Calpain-2 participates in the process of calpain-1 inactivation

Abstract

Calpain-1 and calpain-2 are highly structurally similar isoforms of calpain. The calpains, a family of intracellular cysteine proteases, cleave their substrates at specific sites, thus modifying their properties such as function or activity. These isoforms have long been considered to function in a redundant or complementary manner, as they are both ubiquitously expressed and activated in a Ca2+-dependent manner. However, studies using isoform-specific knockout and knockdown strategies revealed that each calpain species carries out specific functions in vivo. To understand the mechanisms that differentiate calpain-1 and calpain-2, we focused on the efficiency and longevity of each calpain species after activation. Using an in vitro proteolysis assay of troponin T in combination with mass spectrometry, we revealed distinctive aspects of each isoform. Proteolysis mediated by calpain-1 was more sustained, lasting as long as several hours, whereas proteolysis mediated by calpain-2 was quickly blunted. Calpain-1 and calpain-2 also differed from each other in their patterns of autolysis. Calpain-2–specific autolysis sites in its PC1 domain are not cleaved by calpain-1, but calpain-2 cuts calpain-1 at the corresponding position. Moreover, at least in vitro, calpain-1 and calpain-2 do not perform substrate proteolysis in a synergistic manner. On the contrary, calpain-1 activity is suppressed in the presence of calpain-2, possibly because it is cleaved by the latter protein. These results suggest that calpain-2 functions as a down-regulator of calpain-1, a mechanism that may be applicable to other calpain species as well.

Visualizing Cell Death in Live Retina: Using Calpain Activity Detection as a Biomarker for Retinal Degeneration

“…Furthermore, the calpain activity assay was performed on live retinal explants over a duration of 6 h, whereas calpain-2 staining captures a snapshot of those cells in which calpain-2 was activated precisely at the time-point of fixation. Another reason might be that calpain-2 is inactivated very quickly via autolysis [ 47 , 48 ], which might lead to only small amounts of CMAC being cleaved by calpain-2. Further co-staining experiments, including with other calpain isoforms and other signaling molecules involved in cGMP-dependent cell death, could help to unravel the time course of photoreceptor degeneration more precisely.…”

Programmed Non-Apoptotic Cell Death in Hereditary Retinal Degeneration: Crosstalk between cGMP-Dependent Pathways and PARthanatos?

“…The activity of CNGCs depolarizes photoreceptors to the extent that voltage gated Ca 2+ channels (VGCC) in the soma and synapse open [ 97 ]. This results in further Ca 2+ influx that must be extruded by PMCA at the expense of additional ATP and which, otherwise, could lead to the activation of calpain-type proteases, which may promote cell destruction [ 98 , 99 , 100 ]. Accordingly, the idea that high Ca 2+ may be responsible for cell death [ 48 ], and by extension for photoreceptor degeneration [ 101 ], has motivated several studies attempting to block either CNGC or VGCC for therapeutic purposes.…”

Visualizing cell death in live retina: Using calpain activity detection as a biomarker for retinal degeneration

“…Several recent studies have shown that individual calpain species may serve distinct roles in vivo (Baudry 2019;Franco, Perrin, and Huttenlocher 2004;Santos et al 2012). Furthermore, calpain isoforms show differences regarding the time window of proteolysis, the pattern of autolysis and the concentration of Ca 2+ required for activation (Shinkai-Ouchi et al 2020;Yoshimura et al 1983). Notably, calpain-2 requires 2-3 magnitudes more Ca 2+ compared to calpain-1 to be activated.…”