protein. Such a hit would ordinarily be disregarded, unless there was reason to think either that the database protein was improperly annotated, or that the gel protein was a contaminant.
  • Score A score above ⁇ 10 ⁇ 5 is generally considered a good hit, but this is not always the case.
  • MS-Fit there is no absolute value for a score that makes the hit a certainty. Furthermore, a low score does not necessarily indicate that the hit is not the correct hit; it simply indicates that the identification should not be used without further confirmation. MS/MS analysis has confirmed identifications for PMF hits with scores as low as 50.
  • Mass errors should be somewhat uniform.
  • % Coverage Typical % coverage for MALDI data is 20-50%. Higher % coverage indicates that a larger portion of the protein was accounted for by the peptides observed in the spectrum. Very low % coverage, in combination with a large protein MW was considered to be a spurious hit. Location in the protein ofthepeptides that were matched. When peptides that were all located in one end of the protein was considered to be a truncated form of the protein and explained inconsistencies between the experimental and theoretical MW and pi values.
  • MS/MS is the selection of a single peptide from the tryptic digest by the mass spectrometer, followed by the fragmentation of that peptide within the mass spectrometer and the acquisition of its fragment ion spectrum. Given that the fragmentation pattern of a given peptide is specific to its sequence and that the mass of the intact peptide is known, identification obtained by only one or two peptides in the PMF spectrum is often considered accurate.
  • MS/MS analysis was used for protein identification or to confirm the MS analysis.
  • MS/MS can be used to provide sequence information that can be used for BLAST searching, or identify the presence or location of post-translational modifications.
  • the primary tool used was the Mascot from Matrix Science.
  • the search tool performs a theoretical ⁇ in silico) digestion of the proteins in the database using the selected enzyme, generating a list of theoretical peptides for each protein.
  • the MS/MS peak list is submitted to the database, the parent ion is compared to the results from the in silico digestion.
  • a theoretical fragmentation is carried out on all peptides from the in silico digestion that are within the selected mass tolerance of the parent ion.
  • the ion types that are calculated are determined by the parameters selected in the search. In Mascot, these ion types are determined by the selection of instrument type.
  • the ion types may be individually selected, or they are determined by the instrument type selected.
  • the peak list is then compared against the masses generated by the theoretical fragmentation to determine if the fragmentation pattern of a peptide in the database matches the spectrum. If a matching peptide is found, a ranking or score is generated.
  • the search parameters used for the MS/MS searches are:
  • NCBI Database - NCBI is more complete, but SwissProt is more highly annotated and faster to search. NCBI has more entries for certain species, such as mouse and human.
  • Taxonomy Select the desired species or choose "All.”
  • Peptide Tolerance Parent ion mass accuracy should be 50ppm or better.
  • MS/MS Tolerance - Fragment ion mass accuracy may differ for TOF/TOF and MALDI QTOF data, but is typically better than 0.5 Da.
  • the MALDI QTOF is calibrated for acquiring PMF spectra, and so the calibration at the low end of the mass spectrum (below 500 Da) may have a higher mass error than the rest of the spectrum.
  • the TOF/TOF has separate MS and MS/MS calibrations and should be relatively consistent across the entire mass range.
  • Protein database searches were conducted with the then-current version of BLAST P, see Altschul etal. (1997), supra. Searches employed the default parameters, unless otherwise stated.
  • the scoring matrix was BLOSUM62, with gap costs of 11 for existence and 1 for extension. Results are shown in Master Table 1.
  • Ref ' indicates that NCBF s RefSeq is the source database.
  • the identifier that follows is a RefSeq accession number, not a GenBank accession number.
  • RefSeq sequences are derived from GenBank and provide non-redundant curated data representing our current knowledge of known genes. Some records include additional sequence information that was never submitted to an archival database but is available in the literature. A small number of sequences are provided through collaboration; the underlying primary sequence data is available in GenBank, but may not be available in any one GenBank record. RefSeq sequences are not submitted primary sequences.
  • RefSeq records are owned by NCBI and therefore can be updated as needed to maintain current annotation or to i n c o r p o r a t e a d d i t i o n a l s e q u e n c e i n fo r m a t i o n . " S e e a l s o http ://www.ncbi.nlm.nih. gov/LocusLink/refseq .html
  • RNA isolated from subject mice will be resolved by agarose gel electrophoresis through a 1% agarose, 1 % formaldehyde denaturing gel, transferred to positively charged nylon membrane, and hybridized to a probe labeled with [32P] dCTP that was generated from the aforementioned gene or fragment using the Random Primed DNA Labeling Kit (Roche, Palo Alto, CA), or to a probe labeled, with digoxigenin (Roche Molecular Biochemicals, Indianapolis, IN), according to the manufacturer's instructions.
  • Transgenic expression may be used to confirm the favorable or unfavorable role of the identified mouse or human protein.
  • a mouse is engineered to overexpress the favorable or unfavorable mouse protein in question.
  • a mouse is engineered to express the corresponding favorable or unfavorable human protein.
  • a nonhuman animal other than a mouse such as a rat, rabbit, goat, sheep or pig, is engineered to express the favorable or unfavorable mouse or human protein.
  • Tissue Collection We do not attempt to isolate distinct cells such as neurons and glia in brain tissue or ⁇ -islet cells in pancreas, in part because our rapid dissection procedures are desirable to ensure the integrity of the tissue for multiple applications. We are confident that the loss of cell-type specificity will not interfere with our ability to detect important tissue-specific changes that are attributable to stage and severity of obesity and diabetes.
  • Two-dimensional gel-electrophoresis (2-D gel electrophoresis) is a superior technique for the simultaneous resolution of hundreds of proteins from complex mixtures such as the insulin-sensitive tissues of diabetic mice or those susceptible hyperglycemia-induced complications.
  • Coomassie lacks detection sensitivity ( ⁇ 8-10 ng; 102) and silver stain exhibits anonlinear dynamic range of detection (103).
  • Modified procedures may be used as needed to enrich samples for a particular protein to increase resolution.
  • serum contains labundant proteins like albumin and immunoglobulins, and it may be desirable to remove them to improve visualization of less abundant proteins.
  • modifications to these abundant proteins may be significant, like the hyperglycemia-induced modifications to hemoglobin (HbAl c) .
  • HbAl c hyperglycemia-induced modifications to hemoglobin
  • NCBI was used as a search database since it is more complete than others such as SwissProt. However, SwissProt is better annotated and faster to search.
  • the spot is identified as Apolipoprotein because:
  • the spot identified is a C-terminus fragment of a much larger protein and hence the scores are very low.
  • the spot is identified as Contrapsin because:
  • This Spot had low MS scores but we were able to show that 4 of a total of six peaks that were analyzed identified this spot to be contrapsin E17
  • The- MS-Fit data was considered secondary to the Mascot MS data, hence it was not relied on for identification purposes if none of the top 50 MS-FIT scorers were mouse proteins with high Mascot MS scores. It merely performed a confirmatory role.
  • mice that are hyperinsulineinic/hyperglycemic as a result of the high-fat diet were returned to the control diet with 10% kcal fat (Research Diets #D12450B) and monitored in accordance with the protocols described above.
  • the experiments commenced after prolonged exposure (4 months). Typically, the animals will have been diabetic for at least 2 months.
  • Example 3 We also monitored circulating levels of white adipose tissue (WAT)-specific proteins leptin and adiponectin (also called Acrp30, adipocyte complement related protein 3OkDa) because they are important barometers of obesity. Secretion of leptin is proportional to the body's energy stores in fat depots and it signals to the brain to reduce food intake (34,36,37). Adiponectin gene expression is induced during adipocyte differentiation and its secretion is stimulated by insulin. Adiponectin appears to increase tissue sensitivity to insulin.
  • WAT white adipose tissue
  • leptin also called Acrp30, adipocyte complement related protein 3OkDa
  • Adiponectin gene expression is induced during adipocyte differentiation and its secretion is stimulated by insulin. Adiponectin appears to increase tissue sensitivity to insulin.
  • the master tables reflect applicants' analysis of the proteomics data.
  • Master Tables 101-103 correlate each differentially expressed gel spot with one or more mouse proteins, using Mascot MS (Master Table 101), Mascot MS/MS of up to six protein fragments (Master Table 102), and/or MSFIT (Master Table 103).
  • Col. 1 Final Well ID# (the well into which the protein sample was placed immediately prior to mass spec analysis).
  • Col. 2 Apparent molecular weight (kDa).
  • Col. 6 Accession # of matched mouse protein in sequence database.
  • Col. 7 Name of matched mouse protein in sequence database.
  • Col. 8 Calculated molecular weight (Da) of aligned mouse protein.
  • Col. 9 Calculated pi of matched mouse protein.
  • Col. 10 Match Score (Mascot MS implementation of Probability-based MOWSE score). Higher number is better.
  • Col. 11 E value of match. Lower number (i.e., more negative exponent) is better.
  • Col. 12 # of matched peaks, expressed in form X: Y, where Y is the total number of mass spectrometry peaks for the analyzed protein, and X is the number which could be matched to a predicted peptide fragment of the database mouse protein.
  • Col. 13 % covered. The percentage of the matched protein which corresponds to the predicted peptide fragments with the matched mass peaks.
  • Col. 1 Final Well ID# (see Table 101).
  • Col. 3 fragment size (Da).
  • Col. 4 Accession # of matched mouse protein in sequence database.
  • Col. 5 Name of matched mouse protein in sequence database.
  • Col. 6 Match score. (Mascot MS/MS implementation of Probability-based MOWSE score, a.k.a.
  • Col. 7 E value of match.
  • the final well ID# links this table to Master Table 101. If there any ambiguities, the stated behavior can also be considered. Up to six fragments can be listed for a single well (gel sample). For each fragment, one or more matched mouse proteins are listed.
  • Col. 4 Accession # of matched mouse protein in sequence database.
  • Col. 5 Name of matched mouse protein in sequence database.
  • Col. 6 Calculated molecular weight of matched mouse protein.
  • Col. 7 Calculated pi of matched mouse protein.
  • Col. 8 MOWSE score of match. (MS -FIT implementation of Probability-based MOWSE score).
  • Col.9 Number of matched peaks: Number of Total Peaks. The symbol “//” indicates “of, so “3//10” means 3 matched peaks out of 10 total peaks.
  • the Final Well ID# links this table to Master Tables 10.1 and 102. If there are any ambiguities, the apparent MW and pi can also be considered. For each gel spot, one or more matched mouse proteins are listed.
  • Master Table 1 For each differentially expressed mouse protein identified by Master Tables 101-103 , Master Table 1 identifies:
  • Cols. 2-4 The mouse protein database accession #. The choice of column indicates the source of the mouse protein identification, as follows: col. 2 (Mascot MS), col. 3 (Mascot MS/MS), and col. 4 (MSFIT).
  • the mouse protein accession # acts as the principal link between Master Tables 101-103 and Master Table 1, because the mouse protein accession # from Master Tables 101-103 is used as the query sequence in the search which generates the human accession#s of the human protein homologues, as set forth in Master Table 1.
  • the Final Well ID# acts as a further link, and also helps to visually organize the hits in such away that one can see which mouse accession #s correspond to the same gel spot.
  • the numerical value is the ratio of the greater value to the lesser value. If this ratio is at least two fold, the degree of differential expression is considered strong. Usually only mouse proteins exhibiting at least one strong differential expression behavior are listed in the Master Table; exceptions may be noted in the Examples.
  • a ratio may be given as a negative number. This does not have its usual mathematical meaning; it is merely a flag that in the comparison, the former value was less than the latter one, i.e., the gene was favorable.
  • any negative value should be converted to a positive one by taking its absolute value.
  • Col. 6 A related human protein, identified by its database accession number. Usually, several such proteins are identified relative to each mouse protein. These proteins have been identified by BLASTP searches.
  • Col. 7 The name of the related human protein.
  • Col. 8 The score (in bits) for the alignment performed by the BLASTP program.
  • Col. 9 The E-value for the alignment performed by the BLASTP program.
  • bit score and E-value for the alignment is with respect to the alignment of the mouse protein of cols.2, 3 or 4 to the human protein of col. 6 by BlastP, according to the default parameters.
  • Master Table 1 is divided into three subtables on the basis of the behavior in col. 5. If a protein has at least one significantly favorable behavior, and no significantly unfavorable ones, it is put into Subtable IA. In the opposite case, it is put into Subtable IB. If its behavior is mixed, i.e., at least one significantly favorable and at least one significantly unfavorable, it is put into Subtable 1C. Note that this classification is based on the strongest observed differential expression behaviors for each of the three subject comparisons, C-HI, HI-D and C-D.
  • Emery EM, Schmid TL, Kahn HS, Filozof PP 1993 Areview of the association between abdominal fat distribution, health outcome measures, and modifiable risk factors. Am J Health Promot 7:342-53.
  • Clark MG Rattigan S, Clark DG 1983 Obesity with insulin resistance: experimental insights. Lancet 2:1236-40.
  • Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes. Cell 105:745-55.
  • GH growthhormone
  • Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a component of an intronic splicing enhancer complex that activates the splicing of the alternative exon 6A from chicken beta-tropomyosin pre-mRNA. J Biol Chem 277(19): 16614-23
  • any description of a class or range as being useful or preferred in the practice of the invention shall be deemed a description of any subclass (e.g., a disclosed class with one or more disclosed members omitted) or subrange contained therein, as well as a separate description of each individual member or value in said class or range.

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Abstract

Selon l'invention, des protéines de souris exprimées de façon différentielle dans la peau ont été identifiées, ainsi que des protéines humaines correspondantes, dans des comparaisons entre un tissu adipeux blanc normal et un tissu adipeux blanc présentant un état hyperinsulinémique, entre un tissu adipeux blanc présentant un état hyperinsulinémique et un tissu adipeux blanc présentant un état diabétique de type 2 et entre un tissu adipeux blanc normal et un tissu adipeux blanc présentant un état diabétique de type 2. Les molécules humaines ou des antagonistes de celles-ci peuvent être utilisés en protection contre l'hyperinsulinisme ou le diabète de type 2 ou contre leurs séquelles.
PCT/US2006/025373 2005-06-29 2006-06-29 Diagnostic d'un hyperinsulinisme et d'un diabete de type ii et protection contre ceux-ci a partir de proteines exprimees de façon differentielle dans la peau et identifiees par proteomique WO2007002830A2 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP2265949A2 (fr) * 2008-03-28 2010-12-29 Ohio University Isoformes de protéine pour diagnostic

Citations (2)

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Publication number Priority date Publication date Assignee Title
US6143491A (en) * 1996-11-12 2000-11-07 Millennium Pharmaceuticals, Inc. Therapeutic compositions and methods and diagnostic assays for type II diabetes involving HNF-1
US20030100014A1 (en) * 2001-11-23 2003-05-29 George Jackowski Apolipoprotein biopolymer markers predictive of type II diabetes

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Publication number Priority date Publication date Assignee Title
US6143491A (en) * 1996-11-12 2000-11-07 Millennium Pharmaceuticals, Inc. Therapeutic compositions and methods and diagnostic assays for type II diabetes involving HNF-1
US20030100014A1 (en) * 2001-11-23 2003-05-29 George Jackowski Apolipoprotein biopolymer markers predictive of type II diabetes

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HITTEL ET AL.: 'Proteome Analysis of Skeletal Muscle From Obese and Morbidly Obese Women' DIABETES vol. 54, May 2005, pages 1283 - 1288 *
KAHN ET AL.: 'Perspectives in Diabetes: Islet Amyloid: A Long-Recognized but Underappreciated pathological Feature of Type 2 Diabetes' DIABETES vol. 48, February 1999, pages 241 - 253 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2265949A2 (fr) * 2008-03-28 2010-12-29 Ohio University Isoformes de protéine pour diagnostic
EP2265949A4 (fr) * 2008-03-28 2013-02-20 Univ Ohio Isoformes de protéine pour diagnostic

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