The caspase family of proteases plays a critical role in the execution of apoptosis. However, efforts to decipher the molecular mechanisms by which caspases induce cell death have been greatly hindered by the lack of systematic and broadly applicable strategies to identify their substrates. Here we describe a novel expression cloning strategy to rapidly isolate cDNAs encoding caspase substrates that are cleaved during apoptosis. Small cDNA pools (approximately 100 clones each) are transcribed/translated in vitro in the presence of [ 35 S]methionine; these labeled protein pools are then incubated with cytosolic extracts from control and apoptotic cells. cDNA pools encoding proteins that are specifically cleaved by the apoptotic extract and whose cleavage is prevented by the caspase inhibitor acetylTyr-Val-Ala-Asp chloromethylketone are subdivided and retested until a single cDNA is isolated. Using this approach, we isolated a partial cDNA encoding protein kinase C-related kinase 2 (PRK2), a serine-threonine kinase, and demonstrate that full-length human PRK2 is proteolyzed by caspase-3 at Asp 117 and Asp 700 in vitro. In addition, PRK2 is cleaved rapidly during Fas-and staurosporine-induced apoptosis in vivo by caspase-3 or a closely related caspase. Both of the major apoptotic cleavage sites of PRK2 in vivo lie within its regulatory domain, suggesting that its activity may be deregulated by proteolysis.Caspases are a novel family of cysteine proteases with aspartate specificity that are related to the Caenorhabditis elegans cell death gene product CED-3. Evidence from many laboratories indicates that caspases play a critical role in the execution of apoptosis. Ectopic expression of these proteases induces programmed cell death. Caspases are normally present in cells as catalytically inactive proenzymes and are proteolytically processed and activated during the induction of apoptosis. Moreover, viral, peptide, and dominant negative inhibitors of caspases delay or prevent programmed cell death (reviewed in Ref. 1). Finally, homozygous inactivation of caspase-1 (2) and caspase-3 (3) in mice results in selective defects in apoptosis.Because caspase activation is a crucial event in apoptosis, it is essential to identify the downstream molecular targets of these proteases whose selective proteolysis is likely to underlie the characteristic morphological features of apoptotic cell death. Although a number of structural and signaling proteins have been shown to be cleaved by caspases during programmed cell death (reviewed in Ref. 4), our understanding of the molecular mechanisms by which caspases induce cell death has been greatly hindered by the lack of systematic and broadly applicable strategies to identify these substrates. Given the absence of such methods and the growing number of caspase family members, it seems likely that the majority of apoptotic caspase targets have yet to be identified.In this report, we describe a method to identify caspase substrates directly and rapidly using labeled protein pools derived...
Microalbuminuria (MAU) is a common subclinical disease and related with cardiovascular outcome both in diabetic and non-diabetic patients. However, there is rare data about the effect of MAU on the development of diabetes. Thus, we aimed to investigate whether MAU is associated with the development of incident diabetes. A total of 3385 subjects without diabetes (1503 men and 1882 women; mean age, 53 years) who participated in the Ansung–Ansan cohort study from 2001–2002 (baseline) to 2011–2012 (fifth follow-up visit) were followed for a mean of 8 years. The prevalence of MAU at baseline was 10.8% (365 patients), and the incidence of newly developed diabetes during the follow-up period was 15.3% (56 patients) in subjects with MAU. The hazard ratio (HR) for development of diabetes was 1.43 (95% confidence interval (CI) 1.07–1.91, p-value 0.016), independent of traditional risk factors for diabetes including pre-diabetes, age, obesity, and family history. The impact of MAU on diabetes was also significant in the non-pre-diabetic population (HR 2.08, 95% CI 1.07–4.03, p-value 0.031). In conclusion, our results show that incident MAU is associated with future development of diabetes and could be an early marker for diabetes, even in the non-prediabetic population.
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