Calpastatin is regulated by protein never in mitosis gene A interacting-1 (PIN1) in endothelial cells

Liao

et al. 2018

Front. Cell. Neurosci.

The purpose of the current study was to investigate whether peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) can interact with calpastatin (CAST) and regulate CAST/calpain2, under excessive glutamate conditions, and subsequently regulate necrosis in rat retinal neurons. Glutamate triggered CAST/calpain2-mediated necrosis regulation in primary cultured retinal neurons, as demonstrated by propidium iodide-staining and lactate dehydrogenase assay. Co-IP results and a computer simulation suggested that Pin1 could bind to CAST. Western blot, real-time quantitative polymerase chain reaction, immunofluorescence, and phosphorylation analysis results demonstrated that CAST was regulated by Pin1, as proven by the application of juglone (i.e., a Pin1 specific inhibitor). The retinal ganglion cell 5 cell line, combined with siRNA approach and flow cytometry, was then used to verify the regulatory pathway of Pin1 in CAST/calpain2-modulated neuronal necrosis that was induced by glutamate. Finally, in vivo studies further confirmed the role of Pin1 in CAST/calpain2-modulated necrosis following glutamate excitation, in the rat retinal ganglion cell and inner nuclear layers. In addition, a flash electroretinogram study provided evidence for the recovery of impaired visual function, which was induced by glutamate, with juglone treatment. Our work aims to investigate the involvement of the Pin1-CAST/calpain2 pathway in glutamate-mediated excitotoxicity.

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Pin1 Promotes Regulated Necrosis Induced by Glutamate in Rat Retinal Neurons via CAST/Calpain2 Pathway

Liao

et al. 2018

Front. Cell. Neurosci.

“…3 Although Ca 2ϩ is the main regulator of calpain activity, protease activation does not simply occur in response to any stimuli that increases platelet Ca 2ϩ . Indeed, the regulation of proteolytic activity is complex and involves the association of calpain with a regulatory subunit, the endogenous inhibitor calpastatin, 4 other interacting proteins, 5 and binding to phospholipids. 6,7 Genetic deletion of m-calpain results in embryonic lethality, 8,9 but -calpain Ϫ/Ϫ mice are viable and demonstrate attenuated aggregation and clot retraction but normal bleeding times.…”

Section: Introductionmentioning

confidence: 99%

Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes

Randriamboavonjy

Isaak

Elgheznawy

et al. 2012

Blood

Platelets from patients with diabetes are hyperreactive and demonstrate increased adhesiveness, aggregation, degranulation, and thrombus formation, processes that contribute to the accelerated development of vascular disease. Part of the problem seems to be dysregulated platelet Ca 2؉ signaling and the activation of calpains, which are Ca 2؉ -activated proteases that result in the limited proteolysis of substrate proteins and subsequent alterations in signaling. In the present study, we report that the activation of -and m-calpain in patients with type 2 diabetes has profound effects on the platelet proteome and have identified septin-5 and the integrin-linked kinase (ILK) as novel calpain substrates. The calpain-dependent cleavage of septin-5 disturbed its association with syntaxin-4 and promoted the secretion of ␣-granule contents, including TGF-␤ and CCL5. Calpain was also released by platelets and cleaved CCL5 to generate a variant with enhanced activity. Calpain activation also disrupted the ILK-PINCH-Parvin complex and altered platelet adhesion and spreading. In diabetic mice, calpain inhibition reversed the effects of diabetes on platelet protein cleavage, decreased circulating CCL5 levels, reduced plateletleukocyte aggregate formation, and improved platelet function. The results of the present study indicate that diabetesinduced platelet dysfunction is mediated largely by calpain activation and suggest that calpain inhibition may be an effective way of preserving platelet function and eventually decelerating atherothrombosis development. (Blood. 2012;120(2): 415-423) IntroductionCalpains are Ca 2ϩ -regulated cysteine proteases that are responsible for the limited proteolysis of target proteins, resulting in their modification (eg, activation, inhibition, or altered sensitivity to intracellular signals) rather than in their degradation. 1 In contrast to promiscuous degradative proteases, calpains cleave a relatively restricted set of protein substrates and use complex substrate recognition mechanisms involving primary and secondary structural features of target proteins to alter protein function and cellular signaling. 2 Platelets express -calpain (calpain 1) and m-calpain (calpain 2); named for the micro-and millimolar Ca 2ϩ concentrations, respectively, that are required to activate them in vitro. 3 Although Ca 2ϩ is the main regulator of calpain activity, protease activation does not simply occur in response to any stimuli that increases platelet Ca 2ϩ . Indeed, the regulation of proteolytic activity is complex and involves the association of calpain with a regulatory subunit, the endogenous inhibitor calpastatin, 4 other interacting proteins, 5 and binding to phospholipids. 6,7 Genetic deletion of m-calpain results in embryonic lethality, 8,9 but -calpain Ϫ/Ϫ mice are viable and demonstrate attenuated aggregation and clot retraction but normal bleeding times. 10 Mechanistically, the latter effects were attributed to the tyrosine dephosphorylation of platelet proteins because -calpain Ϫ/Ϫ platelets exh...

“…Scale bar: 20 µm in all panels. (Salamino et al, 1997;Liu et al, 2011). Next, using phos-tag SDS-PAGE, we assessed whether the phosphorylation of CAST changed after glutamate treatment.…”

Section: Effect Of Glutamate On Pin1-cast/calpain2 Pathwaymentioning

confidence: 99%

Pin1 Promotes Regulated Necrosis Induced by Glutamate in Rat Retinal Neurons via CAST/calpain2 Pathway

Wang¹

2018

Preprint

The purpose of the current study was to investigate whether peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) can interact with calpastatin (CAST) and regulate CAST/calpain2, under excessive glutamate conditions, and subsequently regulate necrosis in rat retinal neurons. Glutamate triggered CAST/calpain2-mediated necrosis regulation in primary cultured retinal neurons, as demonstrated by propidium iodidestaining and lactate dehydrogenase assay. Co-IP results and a computer simulation suggested that Pin1 could bind to CAST. Western blot, real-time quantitative polymerase chain reaction, immunofluorescence, and phosphorylation analysis results demonstrated that CAST was regulated by Pin1, as proven by the application of juglone (i.e., a Pin1 specific inhibitor). The retinal ganglion cell 5 cell line, combined with siRNA approach and flow cytometry, was then used to verify the regulatory pathway of Pin1 in CAST/calpain2-modulated neuronal necrosis that was induced by glutamate. Finally, in vivo studies further confirmed the role of Pin1 in CAST/calpain2-modulated necrosis following glutamate excitation, in the rat retinal ganglion cell and inner nuclear layers. In addition, a flash electroretinogram study provided evidence for the recovery of impaired visual function, which was induced by glutamate, with juglone treatment. Our work aims to investigate the involvement of the Pin1-CAST/calpain2 pathway in glutamate-mediated excitotoxicity.