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Because of their unique capacity to cross-present Ags to CD8+ T cells, mouse lymphoid tissue-resident CD8+ dendritic cells (DCs) and their migratory counterparts are critical for priming antiviral T cell responses. High expression of the dsRNA sensor TLR3 is a distinctive feature of these cross-presenting DC subsets. TLR3 engagement in CD8+ DCs promotes cross-presentation and the acquisition of effector functions required for driving antiviral T cell responses. In this study, we performed a comprehensive analysis of the TLR3-induced antiviral program and cell-autonomous immunity in CD8+ DC lines and primary CD8+ DCs. We found that TLR3-ligand polyinosinic-polycytidylic acid and human rhinovirus infection induced a potent antiviral protection against Sendai and vesicular stomatitis virus in a TLR3 and type I IFN receptor-dependent manner. Polyinosinic-polycytidylic acid-induced antiviral genes were identified by mass spectrometry-based proteomics and transcriptomics in the CD8+ DC line. Nanostring nCounter experiments confirmed that these antiviral genes were induced by TLR3 engagement in primary CD8+ DCs, and indicated that many are secondary TLR3-response genes requiring autocrine IFN-β stimulation. TLR3-activation thus establishes a type I IFN-dependent antiviral program in a DC subtype playing crucial roles in priming adaptive antiviral immune responses. This mechanism is likely to shield the priming of antiviral responses against inhibition or abrogation by the viral infection. It could be particularly relevant for viruses detected mainly by TLR3, which may not trigger type I IFN production by DCs that lack TLR3, such as plasmacytoid DCs or CD8- DCs. [ABSTRACT FROM AUTHOR]

The article offers information on the identification of gelatinase substrates at the blood-brain barrier that play a role in astroglial barrier function. Using a sensitive label-free mass spectrometry-based secretome approach, the study identifies potential substrates for MMP-9 and MMP-2 at the cell surface of primary astrocytes, including known substrates and previously unknown MMP-dependent events involved in cell-cell and cell-matrix interactions.

Amyloid-like protein aggregation is associated with neurodegeneration and other pathologies. The nature of the toxic aggregate species and their mechanism of action remain elusive. Here, we analyzed the compartment specificity of aggregate toxicity using artificial p-sheet proteins, as well as fragments of mutant huntingtin and TAR DNA binding protein-43 (TDP-43). Aggregation in the cytoplasm interfered with nucleocytoplasmic protein and RNA transport. In contrast, the same proteins did not inhibit transport when forming inclusions in the nucleus at or around the nucleolus. Protein aggregation in the cytoplasm, but not the nucleus, caused the sequestration and mislocalization of proteins containing disordered and low-complexity sequences, including multiple factors of the nuclear import and export machinery. Thus, impairment of nucleocytoplasmic transport may contribute to the cellular pathology of various aggregate deposition diseases. [ABSTRACT FROM AUTHOR]

Small-molecule-induced protein degradation has emerged as a promising pharmacological modality for inactivating disease-relevant protein kinases. DYRK1A and DYRK1B are closely related protein kinases that are involved in pathological processes such as neurodegeneration, cancer development, and adaptive immune homeostasis. Herein, we report the development of the first DYRK1 proteolysis targeting chimeras (PROTACs) that combine a new ATP-competitive DYRK1 inhibitor with ligands for the E3 ubiquitin ligase component cereblon (CRBN) to induce ubiquitination and subsequent proteasomal degradation of DYRK1A and DYRK1B. The lead compound (DYR684) promoted fast, efficient, potent, and selective degradation of DYRK1A in cell-based assays. Interestingly, an enzymatically inactive splicing variant of DYRK1B (p65) resisted degradation. Compared to competitive kinase inhibition, targeted degradation of DYRK1 by DYR684 provided improved suppression of downstream signaling. Collectively, our results identify DYRKs as viable targets for PROTAC-mediated degradation and qualify DYR684 as a useful chemical probe for DYRK1A and DYRK1B.

Short-chain fatty acids (SCFAs) are immunomodulatory compounds produced by the microbiome through dietary fiber fermentation. Although generally considered beneficial for gut health, patients suffering from inflammatory bowel disease (IBD) display poor tolerance to fiber-rich diets, suggesting that SCFAs may have contrary effects under inflammatory conditions. To investigate this, we examined the effect of SCFAs on human macrophages in the presence of Toll-like receptor (TLR) agonists. In contrast to anti-inflammatory effects under steady-state conditions, we found that butyrate and propionate activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in the presence of TLR agonists. Mechanistically, these SCFAs prevented transcription of FLICE-like inhibitory protein (cFLIP) and interleukin-10 (IL-10) through histone deacetylase (HDAC) inhibition, triggering caspase-8-dependent NLRP3 inflammasome activation. SCFA-driven NLRP3 activation was potassium efflux independent and did not result in cell death but rather triggered hyperactivation and IL-1β release. Our findings demonstrate that butyrate and propionate are bacterially derived danger signals that regulate NLRP3 inflammasome activation through epigenetic modulation of the inflammatory response., Competing Interests: Declaration of interests E.L. is co-founder of IFM Therapeutics, Odyssey Therapeutics, DiosCure Therapeutics, and Stealth Biotech. R.C.C. is a co-inventor on patent applications for NLRP3 inhibitors, which have been licensed to Inflazome Ltd., and is a consultant for BioAge Labs., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Despite the prevalence and importance of multiple goals for organizations, research on how organizations respond to performance on multiple goals continues to be limited and has examined only search intensity as the focal response, ignoring that search may occur in different locations. We extend the research on multiple goals by developing and testing novel theory on the relationship between performance feedback on multiple goals and the locus of search. Drawing upon the behavioral theory of the firm and using panel data from global pharmaceutical firms, we first show that when performance is below aspirations on a primary goal, a firm's propensity to engage in distal search increases along both the technological (i.e., familiar vs. unfamiliar search) and the organizational dimension (i.e., internal vs. external search). However, building on more recent literature that points to the need to consider multiple goals of unequal importance and, specifically, the self-enhancement perspective, we argue and find that performance on a secondary goal modifies this pattern, particularly when performance on a primary goal is unsatisfactory. Under feedback inconsistency, where performance on a primary goal is low but performance on a secondary goal is high, decision-makers decrease distal search to both unfamiliar technological areas and areas external to the organization. Our theory and findings highlight the importance of performance feedback regarding multiple goals in regulating the key locus of search choices and extend research on self-enhancement and learning from performance feedback., (© The Author(s) 2023.)

In humans, blood Classical CD14 + monocytes contribute to host defense by secreting large amounts of pro-inflammatory cytokines. Their aberrant activity causes hyper-inflammation and life-threatening cytokine storms, while dysfunctional monocytes are associated with 'immunoparalysis', a state of immune hypo responsiveness and reduced pro-inflammatory gene expression, predisposing individuals to opportunistic infections. Understanding how monocyte functions are regulated is critical to prevent these harmful outcomes. We reveal platelets' vital role in the pro-inflammatory cytokine responses of human monocytes. Naturally low platelet counts in patients with immune thrombocytopenia or removal of platelets from healthy monocytes result in monocyte immunoparalysis, marked by impaired cytokine response to immune challenge and weakened host defense transcriptional programs. Remarkably, supplementing monocytes with fresh platelets reverses these conditions. We discovered that platelets serve as reservoirs of key cytokine transcription regulators, such as NF-κB and MAPK p38, and pinpointed the enrichment of platelet NF-κB2 in human monocytes by proteomics. Platelets proportionally restore impaired cytokine production in human monocytes lacking MAPK p38α, NF-κB p65, and NF-κB2. We uncovered a vesicle-mediated platelet-monocyte-propagation of inflammatory transcription regulators, positioning platelets as central checkpoints in monocyte inflammation., (© 2024. The Author(s).)

Datasets consist of measurement data and metadata. Metadata provides context, essential for understanding and (re-)using data. Various metadata standards exist for different methods, systems and contexts. However, relevant information resides at differing stages across the data-lifecycle. Often, this information is defined and standardized only at publication stage, which can lead to data loss and workload increase. In this study, we developed Metadatasheet, a metadata standard based on interviews with members of two biomedical consortia and systematic screening of data repositories. It aligns with the data-lifecycle allowing synchronous metadata recording within Microsoft Excel, a widespread data recording software. Additionally, we provide an implementation, the Metadata Workbook, that offers user-friendly features like automation, dynamic adaption, metadata integrity checks, and export options for various metadata standards. By design and due to its extensive documentation, the proposed metadata standard simplifies recording and structuring of metadata for biomedical scientists, promoting practicality and convenience in data management. This framework can accelerate scientific progress by enhancing collaboration and knowledge transfer throughout the intermediate steps of data creation., (© 2024. The Author(s).)

T follicular helper (T FH ) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse T FH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-β (TGF-β) induces robust expression of T FH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4 + T cells in vitro. TGF-β-induced mouse CXCR5 + T FH cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated T FH cells and provide critical help to B cells. The study further reveals that TGF-β-induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-β-containing T helper 17 (T H 17)-inducing conditions also yield separate CXCR5 + and IL-17A-producing cells, highlighting shared and distinct cell fate trajectories of T FH and T H 17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-β-induced T FH cell program, that T FH and T H 17 cells share a common developmental stage, and that c-Maf acts as a switch factor for T FH versus T H 17 cell fates in TGF-β-rich environments in vitro and in vivo.

Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites., Competing Interests: The authors declare no conflict of interest.

S100A8/S100A9 is a proinflammatory mediator released by myeloid cells during many acute and chronic inflammatory disorders. However, the precise mechanism of its release from the cytosolic compartment of neutrophils is unclear. Here, we show that E-selectin-induced rapid S100A8/S100A9 release during inflammation occurs in an NLRP3 inflammasome-dependent fashion. Mechanistically, E-selectin engagement triggers Bruton's tyrosine kinase-dependent tyrosine phosphorylation of NLRP3. Concomitant potassium efflux via the voltage-gated potassium channel K V 1.3 mediates ASC oligomerization. This is followed by caspase 1 cleavage and downstream activation of pore-forming gasdermin D, enabling cytosolic release of S100A8/S100A9. Strikingly, E-selectin-mediated gasdermin D pore formation does not result in cell death but is a transient process involving activation of the ESCRT III membrane repair machinery. These data clarify molecular mechanisms of controlled S100A8/S100A9 release from neutrophils and identify the NLRP3/gasdermin D axis as a rapid and reversible activation system in neutrophils during inflammation., (© 2023. The Author(s).)

The neuronal RNA-binding protein Ptbp2 regulates neuronal differentiation by modulating alternative splicing programs in the nucleus. Such programs contribute to axonogenesis by adjusting the levels of protein isoforms involved in axon growth and branching. While its functions in alternative splicing have been described in detail, cytosolic roles of Ptbp2 for axon growth have remained elusive. Here, we show that Ptbp2 is located in the cytosol including axons and growth cones of motoneurons, and that depletion of cytosolic Ptbp2 affects axon growth. We identify Ptbp2 as a major interactor of the 3' UTR of Hnrnpr mRNA encoding the RNA-binding protein hnRNP R. Axonal localization of Hnrnpr mRNA and local synthesis of hnRNP R protein are strongly reduced when Ptbp2 is depleted, leading to defective axon growth. Ptbp2 regulates hnRNP R translation by mediating the association of Hnrnpr with ribosomes in a manner dependent on the translation factor eIF5A2. Our data thus suggest a mechanism whereby cytosolic Ptbp2 modulates axon growth by fine-tuning the mRNA transport and local synthesis of an RNA-binding protein., (© 2023. The Author(s).)

Cell-selective proteomics is a powerful emerging concept to study heterocellular processes in tissues. However, its high potential to identify non-cell-autonomous disease mechanisms and biomarkers has been hindered by low proteome coverage. Here, we address this limitation and devise a comprehensive azidonorleucine labeling, click chemistry enrichment, and mass spectrometry-based proteomics and secretomics strategy to dissect aberrant signals in pancreatic ductal adenocarcinoma (PDAC). Our in-depth co-culture and in vivo analyses cover more than 10,000 cancer cell-derived proteins and reveal systematic differences between molecular PDAC subtypes. Secreted proteins, such as chemokines and EMT-promoting matrisome proteins, associated with distinct macrophage polarization and tumor stromal composition, differentiate classical and mesenchymal PDAC. Intriguingly, more than 1,600 cancer cell-derived proteins including cytokines and pre-metastatic niche formation-associated factors in mouse serum reflect tumor activity in circulation. Our findings highlight how cell-selective proteomics can accelerate the discovery of diagnostic markers and therapeutic targets in cancer., (© 2023. The Author(s).)

The adaptive immune response is under circadian control, yet, why adaptive immune reactions continue to exhibit circadian changes over long periods of time is unknown. Using a combination of experimental and mathematical modeling approaches, we show here that dendritic cells migrate from the skin to the draining lymph node in a time-of-day-dependent manner, which provides an enhanced likelihood for functional interactions with T cells. Rhythmic expression of TNF in the draining lymph node enhances BMAL1-controlled ICAM-1 expression in high endothelial venules, resulting in lymphocyte infiltration and lymph node expansion. Lymph node cellularity continues to be different for weeks after the initial time-of-day-dependent challenge, which governs the immune response to vaccinations directed against Hepatitis A virus as well as SARS-CoV-2. In this work, we present a mechanistic understanding of the time-of-day dependent development and maintenance of an adaptive immune response, providing a strategy for using time-of-day to optimize vaccination regimes., (© 2023. The Author(s).)

High-grade adult-type diffuse gliomas are malignant neuroepithelial tumors with poor survival rates in combined chemoradiotherapy. The current WHO classification is based on IDH1/2 mutational and 1p/19q codeletion status. Glioma proteome alterations remain undercharacterized despite their promise for a better molecular patient stratification and therapeutic target identification. Here, we use mass spectrometry to characterize 42 formalin-fixed, paraffin-embedded (FFPE) samples from IDH-wild-type (IDHwt) gliomas, IDH-mutant (IDHmut) gliomas with and without 1p/19q codeletion, and non-neoplastic controls. Based on more than 5,500 quantified proteins and 5,000 phosphosites, gliomas separate by IDH1/2 mutational status but not by 1p/19q status. Instead, IDHmut gliomas split into two proteomic subtypes with widespread perturbations, including aerobic/anaerobic energy metabolism. Validations with three independent glioma proteome datasets confirm these subgroups and link the IDHmut subtypes to the established proneural and classic/mesenchymal subtypes in IDHwt glioma. This demonstrates common phenotypic subtypes across the IDH status with potential therapeutic implications for patients with IDHmut gliomas., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Proteins are the main targets of most drugs; however, system-wide methods to monitor protein activity and function are still underused in drug discovery. Novel biochemical approaches, in combination with recent developments in mass spectrometry-based proteomics instrumentation and data analysis pipelines, have now enabled the dissection of disease phenotypes and their modulation by bioactive molecules at unprecedented resolution and dimensionality. In this Review, we describe proteomics and chemoproteomics approaches for target identification and validation, as well as for identification of safety hazards. We discuss innovative strategies in early-stage drug discovery in which proteomics approaches generate unique insights, such as targeted protein degradation and the use of reactive fragments, and provide guidance for experimental strategies crucial for success., (© 2022. Springer Nature Limited.)

KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted and immunotherapies. Among the different PDAC subtypes, basal-like mesenchymal PDAC, which is driven by allelic imbalance, increased gene dosage and subsequent high expression levels of oncogenic KRAS, shows the most aggressive phenotype and strongest therapy resistance. In the present study, we performed a systematic high-throughput combination drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib, which targets KRAS-directed oncogenic signaling in mesenchymal PDAC. This combination treatment induces cell-cycle arrest and cell death, and initiates a context-dependent remodeling of the immunosuppressive cancer cell secretome. Using a combination of single-cell RNA-sequencing, CRISPR screens and immunophenotyping, we show that this combination therapy promotes intratumor infiltration of cytotoxic and effector T cells, which sensitizes mesenchymal PDAC to PD-L1 immune checkpoint inhibition. Overall, our results open new avenues to target this aggressive and therapy-refractory mesenchymal PDAC subtype., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Hepatocellular carcinoma (HCC) can have viral or non-viral causes 1-5 . Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need 6,7 . Here we report the progressive accumulation of exhausted, unconventionally activated CD8 + PD1 + T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8 + PD1 + T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8 + PD1 + CXCR6 + , TOX + , and TNF + T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8 + T cells or TNF neutralization, suggesting that CD8 + T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8 + PD1 + T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.

Secreted proteins such as cytokines, interleukins, growth factors, and hormones have pleiotropic functions and facilitate intercellular communication in organisms. Quantification of these proteins conventionally relies on antibody-based methods, i.e., enzyme-linked immunosorbent assays (ELISA), whose large-scale use is limited by availability, specificity, and affordability.Here, we describe an experimental and bioinformatics workflow to comprehensively quantify cellular protein secretion by mass spectrometry. Secreted proteins are collected in vitro or ex vivo, digested with proteases and the resulting peptide mixtures are analyzed in single liquid chromatography-mass spectrometry (LC-MS/MS) runs. Label-free quantification and bioinformatics analysis is conducted in the MaxQuant and Perseus computational environment. Our workflow allows the quantification of thousands of secreted proteins spanning a concentration range of four orders of magnitude and permits the systems-level characterization of secretory programs as well as the discovery of proteins with unexpected extracellular functions.