A human endogenous retrovirus encoded protease potentially cleaves numerous cellular proteins

Background: A considerable portion of the human genome derives from retroviruses inherited over millions of years. Human endogenous retroviruses (HERVs) are usually severely mutated, yet some coding-competent HERVs exist. The HERV-K(HML-2) group includes evolutionarily young proviruses that encode typical retroviral proteins. HERV-K(HML-2) has been implicated in various human diseases because transcription is often upregulated and some of its encoded proteins are known to affect cell biology. HERV-K(HML-2) Protease (Pro) has received little attention so far, although it is expressed in some disease contexts and other retroviral proteases are known to process cellular proteins. Results:We set out to identify human cellular proteins that are substrates of HERV-K(HML-2) Pro employing a modified Terminal Amine Isotopic Labeling of Substrates (TAILS) procedure. Thousands of human proteins were identified by this assay as significantly processed by HERV-K(HML-2) Pro at both acidic and neutral pH. We confirmed cleavage of a majority of selected human proteins in vitro and in co-expression experiments in vivo. Sizes of processing products observed for some of the tested proteins coincided with product sizes predicted by TAILS. Processed proteins locate to various cellular compartments and participate in diverse, often disease-relevant cellular processes. A limited number of HERV-K(HML-2) reference and non-reference loci appears capable of encoding active Pro. Conclusions:Our findings from an approach combining TAILS with experimental verification of candidate proteins in vitro and in cultured cells suggest that hundreds of cellular proteins are potential substrates of HERV-K(HML-2) Pro. It is therefore conceivable that even low-level expression of HERV-K(HML-2) Pro affects levels of a diverse array of proteins and thus has a functional impact on cell biology and possible relevance for human diseases. Further studies are indicated to elucidate effects of HERV-K(HML-2) Pro expression regarding human substrate proteins, cell biology, and disease. The latter also calls for studies on expression of specific HERV-K(HML-2) loci capable of encoding active Pro. Endogenous retrovirus-encoded Pro activity may also be relevant for disease development in species other than human.Deutsche Forschungsgemeinschaft; HOMFO

A systematic evaluation of semispecific peptide search parameter enables identification of previously undescribed N-terminal peptides and conserved proteolytic processing in cancer cell lines

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the most commonly used technique in explorative proteomic research. A variety of open-source tools for peptide-spectrum matching have become available. Most analyses of explorative MS data are performed using conventional settings, such as fully specific enzymatic constraints. Here we evaluated the impact of the fragment mass tolerance in combination with the enzymatic constraints on the performance of three search engines. Three open-source search engines (Myrimatch, X! Tandem, and MSGF+) were evaluated concerning the suitability in semi- and unspecific searches as well as the importance of accurate fragment mass spectra in non-specific peptide searches. We then performed a semispecific reanalysis of the published NCI-60 deep proteome data applying the most suited parameters. Semi- and unspecific LC-MS/MS data analyses particularly benefit from accurate fragment mass spectra while this effect is less pronounced for conventional, fully specific peptide-spectrum matching. Search speed differed notably between the three search engines for semi- and non-specific peptide-spectrum matching. Semispecific reanalysis of NCI-60 proteome data revealed hundreds of previously undescribed N-terminal peptides, including cases of proteolytic processing or likely alternative translation start sites, some of which were ubiquitously present in all cell lines of the reanalyzed panel. Highly accurate MS2 fragment data in combination with modern open-source search algorithms enable the confident identification of semispecific peptides from large proteomic datasets. The identification of previously undescribed N-terminal peptides in published studies highlights the potential of future reanalysis and data mining in proteomic datasets

A single glycan at the 99-loop of human kallikrein-related Peptidase 2 regulates activation and enzymatic activity

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.(VLID)239213

Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival

Background: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. Method: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Results: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. Conclusion: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma

Structural determinants of specificity and regulation of activity in the allosteric loop network of human KLK8/neuropsin

Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and the hippocampus regions of the brain, where it regulates memory formation by synaptic remodeling. Substrate profiles of recombinant KLK8 were analyzed with positional scanning using fluorogenic tetrapeptides and the proteomic PICS approach, which revealed the prime side specificity. Enzyme kinetics with optimized substrates showed stimulation by Ca2+ and inhibition by Zn2+, which are physiological regulators. Crystal structures of KLK8 with a ligand-free active site and with the inhibitor leupeptin explain the subsite specificity and display Ca2+ bound to the 75-loop. The variants D70K and H99A confirmed the antagonistic role of the cation binding sites. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation of activity by Ca2+ and Zn2+, which are important in neuron and skin physiology. Both cations participate in the allosteric surface loop network present in related serine proteases. A comparison of the positional scanning data with substrates from brain suggests an adaptive recognition by KLK8, based on the tertiary structures of its targets. These combined findings provide a comprehensive picture of the molecular mechanisms underlying the enzyme activity of KLK8.(VLID)276376

Characterization of various cell lines from different ampullary cancer subtypes and cancer associated fibroblast-mediated responses

Ampullary cancer is a relatively rare form of cancer and usually treated by pancreatoduodenectomy, followed by adjuvant therapy. The intestinal subtype is associated with markedly improved prognosis after resection. At present, only few cell lines are available for in vitro studies of ampullary cancer and they have not been collectively characterized

Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. IMPORTANCE Chlamydia trachomatis is a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections. C. trachomatis can develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal "coats" or "cages," whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the inclusion and probably through actin the release of the inclusion. Septins are a group of GTP-binding proteins that can organize into heteromeric complexes and then into large filaments. Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol. Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell