Characterization of the Clostridioides difficile 630Δerm putative Pro-Pro endopeptidase CD1597.

is the leading cause of antibiotic-associated infections worldwide. Within the host, can transition from a sessile to a motile state by secreting PPEP-1, which releases the cells from the intestinal epithelium by cleaving adhesion proteins. PPEP-1 belongs to the group of Pro-Pro endopeptidases (PPEPs), which are characterized by their unique ability to cleave proline-proline bonds.

Covalent Probes To Capture Dup Effector Enzymes.

Upon infection of host cells, releases a multitude of effector enzymes into the cell's cytoplasm that hijack a plethora of cellular activities, including the host ubiquitination pathways. Effectors belonging to the SidE-family are involved in noncanonical serine phosphoribosyl ubiquitination of host substrate proteins contributing to the formation of a Legionella-containing vacuole that is crucial in the onset of Legionnaires' disease. This dynamic process is reversed by effectors called Dups that hydrolyze the phosphodiester in the phosphoribosyl ubiquitinated protein.

Direct glycosylation analysis of intact monoclonal antibodies combining ESI MS of glycoforms and MALDI-in source decay MS of glycan fragments.

Monoclonal antibody (mAb) glycoengineering has the potential to improve the efficacy of biopharmaceuticals by fine-tuning specific biological properties. Glycosylation analysis is key to monitoring the glycoengineering process. Various mass spectrometry (MS)-based methods are available to characterize mAb glycosylation at different structural levels, but comprehensive analysis is typically time-consuming and costly.

Epstein-Barr virus nuclear antigen EBNA3A modulates IRF3-dependent IFNβ expression.

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, persistently infects over 90% of the human adult population and is associated with several human cancers. To establish life-long infection, EBV tampers with the induction of type I interferon (IFN I)-dependent antiviral immunity in the host. How various EBV genes help orchestrate this crucial strategy is incompletely defined.

AMPK activation induces RALDH+ tolerogenic dendritic cells by rewiring glucose and lipid metabolism.

Dendritic cell (DC) activation and function are underpinned by profound changes in cellular metabolism. Several studies indicate that the ability of DCs to promote tolerance is dependent on catabolic metabolism. Yet the contribution of AMP-activated kinase (AMPK), a central energy sensor promoting catabolism, to DC tolerogenicity remains unknown.

Acetylated bacterial proteins as potent antigens inducing an anti-modified protein antibody response.

Objective: Gut-residing bacteria, such as , can acetylate their proteome under conditions of amine starvation. It is postulated that the (gut) microbiome is involved in the breach of immune tolerance to modified self-proteins leading to the anti-modified protein antibodies (AMPAs), hallmarking seropositive rheumatoid arthritis (RA). Our aim was to determine whether acetylated bacterial proteins can induce AMPA responses cross-reactive to modified self-proteins and be recognised by human AMPA (hAMPA).

Hotspot DNA Methyltransferase 3A () and Isocitrate Dehydrogenase 1 and 2 () Mutations in Acute Myeloid Leukemia and Their Relevance as Targets for Immunotherapy.

DNA methyltransferase 3A () and isocitrate dehydrogenase 1 and 2 () are genes involved in epigenetic regulation, each mutated in 7-23% of patients with acute myeloid leukemia. Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five human B-lymphoblastoid cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in or .

Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2.

Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic β-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic β-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of β-cells derive from the insulin precursor molecule proinsulin.

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis.

Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial.

Non-prime- and prime-side profiling of Pro-Pro endopeptidase specificity using synthetic combinatorial peptide libraries and mass spectrometry.

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a protease's function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity.

A transcriptomic based deconvolution framework for assessing differentiation stages and drug responses of AML.

The diagnostic spectrum for AML patients is increasingly based on genetic abnormalities due to their prognostic and predictive value. However, information on the AML blast phenotype regarding their maturational arrest has started to regain importance due to its predictive power for drug responses. Here, we deconvolute 1350 bulk RNA-seq samples from five independent AML cohorts on a single-cell healthy BM reference and demonstrate that the morphological differentiation stages (FAB) could be faithfully reconstituted using estimated cell compositions (ECCs).

Development of Inhibitors, Probes, and PROTAC Provides a Complete Toolbox to Study PARK7 in the Living Cell.

The integration of diverse chemical tools like small-molecule inhibitors, activity-based probes (ABPs), and proteolysis targeting chimeras (PROTACs) advances clinical drug discovery and facilitates the exploration of various biological facets of targeted proteins. Here, we report the development of such a chemical toolbox for the human Parkinson disease protein 7 (PARK7/DJ-1) implicated in Parkinson's disease and cancers. By combining structure-guided design, miniaturized library synthesis, and high-throughput screening, we identified two potent compounds, and , inhibiting PARK7 and in cells by covalently and selectively targeting its critical residue, Cys106.

Virus-Associated CD8 T-Cells Are Not Activated Through Antigen-Mediated Interaction Inside Atherosclerotic Lesions.

Introduction: Viral infections have been associated with the progression of atherosclerosis and CD8 T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8 T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8 T-cells in the atherosclerotic lesion.

Expanding the repertoire reveals recurrent, cryptic, and hematopoietic HLA class I minor histocompatibility antigens.

Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease.

Tregs from human blood differentiate into nonlymphoid tissue-resident effector cells upon TNFR2 costimulation.

Tregs can facilitate transplant tolerance and attenuate autoimmune and inflammatory diseases. Therefore, it is clinically relevant to stimulate Treg expansion and function in vivo and to create therapeutic Treg products in vitro. We report that TNF receptor 2 (TNFR2) is a unique costimulus for naive, thymus-derived Tregs (tTregs) from human blood that promotes their differentiation into nonlymphoid tissue-resident (NLT-resident) effector Tregs, without Th-like polarization.

Revised Model for the Type A Glycan Biosynthetic Pathway in Strain 630Δ Based on Quantitative Proteomics of - Mutant Strains.

The bacterial flagellum is involved in a variety of processes including motility, adherence, and immunomodulation. In the strain 630Δ, the main filamentous component, FliC, is post-translationally modified with an -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.

Erratum: A library of cancer testis specific T cell receptors for T cell receptor gene therapy.

[This corrects the article DOI: 10.1016/j.omto.

Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2.

Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, , comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue.

CD44 acts as a coreceptor for cell-specific enhancement of signaling and regulatory T cell induction by TGM1, a parasite TGF-β mimic.

Long-lived parasites evade host immunity through highly evolved molecular strategies. The murine intestinal helminth, , down-modulates the host immune system through release of an immunosuppressive TGF-β mimic, TGM1, which is a divergent member of the CCP (Sushi) protein family. TGM1 comprises 5 domains, of which domains 1-3 (D1/2/3) bind mammalian TGF-β receptors, acting on T cells to induce Foxp3 regulatory T cells; however, the roles of domains 4 and 5 (D4/5) remain unknown.

Neoantigen Targetability in Progressive Advanced Melanoma.

Purpose: The availability of (neo)antigens and the infiltration of tumors by (neo)antigen-specific T cells are crucial factors in cancer immunotherapy. In this study, we aimed to investigate the targetability of (neo)antigens in advanced progessive melanoma and explore the potential for continued T-cell-based immunotherapy.

Experimental Design: We examined a cohort of eight patients with melanoma who had sequential metastases resected at early and later time points.

RNF26 binds perinuclear vimentin filaments to integrate ER and endolysosomal responses to proteotoxic stress.

Proteotoxic stress causes profound endoplasmic reticulum (ER) membrane remodeling into a perinuclear quality control compartment (ERQC) for the degradation of misfolded proteins. Subsequent return to homeostasis involves clearance of the ERQC by endolysosomes. However, the factors that control perinuclear ER integrity and dynamics remain unclear.

In-Depth Specificity Profiling of Endopeptidases Using Dedicated Mix-and-Split Synthetic Peptide Libraries and Mass Spectrometry.

Proteases comprise the class of enzymes that catalyzes 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 toward 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.