Proteolytic enzymes

About: Proteolytic enzymes is a research topic. Over the lifetime, 23096 publications have been published within this topic receiving 835544 citations.

Conjugation of a magainin analogue with lipophilic acids controls hydrophobicity, solution assembly, and cell selectivity.

Abstract: Our basic understanding of how to combat fungal infections has not kept pace with the recent sharp rise in life-threatening cases found particularly among immuno-compromised individuals Current investigations for new potential antifungal agents have focused on antimicrobial peptides, which are used as a cell-free defense mechanism in all organisms Unfortunately, despite their high antibacterial activity, most of them are not active toward fungi, the reason of which is not clear Here, we present a new approach to modify an antibacterial peptide, a magainin analogue, to display antifungal activity by its conjugation with lipophilic acids This approach has the advantage of producing well-defined changes in hydrophobicity, secondary structure, and self-association These modifications were characterized in solution at physiological concentrations using CD spectroscopy, tryptophan fluorescence, and analytical ultracentrifugation In order of increasing hydrophobicity, the attachment to the magainin-2 analogue of (i) heptanoic acid results in a monomeric, unordered structure, (ii) undecanoic acid yields concentration-dependent oligomers of alpha helices, and (iii) palmitic acid yields concentration-independent alpha-helical monomers, a novel lipopeptide structure, which is resistant to proteolytic digestion Membrane-lipopeptide interactions and the membrane-bound structures were studied using fluorescence and ATR-FTIR in PC/PE/PI/ergosterol (5/25/25/1, w/w) SUV, which constitute the major components of Candida albicans bilayers A direct correlation was found between oligomerization of the lipopeptides in solution and potent antifungal activity These results provide insight to a new approach of modulating hydrophobicity and self-assembly of antimicrobial peptides in solution, without altering the sequence of the peptidic chain These studies also provide a general means of developing a new group of lipopeptide candidates as therapeutic agents against fungal infections

A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, calpain.

Abstract: In autoimmune demyelinating diseases such as multiple sclerosis (MS), the degradation of myelin proteins results in destabilization of the myelin sheath. Thus, proteases have been implicated in myelin protein degradation, and recent studies have demonstrated increased expression and activity of a calcium-activated neutral proteinase (calpain) in experimental allergic encephalomyelitis, the corresponding animal model of MS. In the present study, calpain activity and expression (at translational and transcriptional levels) were evaluated in white matter from human patients with MS and Parkinson’s and Alzheimer’s diseases and compared with that of white matter from normal controls. Western blot analysis revealed that levels of the active form of calpain and calpain-specific degradation products (fodrin) were increased by 90.1% and 52.7%, respectively, in MS plaques compared with normal white matter. Calpain translational expression was up-regulated by 462.5% in MS plaques compared with controls, although levels of the specific endogenous inhibitor, calpastatin, were not altered significantly. At the transcriptional level, no significant changes in calpain or calpastatin expression were detected by reverse transcription–PCR. Using double immunofluorescent labeling, increased calpain expression was observed in reactive astrocytes, activated T cells, and activated mononuclear phagocytes in and adjacent to demyelinating lesions. Calpain activity and translational expression were not increased significantly in white matter from patients with Parkinson’s or Alzheimer’s diseases compared with that of normal controls. Because calpain degrades all major myelin proteins, the increased activity and expression of this proteinase may play a critical role in myelinolysis in autoimmune demyelinating diseases such as MS.

The synthesis of protein with an identification peptide

Abstract: A hybrid polypeptide composed of an identification peptide and a desired functional protein are produced by recombinant DNA techniques. A DNA expression vector is constructed that includes segments of DNA coding for the identification peptide and the desired functional protein. The identification peptide consists of a highly antigenic N-terminal portion and a C-terminal linking portion that connects the identification peptide to the N-terminal of the functional protein. The linking portion of the identification peptide is cleavable at a specific amino acid residue adjacent the functional protein by use of a sequence specific proteolytic enzyme or chemical proteolytic agent. The hybrid polypeptide expressed by the host cells transformed by the cloning vector is removed therefrom and purified by affinity chromatography techniques by use of an immobilized ligand specific to the antigenic portion of the identification peptide. The protein is then cleaved from the isolated hybrid polypeptide with an appropriate proteolic enzyme or chemical agent, thereby releasing the mature functional protein in highly purified, highly active state.

Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.

Abstract: Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.