Biology subjects

Hensbergen, P. J.

Publications and source records attributed to Hensbergen, P. J..

3 recordsLinked to original sources

bioRxiv2022

In-depth specificity profiling of Pro-Pro endopeptidases (PPEPs) using combinatorial synthetic peptide libraries

Proteases comprise the class of enzymes that catalyze the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility towards protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. We developed a new method which combines the high diversity of a combinatorial synthetic peptide library with the sensitivity and detection power of mass spectrometry to determine protease cleavage specificity. We applied this method to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e. Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) which has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.

biochemistry↗

bioRxiv2021

Clostridioides difficile phosphoproteomics shows an expansion of phosphorylated proteins in stationary growth phase

Phosphorylation is a post-translational modification that can affect both house-keeping functions and virulence characteristics in bacterial pathogens. In the Gram-positive enteropathogen Clostridioides difficile the extent and nature of phosphorylation events is poorly characterized, though a protein-kinase mutant strain demonstrates pleiotropic phenotypes. Here, we used an immobilized metal affinity chromatography strategy to characterize serine, threonine and tyrosine phosphorylation in C. difficile. We find limited protein phosphorylation in the exponential growth phase but a sharp increase in the number of phosphopeptides after the onset of stationary growth phase. Among the overall more than 1500 phosphosites, our approach identifies expected targets and phosphorylation sites, including the protein kinase PrkC, the anti-sigma-F factor antagonist (SpoIIAA), the anti-sigma-B factor antagonist (RsbV) and HPr kinase/phosphorylase (HprK). Analysis of high-confidence phosphosites shows that phosphorylation on serine residues is most common, followed by threonine and tyrosine phosphorylation. This work forms the basis for a further investigation into the contributions of individual kinases to the overall phosphoproteome of C. difficile and the role of phosphorylation in C. difficile physiology and pathogenesis. ImportanceIn this manuscript, we present a comprehensive analysis of protein phosphorylation in the Gram-positive enteropathogen Clostridioides difficile. To date, only limited evidence on the role of phosphorylation in regulation in this organism has been published; the current study is expected to form the basis for research on this post-translational modification in C. difficile.

microbiology↗

bioRxiv2021

Malaria parasite evades mosquito immunity by glutaminyl cyclase mediated protein modification

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that Plasmodium sporozoites of QC-null mutants are recognized by the mosquito immune system and melanized when they reach the hemocoel. Sporozoite numbers in salivary glands are also reduced in mosquitoes infected with QC-null or QC catalytically-dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito hemocytes or melanization immune responses. Mutation of a single QC-target glutamine of the major sporozoite surface protein (CSP) also results in immune recognition of sporozoites. These findings reveal QC-mediated post-translational modification of surface proteins as a major mechanism of mosquito immune evasion by Plasmodium sporozoites.

microbiology↗