Search

Biological chemistry 404(2-3), 195 - 207 (2023). doi:10.1515/hsz-2022-0273, Oxalyl-CoA synthetase from Saccharomyces cerevisiae is one of the most abundant peroxisomal proteins in yeast and hence has become a model to study peroxisomal translocation. It contains a C-terminal Peroxisome Targeting Signal 1, which however is partly dispensable, suggesting additional receptor bindings sites. To unravel any additional features that may contribute to its capacity to be recognized as peroxisomal target, we determined its assembly and overall architecture by an integrated structural biology approach, including X-ray crystallography, single particle cryo-electron microscopy and small angle X-ray scattering. Surprisingly, it assembles into mixture of concentration-dependent dimers, tetramers and hexamers by dimer self-association. Hexameric particles form an unprecedented asymmetric horseshoe-like arrangement, which considerably differs from symmetric hexameric assembly found in many other protein structures. A single mutation within the self-association interface is sufficient to abolish any higher-level oligomerization, resulting in a homogenous dimeric assembly. The small C-terminal domain of yeast Oxalyl-CoA synthetase is connected by a partly flexible hinge with the large N-terminal domain, which provides the sole basis for oligomeric assembly. Our data provide a basis to mechanistically study peroxisomal translocation of this target., Published by de Gruyter, Berlin [u.a.]

Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p2D) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p2D is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.

Thrombospondin type-1 domain-containing 7A (THSD7A) is a large extracellular protein that is found in podocyte foot processes of the kidney glomerulus. It has been established as a causative autoantigen in membranous nephropathy. Amongst the predicted 21 thrombospondin repeat domains of its extracellular segment, the highest frequency of autoimmune response has been associated with the two N-terminal domains. Here, we show that antibodies against this THSD7A segment in mice induce typical clinical and morphological signs of membranous nephropathy. The high-resolution structure of these two domains reveals a non-canonical thrombospondin repeat fold that is distinct from the established type 1 thrombospondin repeat. As it shares a conserved disulfide pattern with the canonical fold, we refer to these domains d1 and d2 as type 1A thrombospondin repeats. Both domains comprise a seven layered CC-W-PP-R-W-QQ-CC pattern, which is only partly shared by other THSD7A thrombospondin repeat domains. The two domains form a well-defined V-shaped tandem arrangement. Our findings provide crucial insight into specific structural features of these two domains that are distinct from other regions of THSD7A and hence could cause the high level of antigenicity found for these two domains.

Supplementary Figure S4. Data base analysis of correlation between RAI2 and ESR1 mRNA expression.

The human microbiota plays an important role in human health and contributes to the metabolism of therapeutic drugs affecting their potency. However, the current knowledge on human gut bacterial metabolism is limited and lacks an understanding of the underlying mechanisms of observed drug biotransformations. Despite the complexity of the gut microbial community, genomic and metagenomic sequencing provides insights into the diversity of chemical reactions that can be carried out by the microbiota and poses new challenges to functionally annotate thousands of bacterial enzymes. Here, we outline methods to systematically address the structural and functional space of the human microbiome, highlighting a combination of in silico and in vitro approaches. Systematic knowledge about microbial enzymes could eventually be applied for personalized therapy, the development of prodrugs and modulators of unwanted bacterial activity, and the further discovery of new antibiotics.

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with millisecond time-resolution. Canonical micromesh loops are mounted on an electropneumatic piston, reactions are initiated via the liquid application method (LAMA), and finally intermediate states are cryo-trapped in liquid nitrogen. We demonstrate binding of several ligands in microcrystals of three enzymes, and trapping of reaction intermediates and conformational changes in macroscopic crystals of tryptophan synthase.

Infections caused by Mycobacterium abscessus are difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity of M. abscessus to survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin (RMP). Unlike RMP, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position and a naphthoquinone core, is not modified by purified ArrMab. Additionally, we show that the ArrMab D82 residue is essential for catalytic activity. Thermal profiling of ArrMab in the presence of 5j, RMP, or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin against M. abscessus planktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity against M. abscessus in human macrophages and shows synergistic activity with amikacin and azithromycin.

An overview of a unique life science facility at the European XFEL is provided, and its importance in sample preparation, characterization and analysis prior to measurements at the different European XFEL instruments is discussed. The capabilities that the facility can provide for the research community are highlighted, alongside examples of recent successful studies., The science of X-ray free-electron lasers (XFELs) critically depends on the performance of the X-ray laser and on the quality of the samples placed into the X-ray beam. The stability of biological samples is limited and key biomolecular transformations occur on short timescales. Experiments in biology require a support laboratory in the immediate vicinity of the beamlines. The XBI BioLab of the European XFEL (XBI denotes XFEL Biology Infrastructure) is an integrated user facility connected to the beamlines for supporting a wide range of biological experiments. The laboratory was financed and built by a collaboration between the European XFEL and the XBI User Consortium, whose members come from Finland, Germany, the Slovak Republic, Sweden and the USA, with observers from Denmark and the Russian Federation. Arranged around a central wet laboratory, the XBI BioLab provides facilities for sample preparation and scoring, laboratories for growing prokaryotic and eukaryotic cells, a Bio Safety Level 2 laboratory, sample purification and characterization facilities, a crystallization laboratory, an anaerobic laboratory, an aerosol laboratory, a vacuum laboratory for injector tests, and laboratories for optical microscopy, atomic force microscopy and electron microscopy. Here, an overview of the XBI facility is given and some of the results of the first user experiments are highlighted.

RAI2 has initially been identified as a metastasis-associated gene in breast cancer. Recently, we found that increased RAI2 protein in primary tumors predicts early biochemical relapse of prostate cancer patients. On the molecular level, RAI2 interacts with CtBP1 as transcriptional co-repressors via a non-consensus tandem ALDLS-motif in hormone-dependent cancer cells. In this study, we aim to investigate the cell-biological relevance of this molecular interaction in prostate cancer cells using the CDKN1A gene as an example of a transcriptional target of CtBP-mediated gene regulation. To analyze a molecular relation between the RAI2/CtBP1 interaction and repressor of the CDKN1A gene, we first applied a transactivation assay in 293T cells, transiently transfected with CtBP1 and different RAI2 protein variants. Next, we depleted the RAI2 protein in VCaP prostate cancer cells. We used this cell line model to analyze gene expression and protein concertation of RAI2, CtBPs and p21 under different conditions. We studied the subcellular localization and chromatin binding of the proteins of interest by confocal laser-scanning microscopy and ChIP analysis. The gene reporter assay revealed that combined overexpression of RAI2 and CtBP1 reliefs the repression of the proximal CDKN1A-promotor and that RAI2 with an intact ALDLS tandem motif is required for this process. RAI2 depletion in VCaP cells resulted in a significant reduction of both CtBP1 and CtBP2 and almost complete abolishment of p21 protein levels, which is accompanied by reduced interaction of RAI2 with CtBP1 in nuclei. In parental VCaP cells, genotoxic stress significantly induced CDKNA1 gene expression and p21 protein concentration. In contrast, in RAI2-depleted VCaP cells we did not observe any of these effects, demonstrating the requirement of RAI2 to cause the relief of CDKNA1 repression. RAI2 together with CtBP1 appeared as definite foci in the nuclei of VCaP cells that are co-localized with key factors of polycomb 1 and 2. The ChIP analysis revealed that RAI2-depletion significantly induced binding of CtBPs to the CDKN1A promoter. In contrast, the binding of the polycomb protein LCoR to chromatin is significantly reduced in RAI2-depleted cells. Irrespective of the RAI2 status, the EZH2 binding to the chromatin remained unaltered, whereas a significant increase in H3K27me3 in RAI2-depleted cells was observed. In summary, we found that interruption of the interaction of RAI2 with CtBP1 is leading to increased chromatin binding of CtBP1 and trimethylation of H3K27 of the CDKN1A promotor, which is associated with repression of CDKN1A gene expression. We conclude that the molecular interaction of RAI2 with CtBP1 is a new molecular mechanism of corepression by modulating the histone-modifications of the target gene. Citation Format: Sarah Greimeier, Bettina Steinbach, Simon Sander, Lina Merkens, Nishit Goradia, Matthias Wilmanns, Eric Metzger, Klaus Pantel, Stefan Werner. RAI2 controls polycomb-mediated repression of CDKN1A by its interaction with CtBP1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3724.

Previously, we found that increased Retinoic acid-induced 2 (RAI2) protein concentration in primary tumors predicts early biochemical relapse of prostate cancer patients and described a co-repressor function of RAI2 related to androgen receptor activity (1). The presence of a non-canonical tandem ALDLS motif on RAI2 protein prompted the present investigation on a possible role of a dual interaction of RAI2 with C-terminal binding proteins (CtBPs) in relation to prostate cancer progression. We analyzed RAI2 gene expression as well as CtBP1 protein localization and constitution in circulating tumors cells (CTCs) isolated from metastatic prostate cancer patients by semi-quantitative PCR analysis and immune-fluorescence staining. We employed an integrated structural biology approach combining biophysical techniques like isothermal calorimetry, circular dichroism, size exclusion chromatography with high-resolution structural biology techniques including X-ray crystallography and single particle cryo-electron microscopy. We established RAI2-depleted VCaP cells and used this model for validation of the obtained results. RAI2 gene expression is significantly increased in CTCs isolated from patients with castration-resistant disease in comparison to CTCs isolated from patients with hormone-sensitive, aggressive or neuroendocrine variant prostate cancer. We detected CtBP1 protein foci in the nuclei of individual CTCs. We show that RAI2 induces CtBP polymerization. Using single particle cryo-electron microscopy, we obtained crucial insights into the structural organization of this filament. RAI2 inactivation in VCaP cells caused a significant reduction of both CtBP proteins, which is accompanied by the disappearance of CtBP protein aggregates in nuclei. In addition, we observed the induction of neuroendocrine features, marked by the induction of Synaptophysin and Chromogranin A, a higher nucleus to cytoplasm ratio and smaller cell size of RAI2-depleted cells. In summary, we report tandem motifs-induced polymerization of CtBP’s for the first time. We found that elevated RAI2 gene expression is a characteristic of castration-resistant prostate cancer. Because RAI2 depletion resulted in loss of CtBP aggregates and the induction of neuroendocrine traits, we conclude that RAI2-driven CtBP polymerization sustains epithelial differentiation in castration-resistant prostate cancer. These findings holds potential for the diagnosis of castration-resistant disease and inhibiting the progression from castration-resistant to neuroendocrine prostate cancer. 1. Besler K, Weglarz A, Keller L, von Amsberg G, Bednarz-Knoll N, Offermann A, et al. Expression Patterns and Corepressor Function of Retinoic Acid-induced 2 in Prostate Cancer. Clin Chem 2022;68:973-83 Citation Format: Stefan Werner, Nishit Goradia, Edukondalu Mullapudi, Lina Merkens, Sarah Greimeier, Aleksandra Węglarz, Gunhild von Amsberg, Harriet Wikman, Klaus Pantel, Matthias Wilmanns. Retinoic acid-induced 2 driven polymerization of c-terminal binding proteins sustains epithelial differentiation in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1447.

The identification and characterization of enzyme function is lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study where the high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacyl catalysis intermediate allowed its functional characterization as long-chain monacylglycerol lipase. Different from most other lipases, this enzyme uses a minimal helix–β-hairpin–helix lid domain that positions the substrate through a hydrophobic tunnel directly to the enzyme’s active site. Knowledge about the molecular details of the substrate binding site and hydrolysis allowed us to boost the enzymatic activity by adjusting protein/substrate interactions. This enzyme serves as a prototype for non-conventional esterases and lipases that share similar helix–β-hairpin–helix lid domains of variable size. Taken together, our data provide crucial insight to advance our knowledge of unusual esterases and lipases, facilitating future biotechnology applications.

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long (>100s) residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.

Structural and biophysical characterization of molecular mechanisms of disease‐causing pathogens, such as Mycobacterium tuberculosis, often requires recombinant expression of large amounts highly pure protein. For the production of mycobacterial proteins, overexpression in the fast‐growing and non‐pathogenic species Mycobacterium smegmatis has several benefits over the standard Escherichia coli expression strains. However, unlike for E. coli, the range of expression vectors currently available is limited. Here we describe the development of the pMy vector series, a set of expression plasmids for recombinant production of single proteins and protein complexes in M. smegmatis. By incorporating an alternative selection marker, we show that these plasmids can also be used for co‐expression studies. All vectors in the pMy vector series are available in the Addgene repository (www.addgene.com).

Retinoic acid-induced protein 2 is a human protein of 530 residues encoded by the RAI2 gene (Q9Y5P3; RAI2_HUMAN). RAI2 is a novel tumor suppressor protein whose depletion in breast cancer cell lines results in the downregulation of several genes associated with differentiation along with increased invasiveness and aggressive tumor phenotype of the cells. The role of the protein is specified to be a transcriptional regulator that promotes chromosomal stability and hence controls the expression of several regulators of cancer and metastasis. Structurally, RAI2 remains an unknown entity and, hence, to obtain a detailed view on the structure function relationship we report the 1H, 13C, and 15N resonance assignments for the backbone and side chain nuclei of the C-terminal region (a.a. 303-451 of UniProt Q9Y5P3) of RAI2.

Interrupted dimeric coiled coil segments are found in a broad range of proteins and generally confer selective functional properties such as binding to specific ligands. However, there is only one documented case of a basic-helix–loop–helix leucine zipper transcription factor—microphthalmia-associated transcription factor (MITF)—in which an insertion of a three-residue stammer serves as a determinant of conditional partner selectivity. To unravel the molecular principles of this selectivity, we have analyzed the high-resolution structures of stammer-containing MITF and an engineered stammer-less MITF variant, which comprises an uninterrupted symmetric coiled coil. Despite this fundamental difference, both MITF structures reveal identical flanking in-phase coiled coil arrangements, gained by helical over-winding and local asymmetry in wild-type MITF across the stammer region. These conserved structural properties allow the maintenance of a proper functional readout in terms of nuclear localization and binding to specific DNA-response motifs regardless of the presence of the stammer. By contrast, MITF heterodimer formation with other bHLH-Zip transcription factors is only permissive when both factors contain either the same type of inserted stammer or no insert. Our data illustrate a unique principle of conditional partner selectivity within the wide arsenal of transcription factors with specific partner-dependent functional readouts.

Seventy years following the discovery of peroxisomes, their proteome remains undefined. Uncovering the complete peroxisomal proteome, the peroxi-ome, is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover the peroxi-ome of the model eukaryote – Saccharomyces cerevisiae. This strategy enabled us to expand the known organellar proteome by ∼40% and paved the way for performing systematic, whole-organellar proteome assays. Coupled with targeted experiments this allowed us to discover new peroxisomal functions. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly-identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

Von Willebrand disease (VWD) is a bleeding disorder with different levels of severity. VWD-associated mutations are located in the von Willebrand factor (VWF) gene, coding for the large multidomain plasma protein VWF with essential roles in hemostasis and thrombosis. On the one hand, a variety of mutations in the C-domains of VWF are associated with increased bleeding upon vascular injury. On the other hand, VWF gain-of-function (GOF) mutations in the C4 domain have recently been identified, which induce an increased risk of myocardial infarction. Mechanistic insights into how these mutations affect the molecular behavior of VWF are scarce and holistic approaches are challenging due to the multidomain and multimeric character of this large protein. Here, we determine the structure and dynamics of the C6 domain and the single nucleotide polymorphism (SNP) variant G2705R in C6 by combining nuclear magnetic resonance spectroscopy, molecular dynamics simulations and aggregometry. Our findings indicate that this mutation mostly destabilizes VWF by leading to a more pronounced hinging between both subdomains of C6. Hemostatic parameters of variant G2705R are close to normal under static conditions, but the missense mutation results in a gain-of-function under flow conditions, due to decreased VWF stem stability. Together with the fact that two C4 variants also exhibit GOF characteristics, our data underline the importance of the VWF stem region in VWF's hemostatic activity and the risk of mutation-associated prothrombotic properties in VWF C-domain variants due to altered stem dynamics.

Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the E2F1 locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.

Ecotin is a homodimeric serine protease inhibitor produced by many commensal and pathogenic microbes. It functions as a virulence factor, enabling survival of various pathogens in the blood. The ecotin dimer binds two protease molecules, and each ecotin protomer has two protease-binding sites: site1 occupies the substrate-binding groove, whereas site2 engages a distinct secondary region. Owing to the twofold rotational symmetry within the ecotin dimer, sites 1 and 2 of a protomer bind to different protease molecules within the tetrameric complex. Escherichia coli ecotin inhibits trypsin-like, chymotrypsin-like, and elastase-like enzymes, including pancreatic proteases, leukocyte elastase, key enzymes of blood coagulation, the contact and complement systems, and other antimicrobial cascades. Here, we show that mannan-binding lectin-associated serine protease-1 (MASP-1) and MASP-2, essential activators of the complement lectin pathway, and MASP-3, an essential alternative pathway activator, are all inhibited by ecotin. We decipher in detail how the preorganization of site1 and site2 within the ecotin dimer contributes to the inhibition of each MASP enzyme. In addition, using mutated and monomeric ecotin variants, we show that site1, site2, and dimerization contribute to inhibition in a surprisingly target-dependent manner. We present the first ecotin:MASP-1 and ecotin:MASP-2 crystal structures, which provide additional insights and permit structural interpretation of the observed functional results. Importantly, we reveal that monomerization completely disables the MASP-2-inhibitory, MASP-3-inhibitory, and lectin pathway-inhibitory capacity of ecotin. These findings provide new opportunities to combat dangerous multidrug-resistant pathogens through development of compounds capable of blocking ecotin dimer formation.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in Mycobacterium smegmatis , to ESX-5 structures in Mycobacterium xenopi and Mycobacterium tuberculosis . In addition, ESX-4, considered until recently as a non-functional system in both pathogenic and non-pathogenic mycobacteria, has been proposed to play an important role in the virulence of Mycobacterium abscessus ; an increasingly recognized opportunistic NTM causing severe lung diseases. These major findings have led to important new insights into the functional mechanisms of these biological systems, their implication in virulence, nutrient acquisitions and cell wall shaping, and will be discussed in this review.

Infections caused byMycobacterium abscessusare difficult to treat due to its intrinsic resistance to most antibiotics. Formation of biofilms and the capacity ofM. abscessusto survive inside host phagocytes further complicate eradication. Herein, we explored whether addition of a carbamate-linked group at the C25 position of rifamycin SV blocks enzymatic inactivation by ArrMab, an ADP-ribosyltransferase conferring resistance to rifampicin. Unlike rifampicin, 5j, a benzyl piperidine rifamycin derivative with a morpholino substituted C3 position, is not modified by purified ArrMab. Additionally, we show that the ArrMabD82 residue is essential for catalytic activity. Thermal profiling of ArrMabin the presence of 5j, rifampicin or rifabutin shows that 5j does not bind to ArrMab. We found that the activity of 5j is comparable to amikacin againstM. abscessusplanktonic cultures and pellicles. Critically, 5j also exerts potent antimicrobial activity againstM. abscessusin human macrophages and shows synergistic activity with amikacin and azithromycin.

Non-tuberculous mycobacteria (NTM) are a large group of micro-organisms comprising more than 200 individual species. Most NTM are saprophytic organisms and are found mainly in terrestrial and aquatic environments. In recent years, NTM have been increasingly associated with infections in both immunocompetent and immunocompromised individuals, prompting significant efforts to understand the diverse pathogenic and signalling traits of these emerging pathogens. Since the discovery of Type VII secretion systems (T7SS), there have been significant developments regarding the role of these complex systems in mycobacteria. These specialised systems, also known as Early Antigenic Secretion (ESX) systems, are employed to secrete proteins across the inner membrane. They also play an essential role in virulence, nutrient uptake and conjugation. Our understanding of T7SS in mycobacteria has significantly benefited over the last few years, from the resolution of ESX-3 structure in

Science advances 7(26), eabg9923 - (2021). doi:10.1126/sciadv.abg9923, The ESX-5 type VII secretion system is a membrane-spanning protein complex key to the virulence of mycobacterial pathogens. However, the overall architecture of the fully assembled translocation machinery and the composition of the central secretion pore have remained unknown. Here, we present the high-resolution structure of the 2.1-megadalton ESX-5 core complex. Our structure captured a dynamic, secretion-competent conformation of the pore within a well-defined transmembrane section, sandwiched between two flexible protein layers at the cytosolic entrance and the periplasmic exit. We propose that this flexibility endows the ESX-5 machinery with large conformational plasticity required to accommodate targeted protein secretion. Compared to known secretion systems, a highly dynamic state of the pore may represent a fundamental principle of bacterial secretion machineries., Published by Assoc., Washington, DC [u.a.]

The frequent von Willebrand factor (VWF) variant p.Phe2561Tyr is located within the C4 domain, which also harbors the platelet GPIIb/IIIa-binding RGD sequence. To investigate its potential effect on hemostasis, we genotyped 865 patients with coronary artery disease (CAD), 915 with myocardial infarction (MI), and 417 control patients (Ludwigshafen Risk and Cardiovascular Health Study) and performed functional studies of this variant. A univariate analysis of male and female carriers of the Tyr2561 allele aged 55 years or younger revealed an elevated risk for repeated MI (odds ratio, 2.53; 95% confidence interval [CI], 1.07-5.98). The odds ratio was even higher in females aged 55 years or younger, at a value of 5.93 (95% CI, 1.12-31.24). Cone and plate aggregometry showed that compared with Phe2561, Tyr2561 was associated with increased platelet aggregate size both in probands’ blood and with the recombinant variants. Microfluidic assays revealed that the critical shear rate for inducing aggregate formation was decreased to 50% by Tyr2561 compared with Phe2561. Differences in C-domain circular dichroism spectra resulting from Tyr2561 suggest an increased shear sensitivity of VWF as a result of altered association of the C domains that disrupts the normal dimer interface. In summary, our data emphasize the functional effect of the VWF C4 domain for VWF-mediated platelet aggregation in a shear-dependent manner and provide the first evidence that a functional variant of VWF plays a role in arterial thromboembolism.