Structural Biochemistry/Diabetes

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INTRODUCTION[edit]

Mellitus diabetes is categorized as a group of chronic (lifelong) diseases in which high levels of sugar (glucose) exist in human blood. It is the 9th leading cause of death in the world, killing more than 1.2 million people each year. Diabetes is categorized in many groups: type 1, type 2, gestational, prediabetes and a few more. However, the two main groups are type 1 and type 2, type 2 being more common. Different symptoms correlate to different types of diabetes, but generally, diabetes exhibit similar symptoms such as the following:



Type 1 Symptoms include:
- Frequent urination
- Unusual thirst
- Extreme hunger
- Unusual weight loss
- Extreme fatigue and irritability
Type 2 Symptoms include:
- Any of the type 1 symptoms
- Frequent infections
- Blurred vision
- Cuts/bruises that are slow to heal
- Tingling/numbness in the hands/feet
- Recurring skin, gum, or bladder infections


Many complications arise and are caused by diabetes. People may experience problems in their vision which potentially leads to blindness, numbness in their feet and hands, especially in the legs, hypertension (high blood pressure), their mental health, hearing loss, and others that are gender related. Although there is no cure for type 1 diabetes, maintaining an idea body weight with an active lifestyle and a healthy diet can prevent and sustain type 2 diabetes.

TYPE 1 DIABETES[edit]

In type 1 diabetes, the pancreas fails to produce little or no insulin. Insulin is a hormone that is produced by beta cells located in the pancreas. It functions as a transporter of sugar (glucose) into cells throughout the body. Glucose travels through the hemoglobin, an enzyme found in blood, and is stored away and later used for energy by the body’s organs. The failure to produce insulin results with a lack of an appropriate amount needed for the human body to function at a normal pace. Without enough insulin, sugar accumulates in the bloodstream, thus raising the blood’s sugar level – this event is called hypertension.

There is no exact cure for type 1 diabetes and the exact cause is still unknown. Researchers believe it is an autoimmune disorder, a condition in which the immune system mistakenly damages healthy tissues. In this case, the pancreas would have been attacked, preventing its function to produce insulin. Type 1 diabetes shows hereditary correlation, meaning this disease can be passed down through families. Also, the adolescent group is the most often diagnosed group of people.

TYPE 2 DIABETES[edit]

In type 2 diabetes, fat, liver, and muscle cells become insulin resistant. Those cells do not respond to insulin correctly and fails to obtain the sugar (glucose) that is being transported. Because glucose is crucial for the cells’ functions, the pancreas would sustain the equilibrium. That means, if the cells do not intake the glucose, the pancreas would automatically create more insulin to make sure the cells have enough. The remaining sugar in the bloodstream would accumulate, resulting with hyperglycemia.

Maintaining a healthy diet is essential in preventing type 2 diabetes. Low activity level, poor diet, and excess body weight increases the risk of type 2 diabetes because increased fat levels slows down the ability to properly use insulin.

Treatment[edit]

Although there is no known cure for diabetes, it can be managed with certain precautions.

Type 1 & 2 Diabetes Management[edit]

Those with type 1 & 2 diabetes, who want to maintain a healthy lifestyle, exercising regularly and maintaining a healthy weight, eating healthy foods, and most importantly monitoring their blood sugar levels.

Diabetics should try to maintain their blood sugar levels between these readings: Daytime glucose levels: between 80 and 120 mg/dL (4.4 to 6.7 mmol/L) Nighttime glucose levels: between 100 and 140 mg/dL (5.6 to 7.8 mmol/L)

People with type 1 diabetes need insulin to survive, therefore, they typically inject themselves with insulin using either a fine needle and syringe, an insulin pen,or an insulin pump.

TESTING[edit]

To determine if one is diagnosed with diabetes, a few tests must be done.

- Fasting Blood Glucose (Type 1, 2) - Blood test must be higher than 126 mg/dL twice

- Random (nonfasting) Blood Glucose (Type 1) - Blood test is higher than 200 mg/dL - Must be confirmed with fasting test

- Oral Glucose Tolerance Test (Type 1, 2) - Blood level is higher than 200 mg/dL after 2 hours

- Hemoglobin A1c (Type 1, 2) - Normal: <5.7% - Pre-diabetes: between 5.7%-6.4% - Diabetes: >6.5%

- Ketone (Type 1) - Done with urine or blood sample (If sugar level is > 240 mg/dL, when ill (ex. pneumonia, stroke, etc.), when nauseated, vomiting, when pregnant)

STATISTICS[edit]

Out of 25.8 million people in the United States, 8.3% of all children and adults are diagnosed with diabetes. It is the leading cause of nontraumatic lower-limb amputations, kidney failure, and blindness in the United States and contributes greatly to heart disease and stroke.

CLINICAL RESEARCH[edit]

Currently, thousands of laboratories are focusing on the study of diabetes: type 1, type 2, its relationship with heart, kidney diseases, obesity, and many more.

One specific research was done by Karolina I. Woroniecka and company in the Albert Einstein College of Medicine of Yeshiva University. Their topic mainly focuses on the relationship between diabetes and kidney failure, known as diabetic kidney disease (DKD), which is the prominent cause of kidney failures in the United States. The research topic is called “Transcriptome Analysis of Human Diabetic Kidney,” and was published in September 2011. Its objective was to provide a collection of gene-expression changes in human diabetic kidney biopsy samples after being treated. Gene-expression is defined as the translation of information from a gene into a messenger RNA and then to a protein. Transcriptome analysis is often used to obtain insight into disease pathogenesis, molecular classification, and the identification of biomarkers, indicates the presence of some sort of phenomenon, used for future studies and treatments. This study was able to catalog gene expression regulation, identify genes and pathways that may either play a role in DKD or serve as biomarkers.

44 dissected human kidney samples were used in this experiment, portioned out according to their racial status and glomerular filtration rate (25-35 mL/min). A glomeruli is a cluster of capillaries around the end of a kidney tubule. Their method included a series of statistical equations to identify expressed transcripts found in both the control and the diseased samples. Also, algorithms helped the study by defining the regulated pathways.

The human kidneys were obtained from donors and leftover portions of kidney biopsies. The samples were manually microdissected and only the samples without any degradation were further used through the amplification of the RNA. Before any treatment, the raw samples were normalized using the RMA16 algorithm. Its purpose is to obtain a stabilized set of data and reduce any inconsistencies in their patterns. This is where the Benjamin-Hochberg testing was used at a p value < 0.05. After, the oPOSSUM software determines the overrepresented transcription factor binding sites (TFBSs) within a catalog of coexpressed genes and is then compared to a control set. The differentially expressed transcripts that comply with the statistical conditions undergo analysis that uses a ratio to determine the top canonical pathways – the Fischer exact test is used at p value < 0.05. Immunostaining is a major component in the visualization and final step of the procedure. This procedure requires the use of a specific antibody to detect a specific protein in a sample. The following primary antibodies were used: C3, CLIC5, and podocin. The Vectastain ABC Elite kit was used for the secondary antibodies to bind to the proteins and then, 3,3”diaminobenzidine was applied for visualizations. Immunostaining is typically scored on a scale of 0-4, correlating to the amount of activity on that specific protein.

Results from this experiment identified 1,700 differently expressed probesets in DKD glomeruli and 1,831 probesets in diabetic tubuli (seminiferous tubules); probeset is a collection of more than two probes and is designed to measure a single molecular species. There were 330 probesets that were commonly expressed in both compartments. Pathway analysis emphasized the regulation of many genes that factored into the signaling in DKD glomeruli. Some molecules included Cdc42, integrin, integrin-linked kinase, and others. Strong enhancements for the inflammation-related pathways were shown in the tubulointerstitial compartment. Lastly, the canonical signaling pathway was regulated in both the DKD glomeruli and tubuli, which are associated with increased glomerulosclerosis.

With ongoing research about diabetes-linked diseases, results that are obtained contribute to the overall understanding of the biochemical processes and issues. Dr. Karolina I. Woroniecka and company are one of the many research teams throughout the world that dedicate their jobs to saving or improving people’s well-being. This study is one of the many that contribute to the complications of diabetic-related kidney diseases. However there are many more studies that relate to diabetes, such as obesity and heart attack/failure.

Hiroaki Masazuki and company conducted a project relating obesity and diabetes, “Glucose Metabolism and Insulin Sensitivity in Transgenic Mice Overexpressing Leptin with Lethal Yellow Agouti Mutation.” This article was published in August 1999 from the Department of medicine and Clinical Sciences, Kyoto University Graduate School of Medicine at Kyoto, Japan. The objective of this research project was to determine the usefulness of leptin for the treatment of obesity-related diabetes. Leptin is an adipocyte-derived blood-borne satiety factor that increases glucose metabolism by decreasing food intake and increasing energy expenditure. Two different types of mice were crossed and examined at weeks 6 and 12 during the experiment. The first type was a transgenic skinny mice overexpressing leptin breed, with allele Tg/+, and the second is a lethal yellow KKAy mice, commonly used as models for obesity-diabetes syndrome, with allele Ay/+. The F1 animals’ metabolic phenotypes were examined, noting everything from body weight to their sensitivity of insulin and concentrations of leptin. This study was able to demonstrate the potential usefulness of leptin along with a long-term caloric restriction for the treatment of obesity-related diabetes. It demonstrated that hyperleptinemia can delay the onset of impaired glucose metabolism and hasten the recovery from diabetes during caloric food restriction in the crossed F1 bred mice, Tg/+ and Ay/+. Hyperleptinemia is defined as increased serum leptin level.

Although leptin may have been found to be potentially useful in treating diabetes, the fact that a caloric food restriction is required suggests that leptin can stimulate glucose metabolism independent of body weight. Other studies have demonstrated that leptin stimulates glucose metabolism in normal-weight nondiabetic mice and also improves impaired glucose metabolism in over-weight diabetic mice with leptin deficiency. Masazuki and company have created transgenic mice models overexpressing leptin (allele Tg/+) that exhibit insulin sensitivity and increased glucose tolerance. A liver-specific promoter controls the overexpression of leptin and insulin sensitivity results with the activation of signaling in the skeletal muscle and liver. In this study, Masazuki and company genetically crossed the transgenic mice and lethal yellow obese mice. The resulting 4 genotypes are: Tg/+: Ay/+, Tg/+, Ay/+, and wild-type +/+. When at week 6, all the mice were at normal body weight and at week 12, the mice with the Ay/+ allele clearly developed obesity. At 9 weeks, +/+, Ay/+, and Tg/+: Ay/+ were placed on a 3 week food restriction diet and analyzed at week 12.

The research design and methods include: measurements of body weight and cumulative food intake, plasma leptin, glucose, and insulin concentrations, glucose and insulin tolerance tests, and caloric food restriction experiments and later statistical analysis were done. Body weights were measured daily since the mice were 4 weeks old and food intake was measured daily over a 2-week period. Blood was sampled from retro-orbital sinus of mice at 9:00AM. Plasma leptin concentrations were determined using radioimmunoassay (RIA) for mouse leptin. Insulin and plasma glucose concentrations were determined by the glucose oxidase method with a reflectance glucometer. The glucose tolerance tests (GTT) were done after an 8 hour fast and injections of 1.0 mg/g glucose. The insulin tolerance tests (ITT) were done after a 2 hour fast and injection of 0.5 mU/g insulin. The blood was then drawn from the mice tail veins at periodic times after injection at 15, 30, 60, and 90 minutes and blood was drawn from before the injections to measure comparable results. The food restriction experiment was based off of the cumulative food intake at week 12. The mice were then provided with 60% of the amount of food consumed. The exact same tests were measured: plasma leptin, glucose, and insulin concentrations were also determined; GTT and ITT were also done. At the end, all these data were analyzed and expressed at ±SE.

The results identified a large difference in body weights with the four genotypes. At week 4, all the mice showed no significant difference in body weight. At week 6 of age, Tg/+ mice gained approximately 20-30% less weight than the control +/+ mice and indicated a sign of developing adiposity compared to +/+, Tg/+: Ay/+, and Ay/+ mice. At this time, the control, Tg/+: Ay/+, and Ay/+ mice showed no drastic difference in body weights. However, by week 12 of age, the mice with the Ay/+ allele developed obese. As for plasma leptin concentrations, 6 week old Tg/+ mice were approximately 12 times those of the control +/+ mice, at week 12, they were 9 times higher. The concentrations in Ay/+ and +/+ mice were roughly equivalent. The concentrations of Tg/+:Ay/+ mice were 8 times higher than those of the +/+ mice and at week 12, they were higher than the Ay/+. At week 12, the body weight of Tg/+ was ~23% less than the control’s. The food intake of Tg/+ reduced significantly after 6 and 12 weeks of age compared to the control litter. The food intake of Ay/+ mice increased by 50% compared to the control litter. The food intake of Tg/+: Ay/+ mice, compared to the +/+ mice, were roughly the same. The food intake of Ay/+ and Tg/+: Ay/+ were approximately the same. At week 6, the plasma glucose concentrations among all 4 genotypes were the approximately the same. At week 12, the glucose levels of Tg/+ and +/+ mice were the same. However, the glucose level of Ay/+ and Tg/+: Ay/+ elevated significantly compared to the control but compared to each other, they were the same. As for plasma insulin concentration levels, the Tg/+ mice greatly decreased compared to the control at week 6. The plasma insulin concentrations in Tg/+: Ay/+ mice were higher than the control. At this point, the Ay/+ mice demonstrated marked hyperinsulinemia compared to the rest of the genotypes. GTTs and ITTs showed that the plasma glucose elevation is significant in Tg/+ compared to the control. 30 minutes after the injection, the glucose concentrations increased greatly in Ay/+ mice compared to the control.

The genotypes’ glucose metabolisms were examined after their food restriction. 60% of their total food intake were given to these mice and after 2 weeks, the body weights of Tg/+ were 17% less and +/+ were 12% less compared to before and the Ay/+, Tg/+: Ay/+ body weights also decreased. The plasma leptin concentrations in Tg/+ mice were higher than those in +/+ mice and Tg/+:Ay/+ were higher than those of Ay/+. The leptin concentrations between Tg/+ compared to Tg/+:Ay/+ and those of +/+ and Ay/+ were approximately the same. After 3 weeks of food restriction, the plasma glucose concentrations among +/+, Ay/+, and Tg/+:Ay/+ were similar. The plasma insulin concentrations, however, in Ay/+ mice were higher than those of +/+ and Tg/+:Ay/+ mice.

The results indicated that glucose tolerance and insulin sensitivity are increased in Tg/+:Ay/+ mice and plasma leptin concentrations in Tg/+:Ay/+ are higher than regular Ay/+ mice. These indicate that overproduction of leptin can prolong the start of impaired glucose metabolism in Tg/+: Ay/+ mice and endogenous leptin cannot in Ay/+ mice. Leptin can apply its anti-diabetic effect in normal weight animals at week 6. At week 12, Tg/+:Ay/+ mice developed resistance to the anti-diabetic action of leptin. In this study, glucose metabolism is somewhat improved in Ay/+ after a long term body weight reduction due to the 3 week food restriction while the metabolism is improved in Tg/+:Ay/+ compared to Ay/+ and control which suggests that hyperleptinemia enhances glucose level when body weight is stable. Persistent hyperleptinemia delays the beginning of impaired glucose metabolism and quickens the recovery from diabetes in Ay/+ mice in combination with food restriction.

Emilie Vander Haar and her team in the University of Minnesota Minneapolis studied “Insulin signaling to mTOR mediated by the Akt/PKB substrate PRAS40.” In this study, they were able to identify PRAS40 as a crucial regulator of insulin sensitivity of the Akt-mTOR metabolic pathway which can potentially help target the treatment of cancers, insulin resistance, and hamartona syndromes. Insulin activates the protein kinases Akt, also known as PKB, and mammalian target of rapamycin (mTOR) which stimulates protein synthesis and cell growth. This study was able to identify PRAS40 as a unique mTOR binding partner and is induced under conditions that inhibit mTOR signaling. Akt phosphorylates PRAS40, which is crucial for insulin to stimulate mTOR. These findings contribute to the clinical studies of type 2 diabetes insulin related pathways.

mTOR is a kinase-related protein that is a key mediator of insulin. Inhibition of mTOR in mammals proves to reduce insulin resistance and extend lifespan. mTORC1 is a nutrient and insulin regulated complex that is formed from mTOR when it interacts with raptor and a G-protein. This complex is involved in the cytoskeleton regulation and Akt phosphorylation; however the interactions and associated proteins in response to insulin have not been identified. In order to do so, Haar and her team used a mass spectroscopy method. An mTOR antibody prepared mTOR immunoprecipitates from T-cells and the proteins that were bound to the regulator were eluted from the precipitates. The mixtures of proteins were trypsinized and the mass spectra were obtained. The highest P scores obtained from the derived peptides illustrated that mass spectroscopy isolated mTOR-binding proteins. Three sequences were obtained and contributed to the finding that Sin1 is crucial in the formation of the mTOR interaction. The PRAS40 peptide sequence was also identified. However, in order to confirm the hypothesis that mTOR binds with PRAS40, T-cells’ precipitates, which carries PRAS40, were analyzed using western blotting. Compared to the control, PRAS40 was found to bind only with mTOR and nothing else. It was shown that PRAS40 binds specifically in the mTOR carboxy-terminal kinase domain. Certain conditions inhibit mTOR signaling increases affinity, binding abilities, of the PRAS40 mTOR interaction. These conditions include depriving leucine or glucose from the media solution, treatment with the glycolytic inhibitor and mitochondrial metabolic inhibitors. The increase in affinity leads to disrupting the raptor-mTOR interaction, which results with destabilizing the PRAS40-mTOR interactions. This tightened bond between the proteins under nutrient deprivation conditions proposes a hypothesis that states PRAS40 has a negative role in regulating mTOR.

In order to further understand the consequence of PRAS40 in mTOR signaling, the regulator was downregulated in 3T3-L1 and HepG2 cells. The phosphorylation process of Akt at Ser 473 and S6K1 (a mTOR substrate) at Thr 389 was studied. PRAS40 silencing led to a significant decrease in Akt phosphorylation in the cell lines which resulted with negative effects on the Akt components. PRAS40 silencing also led to increased levels of S6K1 phosphorylation, and suggests that the PRAS40-knockout mTOR complex is still active in S6k1 phosphorylation. The mechanism was studied and results indicated that PRAS40 silenced cells and resulting activated state of mTOR may contribute to Akt inactivation – a feedback inhibition. That was the first part of PRAS40 analysis, PRAS40 silencing. In the second part, PRAS40 was overexpressed. Increasing the levels of PRAS40 in cells resulted with decreased S6K1 phosphorylation. These results prove that PRAS40 inhibition of mTOR regulation is likely to require mTOR and raptor binding.

After determining the inhibitory function of PRAS40 in mTOR signaling, Haar and her team studied the role PRAS40 plays in the regulation of mTOR. PRAS40 knockdown in mice and human cells weakened the ability of insulin to stimulate phosphorylation. PRAS40 silencing reduces the levels of phosphorylation in both cell types. In order to further study the response of mTOR to insulin, sample cells were treated with insulin. The data collected proposes that PRAS40 silencing detaches mTOR from Akt signals – PRAS40 plays a crucial role in regulating Akt signaling to mTOR. It also demonstrates that Akt phosphorylation of PRAS40 is crucial for mTOR activation through the use of insulin.

The next matter that Haar and her team touched upon was the study that nutrient starvation has dominant effects on PRAS40-mTOR interaction. PRAS40 was hardly released from mTOR when the conditions are deprived of leucine. Also, the amount of 14-3-3, an interaction induced on PRAS40 phosphorylation, bound to mTOR and PRAS40 was significantly reduced under deprived leucine conditions – the interaction was prevented under non-nutrient conditions. 14-3-3 interactions with mTOR and PRAS40 were also prevented under leucine-deprived conditions. In all, these results prove that PRAS40 is a key mediator of Akt signals to mTOR and a negative effector of mTOR signaling. PRAS40 is a crucial regulator of in insulin sensitivity of mTOR signaling, an important role in insulin resistance.

Some methods of this experiment included the use of antibodies in western blotting, plasmid constructions and mutagenesis, the identification of mTOR-interacting proteins, cell culture and transfection, coimmunoprecipitation, chemical crosslinking, and lentiviral preparation, viral infection, and stable cell line generation. Human PRAS40 cDNAs were provided and mouse PRAS40 cDNA samples underwent PCR amplification and then subcloned into mammalian expression vector. All these cloned samples were confirmed by sequencing. PRAS40 Thr 246 was replaced by amino acids: alanine, glutamate, and aspartate. This is done through a site-directed mutagenesis kit. The way that mTOR immunoprecipitates is through the use of an mTOR antibody on cells cultured in 10% fetal bovine. The cell samples were lysed in a buffer and then incubated with 20ul of protein G resin and 4ug of mTOR antibody. The mTOR precipitates were washed with lysis buffer and the binding proteins were eluted by incubation. The mTOR binding proteins were diluted with digestion buffer and then incubated overnight with trypsin. These samples underwent analysis by mass spectrometry. Data would only be considered accurate when the P score is greater or equal to 0.95. For chemical crosslinking experiments, T cells were treated with dithiobis and then harvested and lysed in a buffer. The precipitates were then analyzed using the SDS-PAGE method. In order to measure the cell-size, T cells were infected with lentiviruses, and then selected in the presence of zeocin. The cell samples were trysinized the following day and diluted 10 times. The ViCell cell-size analyzer analyzed the size of 1.0 mL of diluted cell culture sample.

Overall, the study of PRAS40 in regulating mTOR insulin signaling can potentially lead to potential targets for the treatment of different diseases relating to type 2 diabetes, cancers, and insulin resistance. The results indicate that the Akt/PKB substrate, PRAS40, provide negative effects on the signaling of mTOR. The binding suppresses mTOR activation and insulin-receptor substrate-1 (IRS-1 and Akt, therefore, uncoupling the response of mTOR to Akt signals. PRAS40’s interaction with mTOR is induced under certain environmental conditions such as nutrient, leucine and serum deprivation. In general, this project was able to identify that PRAS40 is a crucial mTOR binding partner that intervenes Akt signaling to mTOR.

Endoplasmic Reticulum Stress Stimuli and Beta-Cell Death In Type 2 Diabetes[edit]

Obesity is related with insulin resistance, however Type 2 Diabetes, a complex known for increased levels of blood glucose due to insulin resistance in the muscle and liver tissue as well as impaired insulin secretion from pancreatic beta-cells, solely cultivates in genetically predisposed and insulin resistant subjects with the beginning of beta-cell dysfunction. As research progresses there is clear data that demonstrates that beta-cell failure and death are due to unresolvable endoplasmic reticulum stress, bringing chronic and strong activation of inositol-requiring protein 1. Endoplasmic reticulum stress can start and generate the characteristics of Beta-cell failure and death observed in Type 2 Diabetes.

Glucose Transport Deficiency in Type 2 Diabetes[edit]

GLUT4 glucose transporters migrate to the cell surface in response to insulin signaling, thereby upgrading glucose levels in muscle and fat cells. This is accomplished by stimulating vesicle transport of glucose to where it is needed. Adult onset diabetes is often the result of gradual increase in insulin tolerance in individuals who overeat. This desensitization to the effects of insulin interferes with metabolism because vesicles containing GLUT4 are not able to efficiently fuse with the cell membrane therefore glucose uptake into cells is inhibited. By understanding this pathway, researchers may eventually find a therapeutic workaround to treat those suffering from Type 2 diabetes. Presumably, this could be accomplished by synthesizing molecules that mimic the function of GLUT4 and its auxiliaries to resolve the trafficking problem. Alternatively, the insulin pathway could be targeted.

RESOURCES[edit]

1. http://www.diabetes.org/diabetes-basics/diabetes-statistics/

2. http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001350/

3. http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001356/

4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2682681/

5. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161334/

6. http://diabetes.niddk.nih.gov/dm/pubs/statistics/

7. http://diabetes.diabetesjournals.org/content/60/9/2354.full

8. http://www.mayoclinic.com/health/type-1-diabetes/DS00329/DSECTION=treatments%2Dand%2Ddrugs

9. http://www.mayoclinic.com/health/type-2-diabetes/DS00585/DSECTION=treatments%2Dand%2Ddrugs

10. http://diabetes.diabetesjournals.org/content/48/9/1822.short

11. http://www.ncbi.nlm.nih.gov/pubmed/22482906

12. http://www.nature.com/ncb/journal/v9/n3/pdf/ncb1547.pdf

References[edit]

1.Endoplasmic reticulum stress and type 2 diabetes. Back SH, Kaufman RJ. Annu Rev Biochem. 2012;81:767-93. Epub 2012 Mar 23. Review. PMID: 22443930 [PubMed - indexed for MEDLINE]