A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by... Show moreA group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), pos-sess the unique ability to hydrolyze proline-proline bonds in proteins. Sincea protease’s function is largely determined by its substrate specificity,methods that can extensively characterize substrate specificity are valuabletools for protease research. Previously, we achieved an in-depth characteri-zation of PPEP prime-side specificity. However, PPEP specificity is alsodetermined by the non-prime-side residues in the substrate. To gain a morecomplete insight into the determinants of PPEP specificity, we character-ized the non-prime- and prime-side specificity of various PPEPs using acombination of synthetic combinatorial peptide libraries and mass spec-trometry. With this approach, we deepened our understanding of the P3-P30 specificities of PPEP-1 and PPEP-2, while identifying the endogenoussubstrate of PPEP-2 as the most optimal substrate in our library data. Fur-thermore, by employing the library approach, we investigated the alteredspecificity of mutants of PPEP-1 and PPEP-2. Additionally, we character-ized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displaysa PPEP-1-like prime-side specificity, which was substantiated by the experi-mental data. Intriguingly, another putative PPEP from Clostridioides diffi-cile, CD1597, did not display Pro-Pro endoproteolytic activity.Collectively, we characterized PPEP specificity in detail using our robustpeptide library method and, together with additional structural informa-tion, provide more insight into the intricate mechanisms that govern prote-ase specificity. Show less
The bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the Clostridioides difficile strain 630 Delta erm, the main filamentous... Show moreThe bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the Clostridioides difficile strain 630 Delta erm, the main filamentous component, FliC, is post-translationally modified with an O-linked Type A glycan structure. This modification is essential for flagellar function, since motility is seriously impaired in gene mutants with improper biosynthesis of the Type A glycan. The cd0240-cd0244 gene cluster encodes the Type A biosynthetic proteins, but the role of each gene, and the corresponding enzymatic activity, have not been fully elucidated. Using quantitative mass spectrometry-based proteomics analyses, we determined the relative abundance of the observed glycan variations of the Type A structure in cd0241, cd0242, cd0243, and cd0244 mutant strains. Our data not only confirm the importance of CD0241, CD0242, and CD0243 but, in contrast to previous data, also show that CD0244 is essential for the biosynthesis of the Type A modification. Combined with additional bioinformatic analyses, we propose a revised model for Type A glycan biosynthesis. Show less
Proteases comprise the class of enzymes that catalyzesthe hydrolysisof peptide bonds, thereby playing a pivotal role in many aspects oflife. The amino acids surrounding the scissile bond determine... Show moreProteases comprise the class of enzymes that catalyzesthe hydrolysisof peptide bonds, thereby playing a pivotal role in many aspects oflife. The amino acids surrounding the scissile bond determine thesusceptibility toward protease-mediated hydrolysis. A detailed understandingof the cleavage specificity of a protease can lead to the identificationof its endogenous substrates, while it is also essential for the designof inhibitors. Although many methods for protease activity and specificityprofiling exist, none of these combine the advantages of combinatorialsynthetic libraries, i.e., high diversity, equimolar concentration,custom design regarding peptide length, and randomization, with thesensitivity and detection power of mass spectrometry. Here, we developedsuch a method and applied it to study a group of bacterial metalloproteasesthat have the unique specificity to cleave between two prolines, i.e.,Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-sidespecificity of PPEP-1 and PPEP-2, but also revealed some new unexpectedpeptide substrates. Moreover, we have characterized a new PPEP (PPEP-3)that has a prime-side specificity that is very different from thatof the other two PPEPs. Importantly, the approach that we presentin this study is generic and can be extended to investigate the specificityof other proteases. Show less
Shitut, S.S.; Shen, M.; Claushuis, B.; Derks, R.J.E.; Giera, M.; Rozen, D.E.; ... ; Kros, A. 2022
Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different... Show moreCell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery.Fusion of cells is an important and common biological process that leads to the mixing of cellular contents and the formation of multinuclear cells. Cell fusion occurs when distinct membranes are brought into proximity of one another and merge to become one. Fusion holds promise for biotechnological innovations, for instance, for the discovery of urgently needed new antibiotics. Here, we used antibiotic-producing bacteria that can proliferate without their cell wall as a model to investigate cell-cell fusion. We found that fusion between genetically distinct cells yields heterokaryons that are viable, contain multiple selection markers, and show increased antimicrobial activity. The rate of fusion induced using physical and chemical methods was dependent on membrane fluidity, which is related to lipid composition as a function of cellular age. Finally, by using an innovative system of synthetic membrane-associated lipopeptides, we achieved targeted fusion between distinctly marked cells to further enhance fusion efficiency. These results provide a molecular handle to understand and control cell-cell fusion, which can be used in the future for the discovery of new drugs. IMPORTANCE Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery. We present here wall-deficient cells as a platform for the same. We identify the fluidity of the membrane as an important characteristic for the process of fusion. We demonstrate a cell-specific approach for fusion using synthetically designed peptides yielding cells with modified antibiotic production profiles. Overall, wall-deficient cells can be a chassis for innovative metabolite production by providing an alternative method for cell-cell fusion. Show less
