WO2011087766A2

WO2011087766A2 - Methods for assessing er stress

Info

Publication number: WO2011087766A2
Application number: PCT/US2010/061427
Authority: WO
Inventors: Ling Qi; Liu Yang; Hui Chen
Original assignee: Cornell University
Priority date: 2009-12-22
Filing date: 2010-12-21
Publication date: 2011-07-21
Also published as: WO2011087766A3

Abstract

This disclosure relates to methods for assessing and quantifying ER stress. More particularly, the methods disclosed herein are based on assessing phosphorylation of IRE1α and PERK using a polyacrylamide gel containing a phosphate-binding moiety. The detection method disclosed herein allows for quantitative assessment of the level of stress in the ER and UPR activation, which provides basis for diagnosing ER stress disorders and for therapeutic drug screening.

Description

METHODS FOR ASSESSING ER STRESS

STATEMENT REGARDING GOVERNMENT SUPPORT

[0001] This invention was made with government support from the National Institutes of Health under grant NIH R01DK082582. The U.S. Government has certain rights in this invention.

CROSS REFERENCE TO RELATED APPLICATION

[0002] This application claims the benefit of U.S. Provisional Application No. 61/289,119, filed on December 22, 2009, the entire content of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0003] This disclosure relates to methods for assessing and quantifying ER stress. More particularly, the methods disclosed herein are based on assessing phosphorylation of IRE la and PERK, two sensors of ER stress.

BACKGROUND ART

[0004] Homeostasis in the endoplasmic reticulum (ER) and differentiation of specialized cell types (B cells and adipocytes) are tightly monitored through ER-to-nucleus signaling cascades, called the unfolded protein response (UPR) (Walter, Nature Reviews: Molecular Cell Biology, Vol. 8, pp. 519-529, 2007). Recent studies have linked ER stress and UPR activation to many human diseases including heart complications, neurodegenerative disorders and metabolic syndrome (Walter, supra; Kim et al., Nature Reviews: Drug

Discovery, Vol. 7, pp. 1013-1030, 2008). Indeed, chemical chaperones and antioxidants aiming to reduce ER stress and UPR activation have been shown to be effective in mouse models of obesity and type-1 diabetes (Basseri et al, Journal of Lipid Response, Vol. 50, pp. 2486-2501, 2009; Back et al., Cell Metab, Vol. 10, pp. 13-26, 2009; Malhotra et al., Proc National Academy of ^'Science, Vol. 105, pp. 18525-18530, 2008). Nevertheless, little is known about the UPR under physiological and pathological conditions, largely due to the lack of sensitive experimental systems that can detect mild UPR sensor activation.

[0005] The underlying molecular details of UPR signaling and activation induced by chemical drugs such as thapsigargin (Tg) have been characterized (Walter, supra). Upon drug-induced ER stress, two key UPR sensors, IRE la and PERK, dimerize or oligomerize and undergo trans-autophosphorylation by the cytosolic kinase domain, leading to their activation (Walter, supra; Kim et al, supra). As phosphorylation of IRE la and PERK has been difficult to detect in vivo, downstream effectors and targets of UPR, such as XBP1, eIF2a, CHOP and various ER chaperone genes (e.g. EDEM, ERDJ4, GRP78, etc), have been used as common markers for ER stress or UPR activation. These methods, albeit simple and convenient, are fraught with significant flaws, mainly due to the possibility of integrating signals not directly related to stress in the ER. For example, the PERK pathway of the UPR is a part of integrated stress response, consisted of three other eIF2a kinases (Walter, supra). Activation of any of these kinases leads to eIF2a phosphorylation and induction of ATF4 and CHOP (Walter, supra). Moreover, UPR target genes such as CHOP and ER chaperones are induced by other signaling cascades such as insulin and cytokines/growth factors (Miyata et al, Biochem Biophyc Res Communication, Vol. 365, pp. 826-832, 2008; Brewer et al, EMBO Journal, Vol. 16, pp. 7207-7216, 1997). Thus, the downstream targets are neither faithful nor reliable to reflect the status of ER stress and should not be used alone to assess UPR under in vivo physiological conditions.

SUMMARY OF THE DISCLOSURE

[0006] This disclosure relates to the use of a polyacrylamide gel containing a phosphate- binding moiety in assessing the phosphorylation status of two proteins located at the ER membrane, specifically, IRE la and PERK. The detection method disclosed herein allows for quantitative assessment of the level of stress in the ER and UPR activation, which provides basis for diagnosing ER stress disorders and for screening therapeutic drug useful for treating ER stress disorders. [0007] In one embodiment, a method of assessing ER stress in a biological sample is disclosed. This method involves detection of phosphorylated forms of IREl and PERK using polyacrylamide gels containing a phosphate-binding moiety.

[0008] In another embodiment, a method of quantifying the level of ER stress in a biological sample is disclosed. This method is based on quantifying the level of phosphorylation of IREla using polyacrylamide gels containing a phosphate-binding moiety.

[0009] In still another embodiment, a method of diagnosing or monitoring the progression of an ER stress disorder in a subject is disclosed. This method is based on quantifying the level of phosphorylation of IREl in a biological sample obtained from the subject, and comparing the level to a suitable control level.

[0010] In a further embodiment, a method of screening for a compound useful for treating an ER stress disorder is disclosed. This method involves subjecting a cell or tissue to a candidate compound and determining the effect of the candidate compound on the level of ER stress in said cell or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figures 1A-1F: Visualization and quantitation of ER stress under pharmacological stress. (A) Immunoblots of IREla (upper) and PERK (lower) proteins in Tg-treated MEFs treated with or without PPase or CIP. (B and D) Immunoblots of IREla (B) and PERK (D) using the Phos-tag™ or regular gels. MEFs were treated with 75 nM Tg at indicated period of time. (C) Quantitation of percent of phosphorylated IREl in total IREla protein in Phos- tag™ gels shown in B. (E) Immunoblots of IREl and PERK in wild type MEFs treated with Tg at indicated concentrations for 4 h. (F) Quantitation of percent of phosphorylated IREla in total IREla protein in Phos-tag™ gels in E. HSP90 and CREB, loading controls. Phos- tag™ gels are indicated with a bar at the left-hand side. "0" refers to the non- or

hypophosphorylated forms of the protein whereas "p" refers to the phosphorylated forms of the protein. [0012] Figures 2A-2D: Accumulation of misfolded proteins induces mild ER stress. (A and C) Immunoblots of IREl and PERK in HEK293T cells transfected with the indicated plasmids for 24 h. NHK, the unfolded form of a 1 -antitrypsin; p97-QQ, dominant negative form of p97-WT. ER-dsRed and GFP, negative control plasmids. HSP90, a position and loading control. (B and D) Quantitation of percent of phosphorylated IREla in total IREla protein in Phos-tag™ gels shown in A, C. Values are mean ± SEM *, PO.05 using unpaired two-tailed Student's t-test. Representative data from at least three independent experiments are shown.

[0013] Figures 3A-3D: Many tissues exhibit basal ER stress under feeding conditions. (A) Immunoblots of IREla and PERK in various tissues of wildtype mice. WAT, white adipose tissues; Pane, pancreas; Muscle, gastrocnemius. HSP90, a position and loading control. (B- C) Immunoblots of IREla and PERK in tissue lysates treated with PPase (B) or in pancreatic and WAT lysates prepared from mice injected with CHX (C). (D) Quantitation of percent of phosphoiylated IREla in total IREla protein in various tissues shown in A. Values are mean ± SEM. Representatives from at least two independent experiments are shown.

[0014] Figures 4A-4C: Fasting-refeeding induces mild ER stress in pancreas. (A)

Immunoblots of lysates from the pancreas of wild type mice either fasted or fasted followed by 2 h refeeding (refed). For the PERK blot, a mixture of all 6 samples treated with CIP were included as a control. For the p-PERK blot, Tg-treated MEF cell lysates with or without CIP treatment were included as a control. HSP90, a loading control. (B) Quantitation of the percent of phosphorylated IREla in pancreas under fasting and refeeding conditions shown in A (N=4 mice per cohort). (C) Q-PCR analyses of UPR genes in the pancreas under either fasting or refeeding. Values are mean ± SEM. Xbplt, total Xbpl; Xbpls/Xbplt, splicing efficiency. N=3-4 mice. *, PO.05 using unpaired two-tailed Student's t-test.

Representatives of at least two independent experiments are shown.

DETAILED DESCRIPTION

[0015] This disclosure relates to the use of polyacrylamide gels containing a phosphate- binding moiety in assessing the phosphorylation status of two proteins located at the ER membrane, which are upstream sensors of unfolded protein response (UPR). The methods disclosed herein permit quantitative assessment of the level of stress in the ER and UPR activation, including the level of tissue-specific basal ER stress, as well as the level of ER stress caused by the accumulation of misfolded proteins under physiological or disease conditions. Thus, related methods for diagnosing diseases associated with ER stress and for screening therapeutic compounds useful for treating such diseases are also disclosed.

[0016] Detection of Phosphorylation of IRE la and PERK

[0017] In one aspect, this disclosure provides a method of assessing the phosphorylation status of two proteins, inositol-requiring enzyme 1 ("IREla") and PKR-like ER-kinase ("PERK"), in a biological sample, by using a polyacrylamide gel containing a phosphate- binding moiety.

[0018] Phosphorylation of PERK and IREla can be more sensitively visualized on a polyacrylamide gel containing a phosphate-binding moiety, as compared to regular SDS- PAGE gels. The mobility shifts of phosphorylated proteins relative to unphosphorylated proteins are enhanced as a result of incorporation of a phosphate-binding moiety in the gel. The detection method disclosed herein is particularly advantageous for assessing

physiological UPR where ER stress can be relatively mild and cannot be reliably detected by conventional methods.

[0019] Polyacrylamide gels containing a phosphate-binding moiety have been described in the art, e.g., in Kinoshita et al. (Mol. Cellular Proteomics 5.4: 749-757, 2006) and US 2010/0108515 Al, the entire contents of which are incorporated herein by reference.

Polyacrylamide gels containing a phosphate-binding moiety suitable for use in the present method are further described below.

[0020] Biological samples of interest can be a cell such as a mammalian cell (e.g., a human cell), a population of cells, and tissue(s) or body fluid (such as blood or serum) of an animal (e.g., a mammal such as a human subject). A biological sample can be processed, e.g., subjected to culturing, centrifugation, or washing procedures, to amplify or concentrate cells of interest which are then lysed to obtain a sample suitable for analysis of proteins by electrophoresis (e.g., cell lysate or fractions of cell lysate).

[0021] Proteins in the sample are then separated on a gel containing a phosphate-binding moiety. Protein bands indicative of IREl and PERK can be visualized by an immunoblot, e.g., a Western blot using antibodies directed specifically to IREla and PERK, respectively. Antibodies directed specifically to IREla and PERK are available in the art, including through various commercial sources (e.g., Cell Signaling), and can be readily generated by those skilled in the art as well. When both IREl and PERK are being analyzed, it is preferable to run two gels which are separately blotted with antibodies to IREl and PERK, respectively.

[0022] Phosphorylated PERK appears as a smear above the unphosphorylated band on the gel. On the other hand, phosphorylated IREla appears on the gel as a discrete band(s) having slower mobility relative to the unphosphorylated IREla band. This characteristic band pattern of IREla permits quantitative assessment of the phosphorylation status of IREla. Densities of bands representing phosphorylated and unphosphorylated IREla, respectively, can be determined using any suitable method known in the art, e.g., the Image J software available from the National Institutes of Health and the Image Lab software from Bio-Rad. The approach disclosed herein is capable of sensitive detection of phosphorylation of IREla when phosphorylated IREla accounts for 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater, of total IREla.

[0023] Detection and Quantitation of ER Stress

[0024] The endoplasmic reticulum ("ER") serves a number of functions, including the facilitation of protein folding and the transport of synthesized proteins. The term

"endoplasmic reticulum stress" ("ER stress") refers to an imbalance between the demand for the synthesis of proteins and the folding capacity of the ER to meet that demand. [0025] The unfolded protein response ("UPR") is activated in response to an accumulation of unfolded or misfolded proteins in the ER lumen. The UPR aims to restore normal function of the cell by halting protein translation and activate the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these objectives are not achieved within a certain time period or the disruption is prolonged, the UPR aims to initiate programmed cell death (apoptosis).

[0026] In this disclosure, ER stress is evaluated based on the detection of the phosphorylation status of PERK and IREla on polyacrylamide gels containing a phosphate-binding moiety. PERK and IREla are laiown to be upstream sensors of unfolded protein response. Therefore, activation of these proteins are more reliable sensors of ER stress, as compared to downstream targets which may be involved in integrating signals from other unrelated pathways.

[0027] In some embodiments, ER stress is evaluated based on detection of phosphorylation of one of PERK or IREla. In other embodiments, ER stress is evaluated based on detection of phosphorylation of both PERK and IREla.

[0028] In further embodiments, ER stress is quantified based on the level of phosphorylation of IREla. As supported by the data disclosed herein, the level of phosphorylation of IREla correlates with the level of ER stress. The level of phosphorylation of IREla can be determined based on the ratio of phosphorylated IREl versus unphosphorylated IREla, or the percentage of phosphorylated IREla among the total IREla. An increase in the level of phosphorylation of IREla indicates an increase in ER stress, and a decrease in the level of phosphorylation of IREla indicates a decrease in ER stress.

[0029] In certain embodiments, the method includes comparing the level of ER stress, i.e., based on the level of IREla phosphorylation, with a reference or control. The reference or control may vary depending upon the purpose of the evaluation. In some embodiments, the levels of ER stress are compared among different cell types or tissues in an individual, i.e., any individual (with or without an ER stress disorder). In other embodiments, the levels of ER stress in the same cell type or tissue among different individuals are compared; e.g., a patient under examination suspected of having an ER stress disorder relative to a normal or healthy individual or a normal population. In still other embodiments, the levels of ER stress in a cell or tissue of the same individual are compared, e.g., post-treatment versus pre- treatment, or at different time points after treatment with a therapeutic or candidate compound.

[0030] Diagnosing and/or Monitoring ER Stress Disorder

[0031] In a further aspect, the level of ER stress in certain cells or tissue(s) is quantified and serves as the basis for diagnosing and/or monitoring an ER stress disorder in a subject.

[0032] The term "ER stress disorder" refers to a disorder caused by, or contributed to by, increased ER stress levels. Examples of ER stress disorders include diabetes (e.g., type 1 or type 2 diabetes), and protein conformational diseases (diseases caused by, or contributed to by, protein misfolding) which include Alzheimer's disease, Parkinson's disease, Huntington's disease, cystic fibrosis, sickle cell anemia, Kreutzfeldt- Jakob disease, familial

hypercholesterolaemia, Alphal -antitrypsin (alphal-AT), cirrhosis, emphysema, systemic and cerebral hereditary amyloidoses, immunoglobulin light chain amyloidosis, haemodialysis- related amyloidosis, reactive amyloidosis, Wolcott-Rallison syndrome, and Wolfram syndrome Wolfram syndrome 1 (WFS1).

[0033] Depending upon the disorder, a biological sample is obtained that contains certain cells or tissue(s) from the subject under examination; e.g., a blood sample (containing lymphocytes) or a tissue biopsy sample. Liver or adipose tissues harvested through biopsy in human patients can be the subject of evaluation.

[0034] In some embodiments, an increased level of ER stress as compared to a suitable control (e.g., relative to the level in a normal and healthy individual, tissue or cell) is indicative of an ER stress disorder. In other embodiments, an increased level of ER stress as compared to a suitable control indicates that ER stress is a component of a disease in an individual. In other embodiments, an increase (or decrease) in the level of ER stress at different time points indicates the progress (or regression) of the ER stress disorder in a subject. [0035] Drug Screening

[0036] In a further aspect, a method of screening for molecules that reduce or increase ER stress is disclosed. According to this method, cells or tissues (in culture) or an animal can be subjected to a candidate molecule, and its effect on the ER stress in the cells, tissue(s), or cells in the animal, can be determined. Molecules that reduce ER stress are useful for treating an ER stress disorder. Molecules that increase ER stress (to lead to cell death) are useful for treating cancer.

[0037] In some embodiments, the cells, tissues, or the animal can be treated first with a molecule known to increase ER stress. Subsequently, the cells, tissues or the animal can then be treated with a candidate molecule to determine whether the candidate compound can reduce the ER stress. Alternatively, the cells, tissues, or the animal can be treated with a candidate molecule to determine whether the candidate compound can affect the ER stress under basal conditions.

[0038] Candidate molecules can be a nucleic acid, polypeptide, peptide, or small molecule compound. Small molecule compounds refer to low molecular weight organic compounds, which are generally not polymers. Typically small molecules have a molecular weight of less than 1000 Daltons, or approximately 800 Daltons or less.

[0039] Kits

[0040] The various reagents and molecules described herein can be packaged in a kit for practicing the methods disclosed herein.

[0041] Polyacrylamide Gel Containing A Phosphate-Binding Moiety

[0042] Polyacrylamide gels containing a phosphate-binding moiety suitable for use in the method of this invention include those described by Kinoshita et al. (Mol. Cellular

Proteomics 5.4: 749-757, 2006) and US 2010/0108515 Al, the entire contents of which are incorporated herein by reference. [0043] More specifically, a polyacrylamide gel suitable for use in the method of this invention is characterized in that at least a part of the structure of the gel contains a structure represented by the following formula (I):

wherein M²⁺ represents a transition metal ion; and X represents a linker group.

[0044] In the above-mentioned formula (I), the transition metal ion of M²⁺ is in some embodiments a divalent cation of a transition metal belonging to the fourth period, such as Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺. In specific embodiments, Mn²⁺ or Zn²⁺ is used. In the acrylamide structure (I), a complex part, in which these transition metal ions are coordinated, has an extremely high coordination ability for a phosphoryl group (phosphate monoester group) of a phosphorylated protein.

[0045] The "linker group" is a group bonding the part which interacts with phosphorylated peptide (generally referred to as phosphate binding moiety) and an acrylamide part, and has a function of making the production of a precursor (monomer) of a polyacrylamide compound easy or making the coordination with the phosphorylated peptide easy by increasing the flexibility of the phosphate binding moiety. Examples of the "linker group" are not particularly limited if they have the above-mentioned function, and may include a C1-C6 alkylene group (a straight or branched divalent aliphatic hydrocarbon group having 1-6 carbon atoms), an amino group (— NH— ), an ether group (~0~), a thioether group (~S~), a carbonyl group (~C(=0)~), a thionyl group (~C(=S)~), an ester group, an amido group, a urea group (-NHC(=0)NH-), a thiourea group (^» HC(=S)NH^»), and a C1-C6 alkylene group having the group selected from a group consisting of an amino group, an ether group, a thioether group, a carbonyl group, a thionyl group, an ester group, an amido group, a urea group and a thiourea group at one terminal or both terminals.

[0046] One or more common substituents such as methyl group can be introduced into a pyridine ring of structure (I), so long as the substituent does not change the binding functionality of the phosphate-binding moiety. Further, the substitution site of the linker group in the acrylamide structure (I) is not particularly limited. For example, the linker group may exist at the site shown in the following structure (Γ).

(Γ)

[0047] A polyacrylamide gel having structure (I) in at least part of the acrylamide structure can be made as described in US 2010/0108515 Al, for example. More specifically, an acrylamide compound characterized by formula (II), and/or a complex of this compound with a transition metal, is added as a monomer to a conventional acrylamide mixed solution (i.e., mixture of acrylamide and N,N'-methylenebisacrylamide): )

wherein X represents the above-mentioned linker group.

[0048] In general, the amount of the compound (II) is added to have a mole ratio to acrylamide of about 1 x 10^~7 to 1 x 10^" , more preferably 1 x 10^~6 to 1 x 10^"4 Further, a transition metal compound such as a transition metal salt is added, typically in an amount at least twice the molar equivalent to the compound (II). The transition metal compound is added prior to polymerization of the acrylamide monomer. Specific examples of transition metal salts include nitrates and acetates.

[0049] A compound for formula (II) can be produced using methods known in the art, e.g., as described in US 2010/0108515 Al .

[0050 In one embodiment, the compound of formula (II) is the following specific compound:

[0051] This compound of fonnula (ΙΓ) can be made according to the procedure described in Kinoshita et al. (Mol. Cellular Proteomics 5.4: 749-757, 2006), and is also commercially available, e.g., through Nard Institute, Ltd. (Japan) under the brand name "Phos-tag™". The compound of formula (ΙΓ) is added to the acrylamide gel solution before polymerization at 1- 75 μΜ, preferably 1-50 μΜ, with twice molar amount of a Mn salt.

[0052] In specific embodiments, the compound of formula (ΙΓ) and MnCl₂ are added to a acrylamide gel solution before polymerization at a final concentration of 25 μΜ and 50 μΜ, respectively, for assessing IREla phosphorylation; and at a final concentration of 3.5 μΜ and 7 μΜ, respectively, for assessing PERK phosphorylation. [0053] The following examples further illustrate and by no means limit the methods of this invention.

[0054] Example 1 - Materials And Methods

[0055] Cells and reagents. HEK293T and MEF cells were maintained in DMEM

supplemented with 10% FBS (Hyclone) and 1% penicillin/streptomycin. IRE la, PERK null MEFs (provided by Dr. David Ron and Douglas Cavaener) were cultured in DMEM with 10% FBS. Tg (EMD Calbiochem) and stock CHX (Sigma) were dissolved in DMSO and ethanol, respectively. Cells were treated with Tg at indicated concentrations for the indicated times and immediately snap-frozen in liquid nitrogen. The Phos-tag™ compound was purchased from NARD Institute (Japan).

[0056] Mice and tissues. Wild type C57BL/6 and ob/ob mice on C57BL/6 background were purchased from the Jackson Laboratory. For some experiments, mice were injected with 40 μg CHX per gram body weight (dissolved in 100 μΐ PBS) for 2 hours. Epididymal white adipose tissues (WAT) and pancreas were harvested. Following cervical dislocation, tissues were harvested immediately, snap-frozen in liquid nitrogen and stored at -80°C. Glucose levels were measured in ob/ob and wild type lean mice under ad libitum condition using glucometer.

[0057] Plasmids. NHK, wild type and dominant-negative E305Q/E578Q p97 (p97-QQ) plamids were provided by Dr. Qiaoming Long and Dr. Fenghua Hu (Cornell University).

[0058] Transfection. HEK293T and MEF cells were transfected with plasmids using polyethylenimine (PEI, Sigma) or lipofectamine 2000 (Invitrogen). Cells were snap-frozen in liquid nitrogen 24 h post-transfection followed by Western blot.

[0059] Protein Lysates. Cells were lysed in Tris-based lysis buffer (150 mM NaCl, 50 mM Tris pH7.5, lmM EDTA and 1% Triton X-100). Supernatant was collected after

microcentrifugation at 4°C for 10 min. Frozen tissues were placed in the same lysis buffer supplemented with 1 μΜ DTT and lx protease inhibitors (Sigma), and sonicated twice for 10 sec. Supernatant was collected after microcentrifugation at 4°C for 10 min.

[0060] Tissue nuclear-cytosolic fractionation. Cells in a 6-cm dish were resuspended in 200 μΐ ice-cold hypotonic buffer (10 mM HEPES, pH 7.9; 10 mM KC1, 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT) and allowed to swell on ice for 15 min followed by addition of 10% of NP-40 to a final concentration of 0.6%. Lysates were vortexed vigorously for 15 s prior to centrifugation at top speed for 1 min. Supernatant was transferred to a fresh tube as the cytosolic fraction. Pellets were resuspended in 50 μΐ ice-cold high-salt buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA and 1 mM DTT) and vortexed vigorously for 15 sec every 5min for a total of 20 min. Extracts were spun at 4°C for 5 min and the supernatant (nuclear fractions) was collected.

[0061] Western blot and Phos-tag™ gels. Lysate protein concentrations were measured using the Bradford assay and normalized to 0.5-2 μg/μl using SDS sample buffer. Samples were boiled 5 min prior to loading onto a SDS PAGE gel. 15-30 μg of total cell lysates or 10 μg of nuclear extracts were used in a mini SDS-PAGE. For a Phos-tag™ gel, 5% SDS- PAGE containing the Phos-tag™ compound of formula (ΙΓ) above and manganese chloride (Sigma) were used. Phos-tag™ gels are sensitive to the salt concentration in the samples, thus samples were prepared at relatively high concentrations and were diluted similarly. 25 μΜ Phos-tag™ compound and 50 μΜ manganese chloride were used for IREla blot; and 3.5 μΜ Phos-tag compound and 7 μΜ manganese chloride were used for PERK blot. Phos- tag™ gels were run under the following running conditions: 100 V for 3 h for IREla, and 15 mA for 15 min followed by 5 mA for 9.5 h for PERK. IREla and PERK were generally loaded on separate gels. For both regular and Phos-tag™ gels, gel-running was stopped when the 75 kDa maker ran off the gel and same amounts of lysates were loaded. Phos- tag™ gels were soaked in 1 mM EDTA for 10 min prior to transfer onto a PVDF membrane. Membranes were routinely strip-reprobed for 2-4 times. The IREla blot in the Phos-tag™ gel was routinely reprobed with HSP90 (90kDa vs. 1 lOkDa IREla) as a position and loading control. [0062] Phosphatase treatment. 100 μg cell lysates or tissue lysates were incubated with 2.5 μΐ calf intestinal phosphatase (CIP) or 0.5 μΐ lambda phosphatase ( PPase, New England BioLabs- NEB) in lx NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT) or lx PMP buffer (50mM HEPES, lOOmM NaCl, 2mM DTT, 0.01% Brij35, NEB) with ImM MnCl₂ at 37 or 30°C for 45 or 30 min, respectively. Reaction was stopped by adding 5x SDS sample buffer and incubated at 90°C for 5min.

[0063] Antibodies for Western blot. GRP78 (goat, 1:1,000), XBP1 (XBPlu/s-specific, rabbit, 1 :1,000), CHOP (mouse, 1 :500) and HSP90 (rabbit, 1:5,000) were purchased from Santa Cruz; p-eIF2a, eIF2a, IRE la and (p)-PERK (rabbit) antibodies were purchased from Cell Signaling and used at 1 : 1,000-2,000. Primary antibodies were diluted in 5% milk/TBST or 2% BSA/TBST and incubated with PVDF membrane overnight at 4°C. Secondary antibodies were goat anti-rabbit IgG HRP, goat anti-mouse IgG HRP (Biorad) and donkey anti-goat IgG HRP (Jackson ImmunoResearch), all of which were used at 1 : 10,000.

[0064] RNA extraction and Q-PCR. Total mRNA extractions were carried out using Trizol per supplier's protocol (Molecular Research Center, Inc.) and reverse transcribed using Superscript III kit (Invitrogen). For Q-PCR, cDNA were analyzed using the SYBR Green PCR kit (Applied Biosystems) on the ABIPRISM 7700 Sequence Detector (Perkin Elmer) or the SYBR Green PCR system (provided by Dr. Jeff Pleiss, Cornell University) on the iQ5 Q- PCR machine (Biorad). All Q-PCR data were normalized to ribosomal 132 gene in the corresponding sample.

[0065] RT-PCR for Xbpl splicing. For RT-PCR to detect Xbpl splicing, PCR primers were designed to encompass the splicing sequences in mouse Xbpl mRNA. PCR amplification was carried out using purified Taq polymerase (provided by Dr. Qiaoming Long, Cornell University) with annealing temperature at 58°C for a total of 30 cycles. PCR products were separated by electrophoresis on a 2.5% agarose gel (Invitrogen) containing ethidium bromide (Sigma). Three controls were samples derived from cells treated with 300 nM Tg for 0, 2 and 5 h. [0066] Image quantification. Quantification was done using the NIH ImageJ software where band densities was calculated and subtracted from the background. Data are represented as mean ± SEM from several independent samples.

[0067] Statistical analysis. Results are expressed as mean ± s.e.m. Comparisons between groups were made by unpaired two-tailed Student t-test. <0.05 was considered as statistically significant. All experiments were repeated at least three times and representative data are shown.

[0068] Example 2 - Visualization Of Sensor Phosphorylation And Quantitation Of E Stress.

[0069] Experiments were performed to optimize the separation of phosphorylated IRE la and PERK proteins in a Phos-tag™ -based Western blot, which was reversed by phosphatase treatment (Figure 1A). It was found that Phos-tag™ gels for IRE la were sensitive to salt concentrations. Unequal lysate concentration could lead to the curvature of the samples. Thus, samples were prepared at relatively high concentrations and diluted similarly.

Moreover, IREla blots were routinely reblotted with HSP90 (90kDa vs. 1 lOkDa IREla) as a position and loading control. For most of the experiments shown here, one shifted band representing the hyperphosphorylated IREla was observed. In the pancreas, two

phosphorylated bands were observed, with one being dominant (Figures 3A, 3D). Phos- tag™ gels for PERK, on the other hand, were not hypersensitive to salt concentrations. Due to the unique hyperphosphorylation pattern exhibited by the PERK protein, locating the non- phosphorylated PERK band under physiological conditions sometimes required complete phosphatase digestion. In most experiments, the goal was to assess and compare UPR activation, i.e. to detect changes in the hyperphosphorylation status of PERK under different conditions; therefore, precise location and designation of the non-phosphorylated form of PERK was not critical. To this end, most PERK blots in Phos-tag™ gels are indicated with a "p", to refer to the hyperphosphorylated forms of PERK, whereas the bottom visible band(s) indicated by "0" refers to the non- or hypophosphorylated PERK. [0070] IRE la and PERK hyperphosphorylation patterns were distinct (Figure 1A), possibly reflecting various levels of phosphorylation upon activation. Specifically, p-IREla exhibited one discrete slow-migrating band in the Phos-tag™ gels, a feature that allowed for quantitation of the percent of p-IREla (see below). Upon treatment with Tg, the percent of phosphorylated IRE la increased from 30 min post-treatment, peaked around 4 h and slightly decreased at 8-17 h, with nearly 30, 100 and 80% of IREla undergoing phosphorylation, respectively (Figures 1B-C). Similarly, PERK hyperphosphorylation increased at 30 min, peaked at 4 h and decreased after 8-17 h. In both cases, the dynamic patterns of IREla and PERK phosphorylation were less discernible or not discernible at all in regular gels or using the phospho-specific antibody (Figures IB and ID).

[0071] The temporal dynamic patterns of IREla and PERK phosphorylation (Figures IB and ID) indicate that hyperphosphorylation of these UPR sensors correlated with the amount of stress in the ER. Further supporting this notion, hyperphosphorylation of IREla and PERK increased with Tg concentrations, peaking and subsequently plateauing at 38 nM Tg upon 4 h treatment (Figure IE). Demonstrating the sensitivity and quantitative nature of the detection, about 15% of IREla protein were phosphorylated upon 4 nM Tg treatment and increased to about 50% under 9 nM Tg (Figures 1E-F). In contrast, using a regular gel system, IREla phosphorylation was hardly visible and phosphorylation of PERK was also much less apparent (Figure IE).

[0072] Example 3 - Accumulation Of Misfolded Proteins Induced Mild ER Stress.

[0073] Although ER stress was initially characterized as induced by accumulation of unfolded proteins, it had been difficult to quantitate the levels of stress inflicted by accumulation of misfolded proteins in the ER prior to this application. Terminally-misfolded a 1 -antitrypsin (at) genetic variant-null Hong Kong (NHK) or the dominant-negative mutant of p97 (p97-qq) was ectopically expressed in HEK293T cells (Figures 2A and 2C). NHK is a frequently mutated allele in human al AT deficiency (Sifers et al., J. Biol Chem. 263: 7330-7335, 1988), and p97 is a member of the AAA-ATPase protein family involved in ERAD (Ye et al., Nature 414: 652-656, 2001). In both cases, IREla and PERK were phosphorylated when compared to cells over-expressing control or wild type proteins (Figures 2 A and 2C), indicating the specificity of sensor activation in response to misfolded proteins. IREla phosphorylation nearly tripled in both cases reaching 20-30% (Figures 2B-2D).

[0074] Similar observations were obtained in Selll-deficient MEFs (not shown), in which ERAD was defective (Francisco et al, J. Biol. Chem. 285: 13694-13703, 2010).

[0075] The above results revealed quantitatively the extent of ER stress induced by accumulation of misfolded proteins in the ER, a finding that would have been impossible using regular gel systems under similar running conditions (Figures 2A and 2C).

[0076] Example 4 - Many Tissues Exhibited Basal ER Stress Under Feeding Conditions.

[0077] This example describes experiments performed to analyze levels of basal ER stress in various tissues from adult mice under feeding conditions. Many tissues exhibited slower electrophoretic mobility of IREla and PERK proteins (Figure 3A). The mobility shift of IREla and PERK was specific for phosphorylation as it was reversed by phosphatase treatment (Figure 3B). Phosphorylation of these sensor proteins was caused by signals from the ER as it was attenuated in the presence of a protein translation inhibitor, cycloheximide (CHX) (Figure 3C). Quantitatively, phosphorylated IREla accounted for over 40% of total IREla protein in the pancreas and about 10% in most of the other tissues (Figure 3D). These data were consistent with another report describing an XBP1-GFP reporter mouse, which exhibited basal UPR primarily in the pancreas (Iwawaki et al., Nat. Med. 10: 98-102, 2004). In skeletal muscle, PERK protein was beyond detection limit, and IREla exhibited multiple slower migrating bands (Figure 3 A). These additional slower migrating bands in the IREla blot were not due to phosphorylation as they were resistant to phosphatase treatment.

[0078] Example 6 - Refeeding Induced Mild ER Stress In The Pancreas.

[0079] This example describes experiments conducted to analyze UPR activation during the fasting-refeeding process in the pancreas (20 hr fasting followed by 2 hr feeding). Refeeding significantly increased phosphorylation of both IREla and PERK (percent of p-IREla under fasting vs. refeeding: 8.7 ± 4.3% vs. 29.5 ± 5.4%; PO.05) (Figure 4A-B). This effect was independent of the region of the pancreas sampled (e.g., proximal, middle and distal regions of the pancreas relative to the duodenum showed similar phosphorylation patterns). In support of the importance of the instant method in analyzing mild physiological UPR, similar running conditions in regular gels resulted in a much less apparent mobility-shift for PERK (Figure 4A). This mild PERK phosphorylation was undetectable using the phospho-PERK antibody (Figure 4A). In addition, although IRE la did exhibit a slightly slower mobility shift upon refeeding in regular gels after prolonged gel running conditions, this shift did not reflect the overall phosphorylation status of IRE la as revealed by the Phos-tag™ gel (Figure 4A). Furthermore, phosphorylation of eIF2a, an immediate downstream effector of PERK, did not change (Figure 4A). Finally, while some UPR targets such as CHOP, ERDJ4 and P58IPK were induced upon refeeding (Figure 4C), both the mRNA and protein levels of Grp78, an ER chaperone, were not altered (Figures 4A and 4C). Thus, these data demonstrate that the fasting-feeding cycle acutely stimulates mild UPR activation in the pancreas.

[0080] Discussion

[0081] The data disclosed herein reveal that many tissues and cell types displayed constitutive basal UPR activity, presumably to counter misfolded proteins passing through the ER. The data also show that a fraction of mammalian IRE la and PERK was constitutively active in many tissues, with about 10% IRE la being phosphorylated and activated. In skeletal muscles, IRE la exhibited multiple non-phosphorylated bands while PERK protein was beyond the detection limit.

[0082] As exocrine pancreatic acinar cells account for over 80% of the pancreatic mass, pancreatic ER stress observed under the fasting-feeding cycle likely reflects the acute elevation of protein synthesis in acinar cells in response to food intake. Indeed, mice with XBP1 or PERK deficiency have been reported to exhibit defective development of exocrine pancreas, suggesting an indispensable role for UPR in countering the fluctuating stress associated with food intake. The data disclosed herein also showed a 3 -fold increase of IREla phosphorylation, i.e. UPR, to enhance ER homeostasis in preparation for an upcoming wave of protein synthesis. These results are in line with earlier reports showing that ER in pancreatic acinar cells became dilated within 2-4 h refeeding. It is proposed herein that the proteostasis network in acinar cells is very flexible in order to respond to many variables in the feeding process. The same is likely to be true for pancreatic islet cells.

[0083] There are a number of advantages provided by the method disclosed herein. For example, dynamic ranges of PERK and IRE la phosphorylation can be more sensitively visualized as compared to regular SDS-PAGE gels. This is important for physiological UPR where ER stress can be relatively mild such that conventional methods may no longer be accurate or reliable. Furthermore, the unique pattern of IRE la phosphorylation in a Phos- tag™ gel allows for quantitative assessment of ER stress. It is believed that the disclosure herein is the first demonstration of quantitation of ER stress under physiological or pathological settings (e.g. the fasting-refeeding cycle and the accumulation of misfolded proteins). Moreover, in comparison to using commercially-available phosphorylation-specific antibodies (e.g. P-Ser724A IRE la and P-Thr980 PERK), the instant method provides a more sensitive and more complete view of the overall phosphorylation status of IRE la and PERK proteins, independent of whether these specific residues are indeed phosphorylated under certain physiological conditions.

Claims

WHAT IS CLAIMED IS:

1. A method of assessing ER stress in a cell, said method comprising separating proteins from the cell on a polyacrylamide gel which contains a phosphate-binding moiety, and detecting phosphorylation of at least one of IRE la or PERK on said gel.

2. The method of claim 1 , wherein the proteins are separated on two separate gels which are used for the detection of phosphorylated IRE la and phosphorylated PERK, respectively, and wherein phosphorylation of both proteins indicates the presence of ER stress.

3. The method of claim 1, wherein phosphorylation of IRE la is detected, and the level of phosphorylation is quantitated and correlated with the level of ER stress wherein a higher level of phosphorylation of IRE la indicates a high level of ER stress, and a lower level of phosphorylation of IRE la indicates a lower level of ER stress.

4. The method of claim 3, wherein the quantitation is based on determining the density of the band or bands representing phosphorylated IRE la in relation to the density of the band representing unphosphorylated IREla in a Western blot analysis.

5. The method of claim 1 , wherein the phosphate-binding moiety has the formula (I) or (Γ).

6. The method of claim 5, wherein the polyacrylamide gel containing said phosphate- binding moiety is formed from polymerizing an acrylamide mixed solution containing an acrylamide compound represented by formula (II) and a transition metal complex thereof as a monomer.

7. The method of claim 6, wherein the compound represented by formula (II) is the compound of formula (ΙΓ), and the transition metal is Mn2+.

8. The method of claim 7, wherein the compound of formula (ΪΓ) and M2+ are added to the acrylamide mixed solution at 25 and 50 μΜ, respectively, for detecting phosphorylation of IRE la.

9. The method of claim 1 , wherein said cell is a cell from a biological sample obtained from a mammal.

10. The method of claim 9, wherein said sample is a blood sample or a tissue biopsy sample.

11. A method of diagnosing or monitoring the progression of an ER stress disorder in a subject, comprising obtaining a biological sample from the subject, quantifying the level of phosphorylation of IRE la in cells of said sample, and comparing the level to a control, wherein an increased level of phosphorylation of IRE la indicates the presence or progression of said ER stress disorder.

12. The method of claim 11, wherein the ER stress disorder is selected from the group consisting of diabetes (e.g., type 1 or type 2 diabetes), Alzheimer's disease, Parkinson's disease, Huntington's disease, cystic fibrosis, sickle cell anemia, Kreutzfeldt- Jakob disease, familial hypercholesterolaemia, Alpha 1 -antitrypsin (alphal-AT), cirrhosis, emphysema, systemic and cerebral hereditary amyloidoses, immunoglobulin light chain amyloidosis, haemodialysis-related amyloidosis, reactive amyloidosis, Wolcott-Rallison syndrome, and Wolfram syndrome Wolfram syndrome 1 (WFS1).

13. The method of claim 11, wherein said biological sample is a blood sample or a tissue biopsy sample.

14. A method of screening for a compound that affects ER stress, comprising subjecting a cell or tissue to a candidate compound, determining the change in the level of phosphorylation of IRE la in said cell or tissue in the presence of the compound relative to the absence of the compound.

15. The method of claim 14, wherein said cell or tissue is in a mammal, and said candidate compound is administered to said mammal.

16. The method of claim 15, wherein said candidate compound is a small molecule compound.

17. The method of claim 14, wherein the compound is identified as a compound that reduces ER stress based on a decreased level of phosphorylation of IRE la.

18. The method of claim 14, wherein the compound is identified as a compound that increases ER stress based on an increased level of phosphorylation of IRE la.