EP1040125A1 - Gene a regulation glucosique - Google Patents

Gene a regulation glucosique

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Publication number
EP1040125A1
EP1040125A1 EP98954094A EP98954094A EP1040125A1 EP 1040125 A1 EP1040125 A1 EP 1040125A1 EP 98954094 A EP98954094 A EP 98954094A EP 98954094 A EP98954094 A EP 98954094A EP 1040125 A1 EP1040125 A1 EP 1040125A1
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European Patent Office
Prior art keywords
hmund
polypeptide
nucleotide sequence
sequence
expression
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EP98954094A
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German (de)
English (en)
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Melvin Silverman
Yong Song
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Individual
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Individual
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to an isolated glucose regulated gene and its protein expression product.
  • the invention also relates to methods of modulating the gene for treatment of hyperglycemia, glomerulosclerosis and renal cell apoptosis.
  • Renal failure caused by glomerulosclerosis is a major complication of insulin dependent ("IDDM”) and non insulin dependent (“NIDDM”) diabetes (1, 2). Renal failure is increasing in Europe and North America (3-5), due to a variety of factors, including an aging population, poor dietary habits, and longer survival of juvenile diabetics. About 25% of patients undergoing treatment of end stage renal disease (ESRD) in the US and Canada, have kidney failure (nephropathy) caused by diabetes (1). Although renal failure in diabetes is not well understood, significant advances have been made recently. There still remains a clear need to characterize the processes that cause diabetes related kidney failure. Renal disease occurs more frequently in IDDM than in NIDDM, and there is a strong genetic component associated with the former (1).
  • IDDM insulin dependent
  • NIDDM non insulin dependent
  • Diabetics have chronically elevated blood glucose levels (hyperglycemia). Hyperglycemia contributes to development of microvascular and renal complications. There is no doubt that controlling blood sugar reduces these complications (8).
  • diabetic glomerulosclerosis is caused by expansion of the mesangial matrix (1).
  • the main product of the mesangial matrix, collagen IV, is found throughout the expanded mesangium. This is characteristic of diabetic glomerulosclerosis (16, 17).
  • the mesangial cell is now considered to be involved in initiation of diabetic glomerulosclerosis.
  • Hyperglycemia is a necessary, but not sufficient condition for diabetic renal complications. Nevertheless, if hyperglycemia could be fully understood at the molecular level, this would permit targeted therapeutic intervention to prevent the hyperglycemia- induced component of diabetic complications. It would also help identify genes that afford cell protection or establish cell vulnerability to sustained, elevated glucose.
  • TGF ⁇ transforming growth factor beta
  • DAG-induced activation of MC PKC ⁇ 2 is responsible for the acute and even certain chronic changes associated with diabetic microvascular and renal complications (13).
  • Administration of a specific PKC ⁇ 2 inhibitor-LY333531 appears to prevent the in vivo and in vitro sequelae of hyperglycemia, described above (14).
  • PKC is a serine-threonine phosphorylation kinase. Many different PKC isoforms exist, and their specificity of action is attributable to their intracellular compartmentalization, which varies from cell to cell. All PKC isoforms contain 2 regulatory domains, C1 and C2, which bind DAG and Ca ++ , respectively, in addition to binding a kinase domain. Under resting conditions, the kinase domain is inactive due to its interaction with the C1 domain. When DAG binds to C1, dissociation occurs, allowing ATP to bind to the kinase region. This activates PKC. A drug named LY333531 acts by competing with ATP for binding at the kinase domain. The effect of this drug is to block PKC phosphorylation without affecting intracellular DAG levels (14,15).
  • PKC pathway is not well understood. Whether PKC activation is the dominant dysfunction in diabetic glomerulopathy is undetermined. Also unknown is whether other signaling pathways stimulated by hyperglycemia are capable of interacting with and modifying DAG induced PKC activation. It would be helpful if DAG activation of PKC (via binding to the C1 domain) and its interaction with other metabolic changes in glomeruli and microvasculature during hyperglycemia were characterized. This would lead to new treatments to control and prevent damage to glomerular and microvascular function caused by hyperglycemia and diabetes. (iv) Signaling Proteins that Belong to the Same Superfamily as PKC
  • Unc-13 one of the members of this family, encodes a phorbol ester/ diacylglycerol-binding protein in C. elegans. Initial evaluation suggested it had a role in neurotransmitter release. (20-23). Mammalian homologues (munc13s), munc13-1 , -2, and -3, were originally cloned from rat brain and similar to Unc-13 in that both possess DAG and Ca 2+ binding domains (20).
  • Hmunc13 a gene from human MC, Hmunc13, which is up-regulated by hyperglycemia. Hmunc13 mediates some of the acute and chronic changes in MC produced by exposure to hyperglycemia. These changes result in diabetic microvascular and renal damage, such as glomerulosclerosis and apoptosis.
  • Hmunc13 is a signaling molecule localized to the plasma membrane of renal mesangial cells, cortical epithelial cells and other cells, The topological organization is illustrated schematically in figure 7. There are functional extracellular RGD domains, and intracellular C1 and C2 domains. There is also an intracellular regulatory domain on Hmunc13 that targets and activates a serine threonine catalytic phosphatase subunit to the plasma membrane
  • Function of Hmund 3 and biologically functional equivalent nucleotide sequences The functional role for Hmund 3 involves intracellular signal transduction and regulation of cell attachment and migration. Hmund 3 acts through modulation of phosphatase activity. In this way, Hmund 3 phosphatase activation opposes downstream serine/threonine phosphorylation initiated in response to PKC and integrin activation.
  • Hmund 3 is activated in response to hyperglycemia-induced increases in DAG, causing (i) stimulation of phosphatase activity and, (ii) modulation of DAG-induced PKC ⁇ activation.
  • DAG activated pathways (i) PKC dependent and (ii) Hmund 3 dependent. These two pathways regulate two opposing cell phenotypes, PKC-proiiferation and hmund 3-apoptosis.
  • Hmund 3 and biologically functional equivalent nucleotide sequences is particularly useful for treatment and prevention of renal cell damage.
  • the invention is an isolated nucleotide sequence encoding a glucose regulated munc polypeptide.
  • the nucleotide is preferably from a kidney cell, human cortical epithelial cell or a cell from testis, ovaries, prostate gland, colon, brain and heart, more preferably a mesangial cell or a kidney cortical epithelial cell.
  • the nucleotide sequence preferably comprises a Hmund 3 polypeptide and all or part of the amino acid sequence in sequence (a) in Figure 1 [SEQ ID NO. 1].
  • the nucleotide sequence preferably comprises a Hmund 3 gene having all or part of the nucleotide sequence in Figure 8 [SEQ ID NO. 2].
  • the molecule preferably comprises at least 40% sequence identity to all or part of the nucleotide sequence of Figure 8.
  • the sequence is preferably selected from a group consisting of mRNA, cDNA, sense DNA, anti-sense DNA, single-stranded DNA and double-stranded DNA.
  • the nucleotide encodes an amino acid sequence of the invention.
  • the nucleotide sequence that encodes all or part of a Hmund 3 polypeptide preferably hybridizes to the nucleotide sequence of all or part of Figure 8 under high stringency conditions (e.g. a wash stringency of 0.2X SSC to 2X SSC, 0.1% SDS, at 65°C).
  • the invention also includes an isolated munc polypeptide, with the provisio that the polypeptide is not found in a mammalian central nervous system.
  • the polypeptide of preferably has transmembrane ECM-cell signaling activity and DAG and Ca ++ activated phosphatase activity and more preferably includes all or part of the Hmund 3 amino acid sequence in sequence (a) in Figure 1 [SEQ ID NO: 1].
  • the invention also includes amimetic of the purified and isolated polypeptide.
  • the polypeptide preferably has at least 40% sequence identity to all or part of the amino acid sequence (a) in Figure 1 [SEQ ID NO: 1] .
  • the polypeptide is preferably from a mammalian kidney cell. It is useful for inducing apoptosis and vesicle trafficking.
  • the invention also includes a recombinant DNA comprising a DNA molecule the invention and a promoter region, operatively linked so that the promoter enhances transcription of said DNA molecule in a host cell.
  • the invention also includes a system for the expression of Hmund 3, comprising an expression vector and Hmund 3 DNA inserted in the expression vector.
  • the expression vector preferably comprises a plasmid or a virus.
  • the invention also includes a cell transformed by the expression vector.
  • the invention also includes a method for expressing Hmund 3 polypeptide comprising: transforming an expression host with a Hmund 3 DNA expression vector and cuituring the expression host.
  • the method preferably also includes isolating Hmund 3 polypeptide.
  • the expression host is preferably selected from the group consisting of a plant, plant cell, bacterium, yeast, fungus, protozoa, algae, animal and animal cell.
  • the invention also includes a pharmaceutical composition, including at least all or part of the polypeptide of the invention, and a pharmaceutically acceptable carrier, auxiliary or excipient.
  • the invention also includes a pharmaceutical composition for use in gene therapy, comprising all or part of the nucleotide sequence of any of the invention and a pharmaceutically acceptable carrier, auxiliary or excipient.
  • the pharmaceutical composition for use in gene therapy preferably comprises all or part of an antisense sequence to all or part of the nucleic acid sequence in Figure 8.
  • kits for the treatment or detection of a disease, disorder or abnormal physical state comprising all or part of the nucleotide sequence of the invention.
  • a kit for the treatment or detection of a disease, disorder or abnormal physical state preferably includes all or part of the polypeptide of the invention.
  • the kit may also comprise an antibody to the polypeptide.
  • the disorder is preferably selected from a group consisting of insulin dependent and independent diabetes, glomeruiopathy and renal failure.
  • the invention also includes a NH2- SQRSNDEVREFVKL-COOH specific antibody, preferably a polyclonal antibody.
  • the invention is also a method of medical treatment of a disease, disorder or abnormal physical state, characterized by excessive Hmund 3 expression, concentration or activity, comprising administering a product that reduces or inhibits Hmund 3 polypeptide expression, concentration or activity.
  • the product is preferably an antisense nucleotide sequence to all or part of the nucleotide sequence of Figure 8, the antisense nucleotide sequence being sufficient to reduce or inhibit Hmund 3 polypeptide expression.
  • the antisense DNA is administered in a pharmaceutical composition comprising a carrier and a vector operably linked to the antisense DNA.
  • the disease, disorder or abnormal physical state is preferably selected from a group consisting of insulin dependent diabetes and independent diabetes, glomerulonephritis and ischemic renal injuries.
  • the invention also includes a method of medical treatment of a disease, disorder or abnormal physical state, characterized by reduced Hmund 3 expression, concentration or activity, comprising administering a product that increases Hmund 3 polypeptide expression, concentration or activity.
  • the product is preferably a nucleotide sequence comprising all or part of the nucleotide sequence of Figure 8, the DNA being sufficient to increase Hmund 3 polypeptide expression.
  • the nucleotide sequence is preferably administered in a pharmaceutical composition comprising a carrier and a vector operably linked to the nucleotide sequence.
  • FIG. 1 Protein sequence alignment of Hmund 3 [SEQ ID NO: 1) (GenBank accession number AF020202) with rat mund 3s. (a) Alignment of all four proteins. Only a partial (AA 251-2207) of rat mund 3-3 is shown, (b) Alignment of the first 100 amino acid at the N-terminal of Hmund 3 and rat mund 3-1. Identical residues are boxed. The dotted line above the sequence indicates the C1 domain and the continuous line indicates the C2 domain as proposed by Brose et al. (7).
  • FIG. 1 Expression of Hmund 3 in human MC culture in 5.5 mM D-glucose plus 9.5 mM L-glucose (L(15)) or 19.5 mM L-glucose (L) or 15 mM (D(15)) 25 mM mM D-glucose (D) and as described in Methods.
  • Increased expression of Hmund 3 after 25 mM D- glucose treatment is revealed by Relative RT-PCR (a) and Northern blot (b). All blots are representative of at least 3 different experiments using different total RNA preparations.
  • FIG. 3 Expression of Hmunc 13 (lane 7, 8) or mund 3-2 (lane 9) in human kidney MC (lane 7), cortical epithelial cells (lane 8) or rat kidney MC (lane 9).
  • RT-PCR was performed using a pair of primers for both Hmund 3 and rat mund 3-2 indicated in the Methods which amplified a segment of 193 bp.
  • a pair of primers for GAPDH generated a 453 fragment were used to PCR no RT RNA (lane 1-3) and RT products (lane 4-6) of human kidney MC (lane 1, 4), cortical epithelial cells (lane 2, 5) and rat MC (lane 3, 6).
  • FIG. 4 In vitro translation of Hmund 3. Note that a proportion of the highest MW band (170 kDa) in the absence of microsomal membranes (lane 1) is shifted to higher MW (180 kDa) in the presence of microsomal membranes (lane 2). Lane 3 is the supernatant of derived from the in vitro translation reaction with microsomal membranes as detailed in Methods.
  • Figure 5 Comparison of gene structure of Hmund 3 to various isoforms of rat Mund 3s.
  • Figure 6. Expression of rat mund 3-2 in renal glomerulus of normal (A) or streptozotocin- treated (B) rats detected by in situ hybridization.
  • a PCR fragment of rat mund 3-2 (residues 5487-5669) with a T7 promoter introduced in its sense primer was in vitro transcripted to anti-sense cRNA with DIG-labeled UTP.
  • a section of normal and streptozotocin-treated rat kidneys on the same slide was hybridized with this probe and the signal was detected by Rodamine-conjugated anti-DIG antibody and observed by confocal microscopy.
  • FIG. 11 Double labeling of apoptotic cells and expression of Hmund 3 or C1 less mutant.
  • Hmund 3 (A-C, E-G) and C1 less mutant (D, H) transiently transfected cells were subjected to TUNEL labeled with fluorescein (E-H) and then subjected to anti-HA and anti-mouse IgG-rhodamine labeling for expression of Hmund 3 and C1 less mutant (A-D).
  • Cells were treated with vehicle (A, E) or 0.1 ⁇ M PDBu for 8 h (B, D, F, H) or 16 h (C, G).
  • C1 less mutant transfected cells treated with vehicle exhibit a similar image as D and H (data not shown).
  • Genomic DNA breakdown in Hmund 3 transfected cells by PDBu treatment Genomic DNA obtained from empty plasmid (pCMV), Hmund 3 or C1 less mutant transfected cells treated with vehicle (-) or 0.1 ⁇ M PDBu for 8 h or 16 h was subjected to 2 % agarose gel eiectrophoresis. Molecular size marker (M) is shown.
  • FIG. 13 Expression of rat mund 3-1 in kidney of normal (A) or STZ-treated diabetic (B-D) rat detected by in situ hybridization. Outer cortex (A, B), medulla (C) and a higher power view of outer cortex (D) from diabetic rat kidney are shown. Similar to diabetic rats, staining in the renal medulla for normal rat kidney is less than the cortex (data not shown). Note the increased expression of mund 3-1 in the tubular epithelial cells as well as in certain glomerular cells. Negative controls with sense cRNA showed little staining in both normal and diabetic rat sections (data not shown).
  • FIG. 14 Expression of mund 3-1, mund 3-2 and mund 3-3 in the renal cortex of the normal rat and following 1 day (1d) and 11 day (11d) of hyperglycemia in STZ-treated rats.
  • 18S ribosome RNA (18S) served as a housekeeping gene.
  • FIG. 15 Schematic representation of DAG activated branched signaling pathways involving PKC and Hmund 3. DAG levels are increased by such factors as hyperglycemia, phospholipase C (PLC) ⁇ / ⁇ and phospholipase D (PLD) resulting in activation of both PKC and Hmund 3 and leading to two separate downstream signaling pathways, respectively resulting in proliferation and differentiation (PKC) or apoptosis (Hmund 3).
  • PLC phospholipase C
  • PLD phospholipase D
  • Hmund 3 human mund 3 gene
  • Hmund 3 contributes to the renal and microvascular complications associated with hyperglycemia in diabetes mellitus, through a variety of mechanisms including Hmund 3 linked apoptosis.
  • DDPT-PCR differential display reverse transcription polymerase chain reaction
  • Hmund 3 is detectable in both MC, epithelial and other cells.
  • the presence of a Hmund 3 gene in MC which has similarity to rat mund 3 was very unexpected because rat mund 3 is believed to be localized only in the brain (20).
  • Hmund 3 is a target for regulation by glucose in MC and other cells.
  • the expression of Hmund 3 is up-regulated by hyperglycemia in cultured kidney MC and epithelial cells.
  • Hmund 3 protein is involved in the acute and chronic effects of hyperglycemia in MC and renal epithelial cells, and contributes to the development of diabetic glomerulopathy. Hmund 3 also interacts with the syntaxins.
  • Hmund 3 Protein Three Dimensional Structure
  • Hmund 3 contains 1 C1 domain and 3 C2 domains.
  • the N-terminal segment is more similar to rat mund 3-1 and the C-terminal segment is more similar to rat mund 3-2 which contains 1 C1 and 2 C2 domains.
  • another AUG codon (residue 444-446) after the first C2 domain contains an optimal Kozak sequence (5'-CACCAUGG-3') (27).
  • Hmund 3 mRNA serves as a bifunctional mRNA (27) that encodes two open reading frames, one for an isoform with 3 C2 domains (mund 3-1) and the other with only 2 C2 domains (mund 3-2).
  • a segment of Hmund 3 (aa 309-371) not present in rat mund 3s, has similarity to a segment of the delta isoform of the B' subunit of protein phosphatase 2Ao - a serine threonine phosphatase (28).
  • This B' subunit has been shown to be a regulatory subunit of the multimeric PP2Ao.
  • the catalytic subunit of PP2Ao associates with specific proteins (B') that serve a targeting and regulatory function.
  • Hmund 3 interacts with ECM element receptors-integrins, such as vitronectin recetpor ⁇ v ⁇ 3 and fibronectin receptor ⁇ 5 ⁇ . Such interaction is important for cell survival.
  • ECM element receptors-integrins such as vitronectin recetpor ⁇ v ⁇ 3 and fibronectin receptor ⁇ 5 ⁇ .
  • Over-expression of Hmund 3 in response to DAG prevents engagement of integrins to ECM resulting in apoptosis.
  • Hmund 3 shows a multifunctional role that involves transmembrane ECM-cell signaling, as well as DAG and Ca ++ activated phosphatase activity.
  • Hmunc 13 is upregulated in the streptozotocin treated diabetic rat compared to normal rats (Fig. 6). Thus Hmund 3 is implicated in the pathogenesis of diabetic nephropathy.
  • the invention also includes nucleotide sequences that are biologically functional equivalents of all or part of the sequence in Figure 8.
  • Biologically functional equivalent nucleotide sequences are DNA and RNA (such as genomic DNA, cDNA, synthetic DNA, and mRNA nucleotide sequences), that encode peptides, polypeptides, and proteins having the same or similar Hmund 3 activity as all or part of the Hmund 3 protein shown in Figure 1.
  • Biologically functional equivalent nucleotide sequences can encode peptides, polypeptides, and proteins that contain a region having sequence identity to a region of a Hmund 3 protein or more preferably to the entire Hmunc 13 protein. Identity is calculated according to methods known in the art. The Gap program, described below, is most preferred.
  • Sequence A For example, if a nucleotide sequence (called “Sequence A”) has 90% identity to a portion of the nucleotide sequence in Figure 8, then Sequence A will be identical to the referenced portion of the nucleotide sequence in Figure 8, except that Sequence A may include up to 10 point mutations, such as deletions or substitutions with other nucleotides, per each 100 amino acids of the referenced portion of the nucleotide sequence in Figure 8. Nucleotide sequences biologically functional equivalent to the Hmund 3 sequences can occur in a variety of forms as described below. A) Nucleotide sequences Encoding Conservative Amino Acid Changes in Hmund 3 Protein
  • the invention includes biologically functional equivalent nucleotide sequences that encode conservative amino acid changes within a Hmund 3 amino acid sequence and produce silent amino acid changes in Hmund 3.
  • the invention includes biologically functional equivalent nucleotide sequence that made non conservative amino acid changes within the Hmunc 13 amino acid sequence to the sequences in Figure 8.
  • Biologically functional equivalent nucleotide sequences are DNA and RNA that encode peptides, polypeptides, and proteins having non-conservative amino acid substitutions (preferably substitution of a chemically similar amino acid), additions, or deletions but which also retain the same or similar Hmund 3 activity as all or part of the Hmund 3 protein shown in Figure 1 or disclosed in the application.
  • the DNA or RNA can encode fragments or variants of the Hmund 3 of the invention.
  • the Hmund 3 or Hmund 3 -like activity of such fragments and variants is identified by assays as described above.
  • Fragments and variants of Hmund 3 encompassed by the present invention should preferably have at least about 40%, 60%, 80% or 95% sequence identity or preferably at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity to the naturally occurring nucleotide sequence, or corresponding region. Most preferably, the fragments have at least 99.5% sequence identity to the naturally occurring nucleotide sequence, or corresponding region. Sequence identity (also known as homology) is preferably measured with the Gap program.
  • Nucleotide sequences biologically functionally equivalent to the Hmund 3 in Figure 8 include: (1) Altered DNA.
  • the sequence shown in Figure 8 may have its length altered by natural or artificial mutations such as partial nucleotide insertion or deletion, so that when the entire length of the coding sequence within Figure 8, is taken as 100%, the biologically functional equivalent nucleotide sequence preferably has a length of about 60-120% thereof, more preferably about 80-110% thereof. Fragments may be less than 60%.; or
  • the mutated DNAs created in this manner should preferably encode a protein having at least about 40%, preferably at least about 60%, at least about 80%, and more preferably at least about 90% or 95%, and most preferably 97%, 98% or 99% sequence identity (homology) to the amino acid sequence of the Hmund 3 protein in Figure 1. Sequence identity can preferably be assessed by the Gap program.
  • nucleic acid sequence in Figure 8 is not the only sequences which may code for a protein having Hmund 3 activity.
  • This invention includes nucleic acid sequences that have the same essential genetic information as the nucleotide sequence described in Figure 8.
  • Nucleotide sequences (including RNA) having one or more nucleic acid changes compared to the sequences described in this application and which result in production of a polypeptide shown in Sequence (a) in Figure 1 are within the scope of the invention.
  • Hmund 3 -encoding nucleic acids can be isolated using conventional DNA-DNA or DNA-RNA hybridization techniques.
  • the present invention also includes nucleotide sequences that hybridize to one or more of the sequences in Figure 8 or its complementary sequence, and that encode expression for peptides, polypeptides, and proteins exhibiting the same or similar activity as that of the Hmund 3 protein produced by the DNA in Figure 8 or its variants.
  • Such nucleotide sequences preferably hybridize to one or more of the sequences in Figure 8 under moderate to high stringency conditions (see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
  • Preferable hybridization conditions are high stringency, such as 42°C for a 20- to 30-mer oligonucleotide, 65°C for a 200-500 bp DNA probe or 70°C for a 200-400 bp cRNA probe.
  • the present invention also encompasses nucleotide sequences that hybridize to genomic DNA, cDNA, or synthetic DNA molecules that encode the amino acid sequence of the Hmund 3 protein, or genetically degenerate forms thereof due to the degeneracy of the genetic code, under salt and temperature conditions equivalent to those described in this application, and that code on expression for a peptide, polypeptide, or protein that has the same or similar activity as that of the Hmund 3 protein.
  • a nucleotide sequence described above is considered to possess a biological function substantially equivalent to that of the Hmund 3 genes of the present invention if the protein produced by the nucleotide sequence displays the following characteristics (i) DAG activated transloaction of the protein in vivo from the cytosol to Golgi (as measured by immunocytochemistry, described in the Materials and Methods section), and (ii) the protein activates apoptosis (if the protein is expressed in vivo, the protein's expression is preferably induced by DAG).
  • nucleotide sequences also referred to as a DNA sequence or a nucleic acid molecule; these terms include either a full gene or a gene fragment.
  • nucleotide fragment that includes all or a fragment of a gene when practicing the invention
  • the nucleotide molecules can also be obtained from other sources known in the art such as expressed sequence tag analysis or in vitro synthesis.
  • the DNA described in this application (including variants that are biologically functional equivalents) can be introduced into and expressed in a variety of eukaryotic and prokaryotic host cells.
  • a recombinant nucleotide sequence for the Hmund 3 contains suitable operativeiy linked transcriptional or translational regulatory elements.
  • Suitable regulatory elements are derived from a variety of sources, and they may be readily selected by one with ordinary skill in the art (Sambrook, J, Fritsch, E.E. & Maniatis, T. (1989). Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press. New York; Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). For example, if one were to upregulate the expression of the gene, one could insert the sense sequence and the appropriate promoter into the vector. Promoters can be inducible or constitutive, environmentally - or developmentally-regulated, or cell - or tissue-specific. Transcription is enhanced with promoters known in the art such as CMV, RSV and SV40.
  • the nucleotide sequence may be either isolated from a native source (in sense or antisense orientations), synthesized, or it may be a mutated native or synthetic sequence or a combination of these.
  • regulatory elements include a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the vector employed, other genetic elements, such as selectable markers, may be incorporated into the recombinant molecule. Other regulatory regions that may be used include an enhancer domain and a termination region. The regulatory elements may be from animal, plant, yeast, bacterial, fungal, viral, avian, insect or other sources, including synthetically produced elements and mutated elements.
  • the polypeptide may be expressed by inserting a recombinant nucleotide sequence in a known expression system derived from bacteria, viruses, yeast, mammals, insects, fungi or birds.
  • the recombinant molecule may be introduced into the cells by techniques such as Agrobacterium f ⁇ mefac/ens-mediated transformation, particle-bombardment-mediated transformation, direct uptake, microinjection, coprecipitation, transfection and electroporation depending on the cell type.
  • Retroviral vectors, adenoviral vectors, DNA virus vectors and liposomes may be used.
  • Suitable constructs are inserted in an expression vector, which may also include markers for selection of transformed cells. The construct may be inserted at a site created by restriction enzymes.
  • a cell is transfected with a nucleotide sequence of the invention inserted in an expression vector to produce cells expressing the nucleotide sequence.
  • Another embodiment of the invention relates to a method of transfecting a cell with a nucleotide sequence of the invention, inserted in an expression vector to produce a cell expressing the Hmund 3 protein.
  • the invention also relates to a method of expressing the polypeptides of the invention in a cell.
  • the invention also includes oligonucleotide probes made from the cloned Hmund 3 nucleotide sequences described in this application or other nucleotide sequences of the invention.
  • the probes may be 15 to 30 nucleotides in length and are preferably at least 30 or more nucleotides.
  • a preferred probe is 5'-
  • the invention also includes at least 30 consecutive nucleotides of Hmund 3 in Figure 8.
  • the probes are useful to identify nucleic acids encoding Hmund 3 peptides, polypeptides and proteins other than those described in the application, as well as peptides, polypeptides, and proteins biologically functionally equivalent to Hmund 3.
  • the oligonucleotide probes are capable of hybridizing to one or more of the sequences shown in Figure 8 or the other sequences of the invention under stringent hybridization conditions.
  • a nucleotide sequence encoding a polypeptide of the invention may be isolated from other organisms by screening a library under moderate to high stringency hybridisation conditions with a labeled probe. The activity of the polypeptide encoded by the nucleotide sequence is assessed by cloning and expression of the DNA. After the expression product is isolated the polypeptide is assayed for Hmund 3 activity as described in this application.
  • Biologically functional equivalent Hmund 3 nucleotide sequences from other cells, or equivalent Hmund 3 -encoding cDNAs or synthetic DNAs can also be isolated by amplification using Polymerase Chain Reaction (PCR) methods.
  • Oligonucleotide primers, including degenerate primers, based on the amino acid sequence of the sequences in Figures 8 can be prepared and used in conjunction with PCR technology employing reverse transcriptase (E. S. Kawasaki (1990), In Innis et al., Eds., PCR Protocols, Academic Press, San Diego, Chapter 3, p. 21) to amplify biologically functional equivalent DNAs from genomic or cDNA libraries of other organisms.
  • reverse transcriptase E. S. Kawasaki (1990), In Innis et al., Eds., PCR Protocols, Academic Press, San Diego, Chapter 3, p. 21
  • the oligonucleotides can be used as probes to screen cDNA libraries.
  • the present invention includes not only the polypeptides encoded by sequences presented in this application, but also "biologically functional equivalent peptides, polypeptides and proteins" that exhibit the same or similar Hmund 3 protein activity as proteins described in this application.
  • the phrase "biologically functional equivalent peptides, polypeptides, and proteins” denotes peptides, polypeptides, and proteins that exhibit the same or similar Hmunc 13 protein activity when assayed. Where only one or two of the terms peptides, polypeptides and proteins is referred to below, it will be clear to one skilled in the art whether the other types of amino acid sequence also would be useful.
  • the same or similar Hmund 3 protein activity is meant the ability to perform the same or similar function as the protein produced by Hmund 3.
  • These peptides, polypeptides, and proteins can contain a region or moiety exhibiting sequence identity (homology) to a corresponding region or moiety of the Hmund 3 protein described in the application, but this is not required as long as they exhibit the same or similar Hmund 3 activity.
  • Identity refers to the similarity of two polypeptides or proteins (or nucleotide sequences) that are aligned so that the highest order match is obtained. Identity is calculated according to methods known in the art, such as the Gap program, described below.
  • Sequence A For example, if a polypeptide (called “Sequence A”) has 90% identity to a portion of the polypeptide in sequence (a) in Figure 1 , then Sequence A will be identical to the referenced portion of the polypeptide in sequence (a) in Figure 1 , except that Sequence A may include up to 10 point mutations, such as deletions or substitutions with other amino acids, per each 100 amino acids of the referenced portion of the polypeptide in sequence (a) in Figure 1.
  • Peptides, polypeptides, and proteins biologically functional equivalent to the Hmund 3 proteins can occur in a variety of forms as described below.
  • Peptides, polypeptides, and proteins biologically functionally equivalent to Hmund 3 protein include amino acid sequences containing amino acid changes in the Hmund 3 sequence.
  • the biologically functional equivalent peptides, polypeptides, and proteins have at least about 40% sequence identity (homology), preferably at least about 60%, at least about 75%, at least about 80%, at least about 90% or at least about 95% sequence identity, to the naturally occurring polypeptide, or corresponding region. Most preferably, the biologically functional equivalent peptides, polypeptides, and proteins have at least 97%, 98% or 99% sequence identity to the naturally occurring protein, or corresponding region or moiety.
  • sequence identity is preferably determined by the Gap program. The algorithm of Needleman and Wunsch (1970 J Mol.
  • Biol. 48:443-4583 is used in the Gap program. BestFit is also used to measure sequence identity. It aligns the best segment of similarity between two sequences. Alignments are made using the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482-489.
  • the invention includes peptides, polypeptides or proteins which retain the same or similar activity as all or part of Hmund 3.
  • Such peptides preferably consist of at least 5 amino acids. In preferred embodiments, they may consist of 6 to 10, 11 to 15, 16 to 25 or 26 to 50, 50 to 150, 150 to 250, 250 to 500 or 500 to 750 amino acids of the Hmund 3.
  • Fragments of the Hmund 3 protein can be created by deleting one or more amino acids from the N-terminus, C-terminus or an intemal region of the protein (or combinations of these), so long as such fragments retain the same or similar Hmund 3 activity as all or part of the Hmund 3 protein disclosed in the application.
  • fragments can be natural mutants of the Hmund 3, or can be produced by restriction nuclease treatment of an encoding nucleotide sequence. Fragments of the polypeptide may be used in an assay to identify compounds that bind the polypeptide. Methods known in the art may be used to identify agonists and antagonists of the fragments. Variants of the Hmund 3 protein may also be created by splicing. Variants can also be naturally occurring mutants of the Hmund 3 disclosed in the application. A combination of techniques known in the art may be used to substitute, delete or add amino acids. For example, a hydrophobic residue such as methionine can be substituted for another hydrophobic residue such as alanine.
  • An alanine residue may be substituted with a more hydrophobic residue such as leucine, valine or isoleucine.
  • An aromatic residue such as phenylalanine may be substituted for tyrosine.
  • An acidic, negatively charged amino acid such as aspartic acid may be substituted for glutamic acid.
  • a positively charged amino acid such as lysine may be substituted for another positively charged amino acid such as arginine.
  • Modifications of the proteins of the invention may also be made by treating a polypeptide of the invention with an agent that chemically alters a side group, for example, by converting a hydrogen group to another group such as a hydroxy or amino group.
  • Peptides having one or more D-amino acids are contemplated within the invention. Also contemplated are peptides where one or more amino acids are acetylated at the N-terminus.
  • peptide mimetics i.e. a modified peptide or polypeptide or protein
  • a variety of techniques are available for constructing peptide mimetics (i.e. a modified peptide or polypeptide or protein) with the same or similar desired biological activity as the corresponding protein of the invention but with more favorable activity than the protein with respect to characteristics such as solubility, stability, and/or susceptibility to hydrolysis and proteolysis. See for example, Morgan and Gainor, Ann. Rep. Med. Chem., 24:243-252 (1989).
  • the invention also includes hybrid genes and peptides, for example where a nucleotide sequence from the gene of the invention is combined with another nucleotide sequence to produce a fusion peptide.
  • a nucleotide domain from a molecule of interest may be ligated to all or part of a Hmund 3 nucleotide sequence encoding Hmund 3 protein described in this application.
  • Fusion genes and peptides can also be chemically synthesized or produced using other known techniques.
  • the variants preferably retain the same or similar Hmund 3 activity as the naturally occurring Hmund 3 of the invention.
  • the Hmund 3 activity of such variants can be assayed by techniques described in this application and known in the art of TUNEL and DNA fragmentation assay.
  • Variants produced by combinations of the techniques described above but which retain the same or similar Hmund 3 activity as naturally occurring Hmund 3 are also included in the invention (for example, combinations of amino acid additions, deletions, and substitutions).
  • Fragments and variants of Hmund 3 encompassed by the present invention preferably have at least about 40% sequence identity, preferably at least about 60%, at least about 75%, at least about 80%, at least about 90% or at least about 95% sequence identity, to the naturally occurring protein, or corresponding region or moiety. Most preferably, the fragments have at least 97%, 98% or 99% sequence identity to the naturally occurring polypeptide, or corresponding region. Sequence identity is preferably measured with either the Gap or BestFit programs.
  • the invention also includes fragments of the polypeptides of the invention which do not retain the same or similar activity as the polypeptides but which can be used as a research tool to characterize the polypeptides of the invention.
  • the activity of the Hmund 3 protein is increased by carrying out selective site- directed mutagenesis.
  • protein modelling and other prediction methods we characterize the binding domain and other critical amino acid residues in the protein that are candidates for mutation, insertion and/or deletion.
  • a DNA plasmid or expression vector containing the Hmund 3 gene or a nucleotide sequence having sequence identity is preferably used for these studies using the U.S.E. (Unique site elimination) mutagenesis kit from Pharmacia Biotech or other similar mutagenesis kits that are commercially available.
  • U.S.E. Unique site elimination
  • This approach is useful not only to enhance activity, but also to engineer some functional domains for other properties useful in the purification or application of the proteins or the addition of other biological functions. It is also possible to synthesize a DNA fragment based on the sequence of the proteins that encodes smaller proteins that retain activity and are easier to express. It is also possible to modify the expression of the cDNA so that it is induced under environmental conditions other than hyperglycemia or in response to different chemical inducers or hormones. It is also possible to modify the DNA sequence so that the protein is targeted to a different location. All these modifications of the DNA sequences presented in this application and the proteins produced by the modified sequences are encompassed by the present invention.
  • Hmund 3 or its protein and biologically functional equivalent nucleotide sequences or proteins are also useful when combined with a carrier in a pharmaceutical composition.
  • Suitable examples of vectors for Hmund 3 are described above.
  • the compositions are useful when administered in methods of medical treatment of a disease, disorder or abnormal physical state characterized by insufficient Hmund 3 expression or inadequate levels or activity of Hmund 3 protein.
  • the invention also includes methods of medical treatment of a disease, disorder or abnormal physical state characterized by excessive Hmund 3 expression or levels of activity of Hmund 3 protein, for example by administering a pharmaceutical composition comprising including a carrier and a vector that expresses Hmund 3 antisense DNA.
  • compositions of this invention used to treat patients having degenerative diseases, disorders or abnormal physical states of tissue such as renal and vascular tissue.
  • apoptosis plays a role in renal diseases related to (1) glomerular inflammation (2) tubular ischemia, toxins and ureteric obstruction (E.G. Neilson and W.G. Couser, Immunologic Renal Disease, (1997, 309-329), 8), could include an acceptable carrier, auxiliary or excipient.
  • apoptosis is protective.
  • apoptosis may contribute to cell injury. Regulation of apoptosis plays a critical role in many different renal disease states including both glomerular and tubulointerstitial types of injury.
  • the conditions which may be treated by the compositions include microvascular and renal complications of diabetes and disorders in which renal apoptosis plays a role.
  • the pharmaceutical compositions can be administered to humans or animals by methods such as aerosol administration, intratracheal instillation and intravenous injection. Dosages to be administered depend on patient needs, on the desired effect and on the chosen route of administration. Nucleotide sequences and proteins may be introduced into cells using in vivo delivery vehicles such as liposomes. They may also be introduced into these cells using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into liposomes.
  • compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the nucleotide sequence or protein is combined in a mixture with a pharmaceutically acceptable vehicle.
  • suitable vehicles are described, for example in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA).
  • the pharmaceutical compositions could include an active compound or substance, such as a Hmund 3 gene or protein, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and isoosmotic with the physiological fluids.
  • an active compound or substance such as a Hmund 3 gene or protein
  • the methods of combining the active molecules with the vehicles or combining them with diluents is well known to those skilled in the art.
  • the composition could include a targeting agent for the transport of the active compound to specified sites within tissue.
  • Expression vectors are useful to provide high levels of protein expression.
  • Cell cultures transformed with the nucleotide sequences of the invention are useful as research tools. Cell cultures are used in overexpression and research according to numerous techniques known in the art.
  • a cell line (either an immortalized cell culture or a primary cell culture) may be transfected with a vector containing a Hmund 3 nucleotide sequence (or variants) to measure levels of expression of the nucleotide sequence and the activity of the nucleotide sequence.
  • a polypeptide of the invention may be used in an assay to identify compounds that bind the polypeptide. Methods known in the art may be used to identify agonists and antagonists of the polypeptides.
  • Hmund 3 is a useful research tool.
  • Hmund 3 cDNA is expressed after it is inserted in a mammalian cell expression plasmid (pCMV SPORT, Gibco BRL).
  • Hmund 3 cDNA is inserted in an inducible mammalian cell expression plasmid (pIND, Invitrogen).
  • Hmund 3 cDNA may also be positioned in reverse orientation in piND as a negative control.
  • stable tansfected mouse mesangial, NIH 3T3, MDCK, HEK 293 and OK cell lines are created with an inducible Hmund 3 plasmid.
  • Hmund 3 Gene Therapy Since it is possible that certain diabetics may be protected from development of renal complications by either up or down regulation of Hmund 3, gene therapy to replace or delete Hmund 3 expression could also be used to modify the development/progression of diabetic renal and vascular complications. In addition, the use of anti-sense DNA that inhibits the expression of hmund 3 will allow treatment of diabetic nephropathy in humans.
  • the invention also includes methods and compositions for providing gene therapy for treatment of diseases, disorders or abnormal physical states characterized by insufficient Hmund 3 expression or inadequate levels or activity of Hmund 3 protein (see the discussion of phamaceutical discussions, above).
  • the invention also includes methods and compositions for providing gene therapy for treatment of diseases, disorders or abnormal physical states characterized by excessive Hmund 3 expression or levels of activity of Hmund 3 protein
  • the invention includes methods and compositions for providing a nucleotide sequence encoding Hmund 3 or biologically functional equivalent nucleotide sequence to the cells of an individual such that expression of Hmund 3 in the cells provides the biological activity or phenotype of Hmund 3 protein to those cells. Sufficient amounts of the nucleotide sequence are administered and expressed at sufficient levels to provide the biological activity or phenotype of Hmund 3 protein to the cells.
  • the method can preferably involve a method of delivering a gene encoding Hmund 3 to the cells of an individual having a disease, disorder or abnormal physical state, comprising administering to the individual a vector comprising DNA encoding Hmund 3.
  • the method may also relate to a method for providing an individual with a disease, disorder or abnormal physical state with biologically active Hmund 3 protein by administering DNA encoding Hmund 3.
  • the method may be performed ex v/Vo or in vivo.
  • Gene therapy methods and compositions are explained, for example, U.S. Patent Nos. 5,672,344, 5,645,829, 5,741,486, 5,656,465, 5,547,932, 5,529,774, 5,436,146, 5,399,346 and 5,670,488, 5,240,846.
  • the method may also relate to a method for producing a stock of recombinant virus by producing virus suitable for gene therapy comprising DNA encoding Hmund 3. This method preferably involves transfecting cells permissive for virus replication (the virus containing Hmund 2) and collecting the virus produced.
  • the invention also includes methods and compositions for providing a nucleotide sequence encoding an antisense sequence to Hmund 3 to the cells of an individual such that expression of the antisense sequence prevents Hmund 3 biological activity or phenotype.
  • the methods and compositions can be used in vivo or in vitro. Sufficient amounts of the nucleotide sequence are administered and expressed at sufficient levels to prevent the biological activity or phenotype of Hmund 3 protein to the cells. Similar methods as described in the preceding paragraph may be used with appropriate modifications.
  • the methods and compositions can be used in vivo or in vitro.
  • the evidence for in vitro usefulness is downregulation of Hmund 3 in hyperglycemia conditions can inhibit hyperglycemia induced renal cell injury.
  • the invention also includes compositions (preferably pharmaceutical compositions for gene therapy).
  • the compositions include a vector containing Hmund 3 or a biologically functional equivalent molecule or antisense DNA.
  • the carrier may be a pharmaceutical carrier or a host cell transformant including the vector.
  • Vectors known in the art are adenovirus and herpesvirus vectors.
  • the invention also includes packaging cell lines that produce the vector. Methods of producing the vector and methods of gene therapy using the vector are also included with the invention.
  • the invention also includes a transformed cell, such as an MC cell or other cell described in this application, containing the vector and recombinant Hmund 3 nucleotide sequence or a biologically functional equivalent molecule.
  • the Hmund 3 protein is also useful as an antigen for the preparation of antibodies that can be used to purify or detect other mund 3 or mund 3-like proteins.
  • Monoclonal and polyclonal antibodies are prepared according to other techniques known in the art. For examples of methods of the preparation and uses of monoclonal antibodies, see U.S. Patent Nos. 5,688,681, 5,688,657, 5,683,693, 5,667,781 , 5,665,356, 5,591,628, 5,510,241, 5,503,987, 5,501,988, 5,500,345 and 5,496,705. Examples of the preparation and uses of polyclonal antibodies are disclosed in U.S. Patent Nos. 5,512,282, 4,828,985, 5,225,331 and 5,124,147.
  • Antibodies recognizing Hmund 3 can be employed to screen organisms containing Hmund 3 protein or Hmund 3-like proteins. The antibodies are also valuable for immuno-purification of Hmund 3 or Hmund 3-like proteins from crude extracts.
  • mund 3 is useful in a pharmaceutical preparation to treat diabetes or its complications.
  • Hmund 3 is also useful as a target.
  • Chemical libraries are used to identify pharmacophores which can specifically interact with Hmund 3 either in an inhibitory or stimulatory mode.
  • the Hmund 3 targets that would be used in drug design include - e.g. the DAG binding site or some other functional domain specific to Hmund 3.
  • Modulation of Hmund 3 expression is commercially useful for identification and development of drugs to inhibit and/or enhance Hmund 3 function directly.
  • drugs would be targeted to any of the following sites: the DAG, Ca ++ , phosphatase and RGD domains.
  • the invention also includes methods of screening a test compound to determine whether it antagonizes or agonizes Hmund 3 protein expression. For example, one method involves testing whether a compound inhibits the translocation of Hmund 3 from cytosol to Golgi as well as its apoptotic effect.
  • the invention also includes methods of screening a test compound to determine whether it induces or inhibits Hmund 3 expression. For example, one method involves testing whether a compound inhibits the promoter activity of Hmund 3.
  • Hmund 3 is expressed in MC, human cortical epithelial cells and cells from testis, ovaries, prostate gland, colon, brain and heart.. Experiments to determine where the gene is expressed were done with RT-PCR.
  • the function of Hmund 3 in other cells will be similar to that in renal epithelial cells such as in translocation and apoptosis Hmund 3 has a C1 domain.
  • a region of the C1 domain from C. elegans unc-13 binds to phorbol esters and DAG similar to PKC (21).
  • the C1 domain is similar among C. elegans unc13, rat mund 3s and Hmund 3 (Fig. 1), so the C1 domain in the Hmund 3 can also bind phorbol esters.
  • Hmund 3 is also involved in cell signaling in response to DAG binding.
  • Hmund 3 was up-regulated by high- glucose treatment (25 mM D-glucose). Even 15 mM D-glucose is enough to stimulate the over expression of Hmund 3 as revealed by Northern blot.
  • hyperglycemia increases PKC activity in MC (13, 14, 31).
  • DAG levels are increased when cultured MC are exposed to hyperglycemia (17, 13). Since Hmund 3 and PKC share similar binding capacities for phorbol esters and DAG and both PKC contain C2 domains, Hmund 3 is part of an alternative cascade following DAG binding. Thus Hmund 3 is activated in response to hyperglycemic induced increases in DAG.
  • Hmund 3 does not contain a kinase domain and cannot therefore serve as a downstream regulator by protein phosphorylation (20, 30), nevertheless it is possible that Hmund 3 modulates intracellular events through competitive binding of PKC or by regulation of vesicle trafficking and exocytosis.
  • Hmund 3 has a cytoplasmic distribution under basal conditions, but with PDBu stimulation, Hmund 3 is translocated to the Golgi apparatus. This effect is unlikely to have taken place through activation of endogenous PKC, since the deletion mutant, C1 less mutant (without the DAG binding domain), showed no translocation.
  • mund 3-1 was localized to the presynaptic region in rat brain by immunocytochemistry.
  • green fluorescent protein tagged mund 3-1, -2 and -3 are all translocated to plasma membrane following phorbol ester stimulation.
  • Hmund 3 is translocated to the Golgi apparatus in response to phorbol ester activation compared to translocation of mund 3-1 , -2 and -3 to the plasma membrane.
  • Hmund 3 is a unique isoform of mund 3s.
  • the multiplicity of PKC isoforms and the tissue specificity of PKC functional expression are well known (32).
  • the mund 3 pathway is also composed of tissue specific functionally different isoforms.
  • the mund 3 proteins have no kinase domain (20, 33).
  • the Golgi apparatus is involved in vesicular traffic.
  • a number of SNARE proteins such as yeast Sed5p (34) and mVps45 (35), mammalian syntaxin 6 (36), VAMP4, Syntaxin 13 and mVtib (36), have all been reported to be localized to the Golgi.
  • Rat mund 3-1 has been shown to interact with a number of proteins involved in vesicle docking and trafficking, such as syntaxin (24) and Doc2 (37). Interaction of mund 3-1 and Doc2 was stimulated by DAG and has been suggested to be involved in Ca 2* dependent exocytosis (37).
  • Hmund 3 is a protein that participates in DAG regulated vesicle trafficking and exocytosis. Further studies are required to investigate if Hmund 3 interacts with other Golgi localized SNARE proteins or whether some SNARE proteins co-translocate to the Golgi with Hmund 3 after DAG stimulation. It has also been suggested that PKC plays a role in Golgi budding (for review see 38). For example, a study in S. Cerevisiae implicated DAG as playing an important role in the formation of Golgi budding involving Sec14 (39).
  • Hmund 3 translocates to the Golgi after DAG stimulation, it would also be of interest to determine the role of Hmund 3 is involved in Golgi budding and interaction with Sec14L, the partial mammalian homologue of yeast Sec14 (40). Role of Hmund 3 in Apoptosis
  • Hmund 3 We investigated the localization of Hmund 3 to determine whether exposure to phorbol esters had any effect on its intracellular translocation. In the course of carrying out these studies, we observed that cells transfected with Hmund 3 became rounded up and died following treatment with phorbol 12, 13-dibutyrate (PDBu), a phorbol ester analogue. We examined the mechanism of phorbol ester induced cell death in the transfected cells. We showed that exposure to phorbol ester causes apoptosis through activation of Hmund 3. This shows the interaction between the diabetic state, activation of Hmund 3 and ceil damage.
  • PDBu 13-dibutyrate
  • Hmund 3 Participates in a Signaling Pathway and Counterbalances DAG Activated PKC
  • a model for the cellular activation of Hmund 3 and PKC isoforms Since both mund 3s and PKC have similar binding affinity to phorbol esters, our results showing that cells transfected with Hmund 3 become apoptotic after DAG treatment mean that Hmund 3 participates in a signaling pathway that serves to counterbalance DAG activated PKC. This concept is illustrated schematically in Figure 15.
  • DAG acts as a secondary messenger to activate two alternate pathways - one pathway effected through PKC results in kinase activation and serine/threonine phospholylaton of downstream targets leading to cell proliferation while the other pathway effected through Hmund 3 induces apoptosis, preferably through interaction involving vesicle trafficking.
  • mund 3-1 and mund 3-2 are mainly localized to cortical tubular epithelial cells.
  • in situ hybridization and relative RT-PCR we have also demonstrated that mund 3-1 and mund 3-2 are over-expressed in kidney of STZ-treated diabetic rats.
  • This result in rat kidney is consistent with our in vitro findings, showing that expression of Hmund 3 is up-regulated by high glucose treatment in cultured human mesangial cells. It has been reported that an increase in intracellular DAG levels is only detectable after 2 days of high glucose treatment (46).
  • DDRT-PCR carried out on RNA extracts from MC exposed to high vs. low glucose conditions yielded 10 bands which exhibited differences between high glucose treatment and controls (both normal glucose and osmolarity controls) (data not shown). After the bands had been cut, reamplified, cloned and sequenced, the sequences were compared to the GenBank database. One of the cDNA sequences had identity to a segment (residues 3523- 3863) of rat mund 3-2 (20).
  • rat mund 3-2 is viewed as having a potential signaling function particularly in neurotransmission and in addition has not previously been reported in any tissue outside the brain, we elected to clone the full gene from human kidney and confirm the nature of its regulation by hyperglycemia.
  • Hmund 3 (residues 1-100) is similar to to rat mund 3-1 (Fig. 1b).
  • the next segment (residues 101-391) exhibits considerable variation in Hmund 3 compared to rat mund 3s and unc-13 (7).
  • the C-terminal segment of unc-13s is highly conserved among human, rat and C. elegans (Fig. 1 , ref. 7).
  • the protein segment from residue 392 to 1591 of Hmund 3 is about 93% similar to rat mund 3-2 (residue 766-1985), 79% similar to mund 3-1 (residue 486-1735) and 74% similar to mund 3-3 (residue 1000-2207).
  • the C terminus of renal Hmund 3 has strongest identity to rat mund 3-2 whereas the N-terminal of Hmund 3 has strongest identity to rat mund 3-1.
  • Hmund 3 was up-regulated in the high-glucose (25mM) treated MC compared to osmolarity controls.
  • Northern blot analysis was carried out on cells grown according to the same protocol.
  • Hmund 3 expression was increased in MC after hyperglycemia (Fig. 2b).
  • Hmund 3 expression in MC following exposure to 15 mM D-glucose was also increased relative to osmolarity control but there was no statistically significant difference between 15 mM D-glucose and 25 mM D-glucose treated cells.
  • RT-PCR was performed using a pair of primers specific for both Hmund 3 and rat mund 3-2.
  • Hmund 3 was detected in cultured human kidney cortical epithelial cells and mund 3-2 was also expressed in primary cultured rat MC. Genomic contamination is unlikely since no band was observed in the no RT control for the GAPDH housekeeping gene (Fig. 3).
  • Hmund 3 is expressed as a ⁇ 170 kDa protein (Fig. 4). This is close to the predicted MW (180.5 kDa) from the cDNA clone. A number of less prominent lower molecular weight bands is also present following in vitro translation because of either initiation of translation from internal AUG codons rather than the first interaction site or a premature termination of translation. Also shown in figure 4 is that in the presence of canine pancreatic microsomal membranes, a proportion of Hmund 3 protein is shifted to a higher molecular weight (-180 kDa) suggesting that it is membrane associated and undergoes co- translational processing. Only the full-length protein is associated with the membrane because the partial length in vitro translation products are not observed in the microsomal pellet (Fig. 4, lane 2).
  • Hmund 3 in opossum kidney (OK) cells, a cell line of renal epithelia origin and compare two constructs - an HA tagged Hmund 3 and an HA tagged Hmund 3 deletion mutant lacking the C1 domain (C1 less mutant).
  • Cells employed in the present study were grown on glass cover slips under growth arrested conditions with serum starvation.
  • Transient transfection of OK cells was confirmed by Western blot analysis ( Figure 10). As shown in Figure 10(i), an -180 kDa protein was expressed in the Hmund 3-HA transfected cells and a -175 kDa protein was detected in the C1 less mutant transfected cells. No band was detected in cells transfected with empty plasmid, pCMVSPORT.
  • Hmund 3-HA in transfected OK cells Intracellular localization of Hmund 3-HA in transfected OK cells was monitored by immunocytochemistry (ICC) using cells doubly labeled with anti-HA antibody (Fig. 10(H), upper panels) and wheat germ agglutinin (WGA) (Fig. 10(H), lower panels).
  • ICC immunocytochemistry
  • WGA wheat germ agglutinin
  • FIG. 10(H) inspection of panel A reveals that Hmund 3 exhibits a cytosolic distribution compared to the Golgi apparatus stained with WGA shown in Panel E.
  • a DAG analogue Hmund 3 is translocated to the peri- nuclear area (panel B) and co-localizes with WGA at the Golgi apparatus (compare panels B and F).
  • Hmund 3 Translocation of Hmund 3 from cytosol to the Golgi apparatus after PDBu treatment was also confirmed by immunoblot analysis of a Golgi membrane preparation, following subcellular fractionation. As shown in Figure 10 (iii), after PDBU treatment, Hmund 3 is enriched in Golgi membranes compared to whole cell lysates. .
  • Hmund 3 has functional implications. While attempting to study the effect of prolonged exposure to DAG activation on Hmund 3 transfected cells, we noticed that the cells rounded up and died. However, Hmund 3 transfected cells without PDBu treatment and cells transfected with the C1 less mutant, with or without PDBu treatment, were relatively healthy. This finding was somewhat unexpected since DAG has long been known as a carcinogen and a promoter of cell growth, and led us to investigate the possibility and conclude that treatment with phorbol ester is inducing apoptosis in cells transfected with Hmund 3.
  • Hmund 3 is up-regulated by high glucose treatment in cultured human mesangial (33). Since the main thrust of the present study was to investigate the functional role of Hmund 3, we documented its in vivo expression. Furthermore, confirmation of up-regulation of Hmund 3 by hyperglycemia in an in vivo state is necessary to show the role for this gene in diabetic nephropathy.
  • mund 3-1 was higher in STZ-treated diabetic rat after 11 days of hyperglycemia. Expression of mund 3-1 was significantly higher in certain glomerular cells of diabetic animals. But it is impossible to identify these cells with any certainty at the resolution of confocal microscopy. However, because of our previous in vitro results (33), we determined that mund 3-1 is up-regulated in the mesangial cells. Increased expression level of mund 3-2 was also detected in diabetic rats with similar expression pattern as mund 3-1. Possibly because of low basal expression, we could not obtain satisfactory in situ hybridization data for mund 3-3 in rat kidney.
  • MC basal culture medium (MsBM) and renal epithelial basal medium (REBM) were purchased from Clonetic, San Diego, CA.
  • DNase I and ⁇ Sequence kit were purchased from Pharmacia Biotech, Uppsala, Sweden.
  • TA cloning kit was from Invitrogen, San Diego, CA.
  • RNeasy total RNA preparation kit, QIAshredder and QIAquick Gel Extraction kit were purchased from Qiagen, Chatsworth CA.
  • SP6 RNA polymerase, human cyclophilin template, 18S rRNA primers and competimers were from Ambion, Austin, TX. Vent DNA polymerase was obtained from New England Biolab, Inc, Beverly, MA. Rapid hybridization buffer and ⁇ -[ 32 P]-dATP (specific activity 800 Ci/mmol) were purchased from Amersham, Arlington Heights, IL.
  • [ 35 S]-Methionine (specific activity, 1000 Ci/mmol) was from NEN Life Science Products, Boston, MA.
  • Duralon-UV membranes was purchased from Stratagene, La Jolla, CA.
  • Six percent denatured polyacrylamide solution was purchased from National Diagnostics, Somerville, NJ.
  • Oligonucleotides were synthesized by Gibco BRL.
  • X-ray film was from Kodak, Rochester, NY.
  • Flexi rabbit reticulocyte lysate system and canine pancreatic microsomal membranes were purchased from Promega, Madison, Wl. Other chemicals with cell culture or molecular biology grade were obtained from local suppliers.
  • Human kidney MC and cortical epithelial cells Primary cultures of human kidney MC and cortical epithelial cells were purchased from Clonetic. Human MC were plated onto 25 cm 2 culture flasks and incubated in MsBM containing 5.5 mM D-glucose with 100U/ml penicillin, 100 ⁇ g/ml streptomycin and 5% FBS. Cells were subcultured at 80-90 % confluence. Cortical epithelial cells were grown in REBM supplement with 100U/ml penicillin and 100 g/ml streptomycin. Rat renal MC were prepared and cultured as previously described (53,54).
  • Human MC between passage 5-9 were used in this study. Three parallel experimental conditions were employed: 25 mM D-glucose (hyperglycemia), 5.5 mM D- glucose (low glucose control) and 25 mM L-glucose (osmolarity control). The details are as follows: for high glucose treatment, subconfluent MC were growth-arrested in MsBM + 0.5% FBS overnight and exposed to 5.5 mM or 25 mM D-glucose for 3 days with one change of medium on the second day. In parallel, L-glucose at the final concentration of 19.5 mM was added to the culture medium to serve as an osmolarity control.
  • RNA from human MC and cortical epithelial cells as well as rat MC was prepared using an RNeasy total RNA preparation kit according to manufacturer's instructions. Cell lysates were prepared following homogenization using a QIAshredder.
  • DDRT-PCR was performed by modified methods published by Liang and Pardee (55) and Sokolov and Prockop (56).
  • Total RNA from human kidney MC was incubated with DNase I to remove any contaminating genomic DNA prior to first strand DNA synthesis.
  • Reverse transcription (RT) was carried out by incubating a 20 ⁇ l reaction mixture containing 1 ⁇ g total RNA, 100 ng fully degenerate hexamer, 500 ⁇ M each of dATP, dGTP, dCTP and dTTP and 200 units of reverse transcriptase (Superscript II RNase H ' ) together with the buffer provided by the manufacturer.
  • the reaction mixture was incubated at 42°C for 50 min.
  • the reaction was terminated by heating at 70°C for 15 min.
  • E. coli RNase H (2 units) was then added to the reaction mixture followed by incubation at 37°C for a further 20 min to remove RNA complementary to the cDNA. Demonstration that the RNA was free of genomic DNA was confirmed using a pair of GAPDH specific primers (5'-ACCACAGTCCATGCCATCAC-3' and 5'-
  • PCR was carried out using two 10-mer oligonucleotides, 5'-CAAGCGAGGT-3' and 5'-GTGGAAGCGT-3'. In a total of 12.5 ⁇ l, the reaction mixture contained 1 ⁇ l of RNA with RT, 100 ⁇ M of each of dNTP, 4 ⁇ M of oligonucleotides, 1.5 mM of MgCI 2 , 0.1 mCi/ml of ⁇ -[ 32 P]-dATP and 1.25 unit of Taq DNA polymerase.
  • PCR was carried out using a Perkin Elemer PCR System 2400 (Perkin Elemer, Foster City, CA) starting at 94°C for 1 min, 34°C for 1 min and 72°C for 1 min for 45 cycles.
  • the resulting PCR products were subjected to 6% denatured polyacrylamide gel electrophoresis (PAGE) using radiolabelled 100 bp ladder as size markers. The gels were then dried and exposed to x-ray film overnight.
  • PAGE polyacrylamide gel electrophoresis
  • the resulting clone (pCMV SPORTHmund 3) was sequenced from both strands using standard techniques described above.
  • the primers were SP6, T7 promoters or synthetic oligonucleotides derived from the sequence information. Alignment and analysis of sequences was performed with Genework 2.5.1 (Oxford Molecular Group, Campbell, CA) using a Macintosh computer. Comparisons of similarity were performed using the Gapped BLAST search from GenBank.
  • RT products previously described were subjected to PCR for 30 cycles using a pair of primers (5'-GGAGCAAATCAATGCCTTGG-3' and 5'- TCGGATCCAATGTGCTCTGG-3') specific for Hmund 3, amplifying a 671 bp fragment.
  • 18S rRNA was chosen as a housekeeping gene by using 18S rRNA primers and 18S rRNA competimers with a ratio of 1 :2. These primers amplify a 488 bp fragment.
  • Resulting PCR products were subjected to 1.2 % agarose gel electrophoresis.
  • RT-PCR To determine mund 3 expression in epithelial and rat MC, we employed RT-PCR with a pair of primers (5'-GA(T)GTC(A)CTGAAGGAGCTCTGG-3' and 5'- AGGACA(T)GCACACTGCTTTGG-3' ) targeted to Hmund 3 and rat mund 3-2 both of which yield a 193 bp fragment. RT were performed post DNase I treatment on total RNA extracted from these cells as described above.
  • RNA (10 ⁇ g) extracted from human kidney MC was subjected to 1 % denatured formaldehyde agarose gel electrophoresis as described (36) then transferred to Duralon-UV membranes overnight and exposed to UV light for cross linking.
  • An 32 P- radiolabelled probe of Hmund 3 were generated from a PCR fragment derived from pCMV SPORTHmund 3 (4095 - 4288) with ⁇ -[ 32 P]-dATP using a Klenow Fragment and random hexamers.
  • Membranes were pre-incubated with rapid hybridization buffer at 65°C for 15 min and then incubated with radiolabelled probes at 65°C for 2 hours.
  • membranes were washed first in 2 x SSPE (1 x SSPE contains 150 mM NaCl, 20 mM NaH 2 PO 4 and 1 mM EDTA, pH, 7.4) with 0.1% SDS at room temperature for at least 20 min then twice with 0.1 x SSPE with 0.1% SDS at 65°C for 30 min each.
  • the Phosphor screen Molecular Dynamics, Sunnyvale, CA
  • the blots with Hmund 3 probe were stripped with a boiling solution of 0.1 x SSPE with 0.1% SDS.
  • the stripped membranes were reprobed with a 32 P-labelled human cyclophiiin template. Radioactivity of each band in digital images was analyzed on a PC using ImageQuant 4.0 (Molecular Dynamic). In vitro Translation
  • Translation products were detected by incorporating 1 ⁇ Ci/ ⁇ l of [ 35 S] methionine in the reaction mixture.
  • 1.5 equivalent of canine pancreatic microsomal membranes was added to 10 ⁇ l of in vitro translation reaction.
  • the resulting reaction was centrifuged at 16,000 g for 15 min to pellet microsomes.
  • In vitro translation products were subjected to 8% PAGE.
  • the gel was stained with Commassie brilliant blue then destained. The stained gel was then dried and exposed to x-ray film.
  • a PCR fragment was generated with Vent DNA polymerase, insert of pCMVSPORThmunc13 as a template and a pair of primers (5'-GAATACGGTTCTGGATGAGCT-3' and 5'- ocggccgcTCAAGCGTAGTCTGGGACGTCGTATGGGTAGCTCCCCTCCTCCGTGGAAC G -3') where the HA tag sequence is underlined and a Not I site is shown in lower case.
  • a stop code (5'-TCA-3') was placed between the HA tag and the Not I site.
  • the PCR product was then incubated with 2 units of Taq DNA polymerase at 72 C for 15 min and extracted by phenol/chloroform and ethanol precipitation.
  • the resulting pellet was resuspended and ligated to pCR2.1 by using a TA cloning kit.
  • This plasmid was then digested with Not I and EcoN I, subjected to 1% agarose gel electrophoresis.
  • the insert was purified and ligated to pCMVSPORThmunc13 previously cut with Not I and EcoN I.
  • the resulting construct (hmund 3-HA) was sequenced to confirm the addition of the HA tag.
  • the two PCR fragments were digested with Asc I, ligated with T4 DNA ligase, and the ligated product was subjected to 1% agarose gel electrophoresis to check the size and for purification.
  • the gel purified ligated piece was further digested with Kpn I and BstZ171 and ligated to Kpn I and BstZ171 digested pCMVSPORThmunc13-HA. Plasmids for cell transfection were prepared using a Midi plasmid preparation kit according to manufacturer's instructions.
  • OK cells were grown in MEM supplemented with 10% FBS and 100 U/ml penicillin and 100 ⁇ g/ml streptomycin, and plated in 60 mm or 100 mm culture dishes or on glass cover slips placed in 24 wells culture plates. Cells were transiently transfected
  • Cells were washed at least 8 times with PBST between incubation of anti-HA and anti-mouse IgG-rhodamine or after anti-mouse IgG-rhodamine. Cover slips were then mounted on a glass slide and observed under a confocal scanning microscope. For labeling of the Golgi apparatus, 0.05 mg/ml WGA-FITC was added to the anti-mouse IgG-rhodamine.
  • Cells grown on culture plates were washed 3 times with ice cold Hank's solution and scraped into 0.5 ml cell lysis buffer (50 mM Tris-HCI, 150 mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, 1 mM EDTA and protease inhibitor cocktail, pH 7.5), and then rocked at 4 C for 45 min. The insoluble fraction was removed by centrifugation at 14,000 g for 5 min. Supematants were subjected to 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • the membrane was washed twice with TBS, blocked with TBS containing 0.1 % Tween-20 (TBST) and 1 % normal horse serum for 30 min and then incubated with 0.5 ⁇ g/ml anti-HA in TBST. After washing with TBST for at least 4 times, the membrane fraction was incubated with 0.2 ⁇ g/ml anti-mouse IgG-biotin, washed with TBST and then incubated with the A and B reagent mix in a Vector ABC staining kit according to manufacturer's instructions. The blot was detected by ECL according to the manufacturer's instruction.
  • Golgi membranes were prepared by a sucrose density method reported previously (57) with a protease inhibitor cocktail presented in all buffer solution. The band at the interface of 0.8M and 1.2M sucrose was collected and subjected to 6% SDS PAGE and immunobloting as described above. Protein concentration was determined by Lowry assay with bovine serum albumin as standard using a DC Protein Assay kit following its instruction.
  • TdT terminal deoxynucleotidyl transferase
  • TUNEL mediated dUTP nick end labeling
  • Genomic DNA fragmentation of cells grown on 60 mm culture dishes was analyzed by 2% agarose gel electrophoresis using the procedure described elsewhere (58).
  • RT-PCR Relative reverse transcription polymerase chain reaction
  • RNA from rat kidney cortex was prepared using a TRIzol reagent according to instructions provided by the manufacturer and then treated with DNase I. Confirmation of no genomic DNA contamination in RNA preparations and relative RT-PCR were performed as described elsewhere (33). Primers for amplification of rat mund 3-1 are 5'- CGTGACCAAGATGAGTACTCC-3' (sense) and 5'-CGAAGTCGTGTAGTAAGGCG-3' (anti-sense) yielded a fragment of 195 bp.
  • Primers for rat mund 3-2 are 5'- GAGTCCTGAAGGAGCTCTGG-3' (sense) and 5'-AGGACAGCACACTGCTTTGG-3' (anti-sense) yielded a fragment of 193 bp.
  • Primers for rat mund 3-2 are 5'- GAGTCCTGAAGGAGCTCTGG-3' (sense) and 5'-AGGACAGCACACTGCTTTGG-3' (anti-sense) yielded a fragment of 193 bp.
  • Primers for rat mund 3-2 are 5'- GAGTCCTGAAGGAGCTCTGG-3' (sense) and 5'-AGGACAGCACACTGCTTTGG-3' (anti-sense) yielded a fragment of 193 bp.
  • Primers for rat mund 3-2 are 5'-
  • Templates for in vitro transcription were generated by PCR with primers described above for three different isoforms, except that for anti-sense cRNA, addition of T7 promoter (5'-TAATACGACTCACTATAGGGA-3') was present in the sense strain and for sense cRNA, T7 promoter was present in the anti-sense strain.
  • Anti-sense and sense cRNA for different isoforms were obtained by in vitro transcription. PCR templates (200 ng) were incubated with T7 RNA polymerase (40U), its reaction buffer provided by the manufacturer and DIG RNA labeling mix in a total volume of 40 ⁇ l at 37 C for 90min.
  • RNA Twenty ⁇ l recombinant RNA was purified by using a RNeasy total RNA preparation kit and its yield was estimated by A 6 o- The remaining cRNA was subjected to ethanol precipitation and resuspended in nuclease-free water.
  • Sections were then washed twice with PBS and acetylated with freshly prepared 0.1 M triethanolamine buffer (pH 8.0) containing 0.25% acetic anhydride. Slides were then incubated first with 4x SSPE (1x SSPE containing 150 mM NaCl, 20 mM NaH2PO4 and 1 mM EDTA, pH 7.4) containing 50% formamide at 37 C for 20 min and then overlaid with 75 ⁇ l hybridization buffer (40% fromamide, 10% dextran sulfate, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 10 mg/ml bovine serum albumin, 4x SSPE, 10 mM DTT, 0.4 mg/ml yeast t-RNA and 0.1 mg/ml poly(A) ) containing 50 ng of denatured DIG-labeled cRNA probe.
  • 4x SSPE (1x SSPE containing 150 mM NaCl, 20 mM NaH2PO4 and
  • Slides were incubated in a humid chamber at 42 C overnight. After hybridization, slides were washed at least 4 times in 1x SSPE at 37 C. Sections were incubated with 20 ⁇ g/ml RNase A in NTE buffer (500 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, pH 8.0) at 37 C for 30 min and washed twice with 0.1x SSPE. Slides were washed and blocked in TBS (100 mM Tris-HCI and 150 mM NaCl, pH 7.5) containing 1% blocking reagent and then incubated with 0.02 mg/ml anti-DIG-rhodamine for 1 h.
  • NTE buffer 500 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, pH 8.0
  • Diabetic nephropathy in type I diabetes an epidemiological study. Diabetologia 25: 496-501, 1983
  • Ziyadeh FN The extracellular matrix in diabetic nephropathy. Am J Kidney Diseases 22: 736-744, 1993
  • Derubertis FR, Craven PA Activation of protein kinase C in glomerular cells in diabetes: mechanisms and potential links to the pathogenesis of diabetic glomerulopathy. Diabetes 43: 1-8, 1994

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Abstract

L'invention concerne un gène humain munc13 (Hmunc13), ainsi qu'une protéine du rein et d'autres cellules, dont le rôle est important dans la signalisation cellulaire. Le gène considéré est à régulation glucosique. Il contribue aux complications rénales et microvasculaires associées à l'hyperglycémie dans le diabète sucré, par une variété de mécanismes englobant l'apoptose liée au Hmunc13. L'invention concerne également des séquences nucléotidiques et des protéines tenant lieu d'équivalents fonctionnels du point de vue biologique. L'invention concerne en outre des procédés relatifs à l'utilisation desdites séquences et protéines dans le cadre des traitements médicaux et de la sélection des médicaments.
EP98954094A 1997-12-12 1998-11-19 Gene a regulation glucosique Withdrawn EP1040125A1 (fr)

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US6935297P 1997-12-12 1997-12-12
US69352P 1997-12-12
PCT/CA1998/001061 WO1999031134A1 (fr) 1997-12-12 1998-11-19 Gene a regulation glucosique

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US7033790B2 (en) 2001-04-03 2006-04-25 Curagen Corporation Proteins and nucleic acids encoding same

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See references of WO9931134A1 *

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