Interference of amino‐terminal desmin fragments with desmin filament formation

Abstract

Short polypeptides from intermediate filament (IF) proteins containing one of the two IF-consensus motifs interfere severely with filament assembly in vitro. We now have systematically investigated a series of larger fragments of the muscle-specific IF protein desmin representing entire functional domains such as coil1 or coil 2. "Half molecules" comprising the amino-terminal portion of desmin, such as DesDeltaC240 and the "tagged" derivative Des(ESA)DeltaC244, assembled into large, roundish aggregates already at low ionic strength, DesDeltaC250 formed extended, relatively uniform filaments, whereas DesDeltaC265 and DesDeltaC300 were soluble under these conditions. Surprisingly, all mutant desmin fragments assembled very rapidly into long thick filaments or spacious aggregates when the ionic strength was raised to standard assembly conditions. In contrast, when these desmin mutants were assembled in the presence of wild-type (WT) desmin, their assembly properties were completely changed: The elongation of the two shorter desmin fragments was completely inhibited by WT desmin, whereas DesDeltaC250, DesDeltaC265 and DesDeltaC300 coassembled with desmin into filaments, but these mixed filaments were distinctly disturbed and exhibited a very different phenotype for each mutant. After transfection into fibroblasts and cardiomyocytes, the truncated mutant Des (ESA)DeltaC244 localized largely to the cytoplasm, as revealed by a tag-specific monoclonal antibody, and also partially colocalized there with the collapsed endogenous vimentin and desmin systems indicating its interference with IF-organizing processes. In contrast, in cells without an authentic cytoplasmic IF system such as line SW13, Des(ESA)DeltaC242 entered the nucleus and was deposited in small dot-like structures in chromatin-free spaces without any noticeable effect on nuclear morphology.

Caspase Cleavage of GFAP Produces an Assembly-Compromised Proteolytic Fragment that Promotes Filament Aggregation

“…This study parallels recent studies on a similarly truncated desmin, which contains the first 264 amino acids of desmin (N-desmin). Both N-GFAP and N-desmin have a strong tendency to aggregate in vitro (Bar et al., 2009 ) and in transfected cells (Chen et al., 2003 ), and both appear to function as potent disruptors of IF assembly. These findings are consistent with the observations that a similar N-terminal vimentin fragment comprising the head and conserved 1A domains interferes with normal filament assembly in a dominant-negative fashion (Kural et al., 2007 ; Chang et al., 2009 ).…”

Desminopathies: pathology and mechanisms

“…Desmin also is a specific target of the proteolytic calpain (Ca 2+ -activated cysteine proteinase) system [60]. The limited proteolysis of desmin by calpains results in desmin “head,” “rod,” and “tail” domain cleavage products, which are no longer capable to participate in desmin IF formation, but instead heavily interfere with the proper assembly process [15, 17, 122]. The calpain system has also been attributed to exert a role in the spatiotemporal regulation of desmin protein levels during myotube formation [44].…”

Drawbacks of Dialysis Procedures for Removal of EDTA

“…Later, the denatured proteins are refolded by step-wise dialysis to gradually remove urea, usually in low salt buffer to keep the protein unassembled, as high ionic strength triggers polymerization [8]. In fact, intermediate filament proteins such as desmin, vimentin or glial fibrillary acidic protein (GFAP) purified through these methods, have been widely studied to assess polymerization or association changes induced by increasing ionic strength or by divalent cations, usually at millimolar concentrations [9, 10]. …”