Proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins.

Abstract

The degradation of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins proceeds in two stages in the form of a limited proteolysis. At first, the reaction is very rapid, with the stepwise and complete removal of a peptide (ca. 9,000 daltons) from the N-terminal of vimentin and desmin. This results in the production of a characteristic "staircase" of degradation products, as seen in two-dimensional polyacrylamide gel electrophoresis. The second stage of proteolysis is characterized by the accumulation of peptides which are resistant to further proteolysis; this is due not to product inhibition but to the fact that these peptides are not substrates for the proteinase and therefore do not protect the latter from inactivation (autodigestion). In vitro phosphorylation of the substrates does not affect proteinase activity, probably because the phosphorylation site is located towards the C-terminal of the molecules. The specific and limited proteolysis of vimentin and desmin results in the deletion of the nucleic acid binding and filament assembly site of these proteins, indicating that the Ca2'-activated proteinase plays a role in regulating the function(s) of these intermediate filament proteins, rather than their simple turnover during the cell cycle.Intermediate (10-nm) filaments are a heterogeneous class of protein fibrils which can be subdivided into five groups based on their subunit composition and cellular origin (1,2,7,17 helix (particularly at the amino terminal) interspersed with regions of non-a-helix (11,32,33). Most recently, it has been shown that there is ca. 70% amino acid sequence homology at the carboxy terminal between vimentin and desmin (11-13). In addition, both proteins bind to nucleic acids (38).Vimentin and desmin are also very susceptible to proteolysis in situ (17). This is due to the activity of a highly specific, Ca2'-activated, neutral proteinase for which we have recently described the purification and characterization (18,19). The proteinase appears to be coconserved during evolution with the substrate vimentin (20) and is present in reduced amounts in cells in which little or no vimentin is synthesized (36). Together with the fact that the Ca2+-activated proteinase does not significantly degrade other proteins (18,19), these results indicate that the proteinase plays an important role in the regulation of the function of vimentin and desmin. To investigate this, we have analyzed in detail the effect of the proteinase on vimentin and desmin. We show that there is only a limited proteolysis of a 9-kilodalton (kd) fragment from the N-terminal, resulting in the accumulation of large peptides which are resistant to further proteolysis. This accumulation is due not to product inhibition of proteolytic activity but to the fact that these peptides are poor substrates,

The Calpain System

“…IIB) is to ensure that proteolytically active calpain is not present in the circulatory system. On the other hand, Vimentin Rapidly degraded to two 40-and 42-kDa fragments with stable 27-, 22-, 20-, 16-, and 15-kDa fragments being produced subsequently; pattern and effects of calpain digestion are similar to those observed for calpain degradation of desmin (318). Murine vimentin is cleaved at 10 sites that are all in the nonhelical region of the molecule; 9 of these sites are in the NH 2 -terminal part of the molecule to produce the stable 42-kDa fragment (116).…”

“…Rapidly degraded to a 32-to 37-kDa stable fragment; an 18-kDa fragment appears after longer digestion (336); a 9-kDa fragment is removed from the NH 2 terminus leaving the central rod domain; calpain degradation destroys the ability of desmin to self-assemble and to bind nucleic acids (318). Dystrophin…”

Developmentally regulated cytokeratin gene in Xenopus laevis.

“…The presence of structural similarity between the generally variable C-terminal regions of two distinct proteins suggests that this segment may serve a similar function in these molecules. Since the terminal domains of IF polypeptides may be involved in the end-to-end linkage of subunits (13,36,49), sequence relationships within these domains may influence subunit specificity of filament assembly.…”

Molecular mechanisms involved in development of cerebral vasospasm

“…3,9,16,23,25,50 We suggest, therefore, that the uncoupling of the contractile units from the cytoskeleton is more severe in vasospasm than in KCland serotonin-induced vasocontraction, probably due to progressive proteolytic mechanism with -calpain after SAH, making relaxation difficult in response to vasodilator agents.…”