Aspergillus fumigatus is a world-wide saprophyte filamentous fungus which released conidia, its infectious morphotype, in the atmosphere. These conidia are inhaled daily by humans and can colonize the respiratory tract, where they may develop into hyphae, the invasive morphotype. We previously showed that bronchial epithelial cells (BECs) restrict A. fumigatus virulence by inhibiting conidial germination and filament formation through a process requiring PI3K signaling and the conidial fucose-specific lectin FleA. In the present study, we are looking to identify host factors and cellular partners involved in the BEC antifungal response and to define the molecular interactions underpinning FleA recognition. For this, we analyzed transcriptome of BECs infected with A. fumigatus in the presence or absence of the PI3K inhibitor LY294002. Functional involvement of candidate genes was assessed by siRNA knockdown and readouts of fungal filamentation (microscopic scoring and galactomannan release). FleA-interacting host proteins were identified by biotin-FleA affinity co-precipitation coupled to Tandem mass spectrometry, and validated by surface plasmon resonance and biolayer interferometry. The spatiotemporal dynamics of FleA and candidate partners were analyzed by confocal microscopy and proximity ligation assay. We demonstrated that BEC antifungal activity involves at least two complementary pathways: a PI3K/laminin-332 axis promoting conidial adhesion, and a FleA-dependent pathway engaging ITGB1 and MRC2 consistent with lectin uptake and trafficking toward LAMP1-positive compartments. These findings nominate FleA-host receptor interactions as attractive targets for anti-adhesive strategies against A. fumigatus.

The rise of multidrug-resistant tuberculosis (TB) has increased the need for new antitubercular (anti-TB) drugs and the identification of novel drug targets. One promising target is Mycobacterium tuberculosis (Mtb) cytochrome P450 enzymes (P450s). This study focuses on the characterization of CYP135B1, a prevalent Mtb P450. Using a combination of microbiology, genomics, bioinformatics, docking, spectroscopy, and mass spectrometry, researchers successfully expressed, purified, and characterized CYP135B1. A 3D model was built with AlphaFold 3. The enzyme displayed typical features of P450 proteins and showed strong binding to imidazole derivatives. Notably, CYP135B1 metabolized the anti-TB drug SQ109 by inserting oxygen into its geranyl moiety in a manner distinct from CYP124A1. However, genetic studies using a ΔCYP135B1 mutant strain revealed that CYP135B1 is not required for SQ109's antibacterial activity, as its deletion did not affect drug efficacy despite CYP135B1 metabolizes SQ109.

This study aimed to identify dysregulated proteins in Schirmer strip samples (ScS) from Sjögren's syndrome dry eye (SSDE) patients to uncover key biological processes using untargeted proteomics. The focus then shifted to two dysregulated proteins, PRDX6 and CXCL17, due to their roles in oxidative stress and mucosal immunity, with the goal of exploring their potential as therapeutic targets. Their involvement was further investigated in vitro and validated in a larger SSDE cohort, assessing their relationship with clinical signs. ScS from 12 SSDE patients and 6 healthy controls underwent untargeted proteomic analysis. PRDX6 and CXCL17 levels in ScS from 39 SSDE patients were quantified using ELISA. In vitro, human corneal epithelial cells (HCEc) were exposed to hyperosmolarity or IFN-γ, and PRDX6 and CXCL17 expression was assessed by RT-qPCR for gene expression and by ELISA and immunocytochemistry for protein expression. Untargeted proteomics identified 111 dysregulated proteins in SSDE, highlighting alterations in oxidative stress, cell metabolism, cytoskeleton organization, and programmed cell death. Targeted proteomics showed positive correlations between PRDX6 levels and TBUT/Schirmer tests, and negative correlations with OSDI/Oxford scores. CXCL17 levels negatively correlated with the Oxford score. In vitro, PRDX6 and CXCL17 expression increased under hyperosmotic or inflammatory stress, displaying inverse trends compared to ScS from SSDE patients. This study elucidates the biological processes driving epithelial cell alterations in SSDE, focusing on oxidative stress and mucosal homeostasis. It underscores the significant roles of PRDX6 and CXCL17 in these processes, suggesting their potential as biomarkers or therapeutic targets for SSDE.

LMNA gene mutations are responsible for a wide spectrum of disorders called laminopathies, the majority of which affecting striated muscles. Among them, Emery-Dreifuss muscular dystrophy (EDMD) and limb-girdle muscular type 1B (LGMD1B) show skeletal muscle involvement of different severity but share the same cardiac involvement, i.e., dilated cardiomyopathy with conduction system disease (DCM-CD) that can also be present in an isolated manner. Clinical heterogeneity is well known among the LMNA mutation carriers. Modifier genes have been suggested to explain such variability. The LMNA mutation (p.Gln6*), identified in a large French family (named here EMD1), is associated with a wide range of age at onset of myopathic symptoms (AOMS). According to this latter, three phenotypic subgroups have been described within the family: AOMS before 20 years (early AOMS), AOMS after 30 years (late AOMS) and isolated cardiac disease without musculo-skeletal symptoms. Our objective was to identify genetic modifiers underlying the intrafamilial phenotypic variability within EMD1 family. Whole genome sequencing (WGS) was performed in 16 LMNA-mutation carriers exhibiting the 3 phenotypic subgroups in EMD1 family. Among the 12 million variants annotated, 2 splice variants with a potential aggravating effect and 1 intronic variant with a potential protective effect have been identified and are currently under functional validation. Moreover, 4 structural variants have been detected only in early AOMS patients. An identity by descent analysis specific to phenotypic subgroups was performed and identified one region shared on chromosome 1, containing the LMNA gene. Our results suggest that a single genetic modifier may not be solely responsible for phenotypic variability in this family, but that a combination of several factors is more likely.

In the central nervous system, the formation of myelin by oligodendrocytes (OLs) relies on the switch from the polymerization of the actin cytoskeleton to its depolymerization. The molecular mechanisms that trigger this switch have yet to be elucidated. Here, we identified P21-activated kinase 1 (PAK1) as a major regulator of actin depolymerization in OLs. Our results demonstrate that PAK1 accumulates in OLs in a kinase-inhibited form, triggering actin disassembly and, consequently, myelin membrane expansion. Remarkably, proteomic analysis of PAK1 binding partners enabled the identification of NF2/Merlin as its endogenous inhibitor. Our findings indicate that Nf2 knockdown in OLs results in PAK1 activation, actin polymerization, and a reduction in OL myelin membrane expansion. This effect is rescued by treatment with a PAK1 inhibitor. We also provide evidence that the specific Pak1 loss-of-function in oligodendroglia stimulates the thickening of myelin sheaths in vivo. Overall, our data indicate that the antagonistic actions of PAK1 and NF2/Merlin on the actin cytoskeleton of the OLs are critical for proper myelin formation. These findings have broad mechanistic and therapeutic implications in demyelinating diseases and neurodevelopmental disorders.

IntroductionLes pathologies pulmonaires distales telles que la fibrose pulmonaire ou les lésions pulmonaires aiguës sont souvent associées à une hypoxie alvéolaire locale. Nous avons précédemment montré que l’exposition des cellules épithéliales alvéolaires de rat de type 2 (CEA2) à l’hypoxie stimule l’apoptose ou la transition épithélio-mésenchymateuse et peut aggraver ces lésions pulmonaires. De plus, la culture avec des cellules souches mésenchymateuses humaines (CSMh) ou leur milieu conditionné (mc-CSMh) protège les CEA2 de rat des effets délétères de l’hypoxie. Cependant, les facteurs paracrines et les cascades de signalisation précises impliquées dans les effets anti-apoptotique, anti-oxydant et anti-fibrotique des CSMh restent à élucider.MéthodesAfin de caractériser les effets protecteurs paracrines des CSMh sur les CEA2 hypoxiques, nous avons comparé le protéome des CEA2 de rat soumises ou non à l’hypoxie et cultivées en présence ou non de mc-CSMh via une approche protéomique « label free ».RésultatsPlusieurs milliers de protéines ont été quantifiées. Bien qu’une majorité de protéines modulées par l’hypoxie soient impliquées dans le métabolisme, illustrant le shift métabolique des cellules hypoxiques (effet Warburg), un pool important de protéines régule la mort cellulaire et l’équilibre redox, mais aussi l’inflammation ou la fibrose. Les résultats de cette étude confirment les effets délétères de l’hypoxie, caractérisés à la fois par une diminution des protéines favorisant la survie cellulaire et par une augmentation des protéines pro-oxydantes, pro-apoptotiques, pro-inflammatoires et pro-fibrotiques. Enfin, les données obtenues indiquent que les effets cytoprotecteurs du mc-CSMh sont associés à une diminution des protéines pro-apoptotiques, pro-inflammatoires et pro-fibrotiques, et à un enrichissement en facteurs anti-apoptotiques et anti-oxydants, contrecarrant ainsi les effets les plus délétères de l’hypoxie.ConclusionCette étude fournit de nouvelles pistes pour comprendre l’effet cytoprotecteur paracrine des CSMh sur les CEA2 hypoxiques de rat. Une meilleure compréhension des mécanismes moléculaires sous-jacents à l’effet anti-apoptotique, anti-oxydant et anti-fibrotique des CSMh in vitro est une étape importante et nécessaire pour envisager par la suite des thérapies ciblées chez l’homme, et représente un enjeu thérapeutique majeur.

In the central nervous system (CNS), myelin formation by oligodendrocytes (OLs) relies on actin dynamics. Actin polymerization supports the ensheathment step, when the OL process contacts the axon, while a drastic shift to actin depolymerization is required to enable the following step of wrapping and expansion of myelin membranes. The molecular mechanisms triggering this switch, essential for proper myelination, have yet to be elucidated. Here, we identify P21-activated kinase 1 (PAK1) as a major regulator of actin depolymerization in OLs. We show that PAK1 accumulates in OLs in a kinase inhibited form, triggering actin disassembly and, consequently, myelin expansion. Remarkably, we identify NF2/Merlin as an endogenous inhibitor of PAK1 by proteomics analysis of its binding partners. We found that Nf2 knockdown in OLs results in PAK1 activation and impairs myelin formation, and that pharmacological inhibition of PAK1 in Nf2 -knockdown OLs rescues these defects. Moreover, we demonstrate that modulating PAK1 activity in OLs controls myelin expansion and provide compelling evidence indicating that specific Pak1 loss-of-function in oligodendroglia stimulates the thickening of myelin sheaths in vivo . Overall, our data indicate that PAK1-NF2/Merlin duo plays a key role in actin cytoskeleton remodeling in OLs, required for proper myelin formation. These findings have broad mechanistic and therapeutic implications for demyelinating diseases and neurodevelopmental disorders. Significance Remodeling actin cytoskeleton plays a crucial role in myelin formation by oligodendrocytes (OLs). Recent studies have shown that expansion and wrapping of myelin membranes around axons depends on actin depolymerization. However, the molecular mechanisms triggering this key step in myelination are not fully elucidated. Using genetic and pharmacological tools as well as proteomics analyses, we found that PAK1 (P21 Activated Kinase 1) kinase activity is maintained inhibited by NF2/Merlin in OLs to allow actin depolymerization and, consequently, myelin membrane expansion. Pak1 loss-of-function in OLs leads to an increase in myelin thickness in the white matter of adult mice, confirming the role of PAK1 inactivation in myelin membrane expansion.

The tear film forms a protective barrier between the ocular surface and the external environment. Despite its small volume, recent advancements in preanalytical and analytical procedures have enabled its in-depth analysis using multiple approaches. However, the diversity of tear film collection methods and the lack of standardization in pre-analytical methods represent the main obstacles to reproducible results and comparison among different studies. In this study, we first improved the pre-analytical procedures for the extraction of various molecular entities from Schirmer strips (ScS). Subsequently, our investigation focused on analyzing the molecular variances that might occur between two primary tear collection methods: capillary tube (CT) and ScS. Additionally, we examined different parts of the ScS to underscore these variations, which could serve as crucial factors for developing a standardized, optimized protocol for sample processing. Our results show that the inclusion of surfactants in the extraction process enhanced both the yield of protein extraction and the number of proteins identified in ScS, by effectively lysing the cells and improving the solubility of several intracellular proteins. In addition to proteins, nucleic acids could also be recovered for gene expression analyses, particularly from the bulb region of the ScS which is placed in the cul-de-sac. Despite their diluted nature, extracts from ScS remain a suitable material for retrieving tear proteins such as IL-17A at levels as low as the fg/mL range, thanks to highly sensitive immunoassays. Collection methods can affect measured tear protein levels. Lactoferrin is found in higher percentages in capillary electrophoresis analysis of tears collected using ScS compared to tears collected by CT (39.6 ± 4.8% versus 31 ± 4.4%).

The ocular surface (OS) enzymes are of great interest due to their potential for novel ocular drug development. We aimed first to profile and classify the enzymes of the OS to describe major biological processes and pathways that are involved in the maintenance of homeostasis. Second, we aimed to compare the enzymatic profiles between the two most common tear collection methods, capillary tubes (CT) and Schirmer strips (ScS). A comprehensive tear proteomic dataset was generated by pooling all enzymes identified from nine tear proteomic analyses of healthy subjects using mass spectrometry. In these studies, tear fluid was collected using CT (n = 4), ScS (n = 4) or both collection methods (n = 1). Classification and functional analysis of the enzymes was performed using a combination of bioinformatic tools. The dataset generated identified 1010 enzymes. The most representative classes were hydrolases (EC 3) and transferases (EC 2). Phosphotransferases, esterases and peptidases were the most represented subclasses. A large portion of the identified enzymes was common to both collection methods (n = 499). More enzymes were specifically detected in the ScS-extracted proteome. The major pathways in which the identified enzymes participate are related to the immune system and protein, carbohydrate and lipid metabolism. Metabolic processes for nucleosides, cellular amides, sugars and sulfur compounds constituted the most enriched biological processes. Knowledge of these molecules highly susceptible to pharmacological manipulation might help to predict the metabolism of ophthalmic medications and develop novel prodrug strategies as well as new drug delivery systems. Combining such extensive knowledge of the OS enzymes with new analytical approaches and techniques might create new prospects for understanding, predicting and manipulating the metabolism of ocular pharmaceuticals. Our study reports new, essential data on OS enzymes while also comparing the enzyme profiles obtained via the two most popular methods of tear collection, capillary tubes and Schirmer strips.

While low concentrations of high-density lipoprotein-cholesterol (HDL-C) are widely accepted as an independent cardiovascular risk factor, HDL-C-rising therapies largely failed, suggesting the importance of both HDL functions and individual subspecies. Indeed HDL particles are highly heterogeneous, with small, dense pre-beta-HDLs being considered highly biologically active but remaining poorly studied, largely reflecting difficulties for their purification. We developed an original experimental approach allowing the isolation of sufficient amounts of human pre-beta-HDLs and revealing the specificity of their proteomic and lipidomic profiles and biological activities. Pre-beta-HDLs were enriched in highly poly-unsaturated species of phosphatidic acid and phosphatidylserine, and in an unexpectedly high number of proteins implicated in the inflammatory response, including serum paraoxonase/arylesterase-1, vitronectin and clusterin, as well as in complement regulation and immunity, including haptoglobin-related protein, complement proteins and those of the immunoglobulin class. Interestingly, amongst proteins associated with lipid metabolism, phospholipid transfer protein, cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase were strongly enriched in, or restricted to, pre-beta-HDL. Furthermore, pre-beta-HDL potently mediated cellular cholesterol efflux and displayed strong anti-inflammatory activities. A correlational network analysis between lipidome, proteome and biological activities highlighted 15 individual lipid and protein components of pre-beta-HDL relevant to cardiovascular disease, which may constitute novel diagnostic targets in a pathological context of altered lipoprotein metabolism.

Objectives Inflammatory bowel disease (IBD) results from a combination of genetic predisposition, dysbiosis of the gut microbiota and environmental factors, leading to alterations in the gastrointestinal immune response and chronic inflammation. Caspase recruitment domain 9 (Card9), one of the IBD susceptibility genes, has been shown to protect against intestinal inflammation and fungal infection. However, the cell types and mechanisms involved in the CARD9 protective role against inflammation remain unknown. Design We used dextran sulfate sodium (DSS)-induced and adoptive transfer colitis models in total and conditional CARD9 knock-out mice to uncover which cell types play a role in the CARD9 protective phenotype. The impact of Card9 deletion on neutrophil function was assessed by an in vivo model of fungal infection and various functional assays, including endpoint dilution assay, apoptosis assay by flow cytometry, proteomics and real-time bioenergetic profile analysis (Seahorse). Results Lymphocytes are not intrinsically involved in the CARD9 protective role against colitis. CARD9 expression in neutrophils, but not in epithelial or CD11c+cells, protects against DSS-induced colitis. In the absence of CARD9, mitochondrial dysfunction increases mitochondrial reactive oxygen species production leading to the premature death of neutrophilsthrough apoptosis, especially in oxidative environment. The decreased functional neutrophils in tissues might explain the impaired containment of fungi and increased susceptibility to intestinal inflammation. Conclusion These results provide new insight into the role of CARD9 in neutrophil mitochondrial function and its involvement in intestinal inflammation, paving the way for new therapeutic strategies targeting neutrophils.

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.

Aspergillus fumigatus est un champignon opportuniste présent dans l’environnement. Il se propage sous forme de spores qui une fois inhalées peuvent pénétrer dans le tractus respiratoire où elles sont normalement éliminées par les défenses immunitaires innées. Chez les personnes souffrant de déficit immunitaire, les spores peuvent germer et former des filaments. Nous avons montré que les cellules épithéliales bronchiques, premières cellules du tractus respiratoire à entrer en contact avec les pathogènes, reconnaissent la lectine FleA des spores, et ainsi inhibent la formation des filaments (Richard et al., 2018). Dans la présente étude, nous avons recherché les récepteurs des cellules épithéliales bronchiques capables de reconnaître la lectine FleA, encore inconnus à l’heure actuelle. Les cellules épithéliales bronchiques (lignée BEAS-2B) incubées avec la lectine FleA biotinylée ont été lysées afin d’identifier, par immunoprécipitation, les complexes protéiques interagissant avec elle. Ces complexes ont été analysés par spectrométrie de masse MALDI-TOF. P ouridentifier le rôle de ces protéines dans l’activité antifongique, les cellules ont été transfectées avec des siARN des protéines identifiées et infectées par A. fumigatus. L’immunoprécipitation a permis d’identifier plusieurs protéines se liant à la lectine FleA, telles que les intégrines α3 (ITGA3) et β1 (ITGB1), le C-type mannose receptor 2 (MRC2) et le lysosome-associated membrane glycoprotein 1 (LAMP1). En utilisant des siARN spécifiques, nous avons mis en évidence que les intégrines ITGA3 et ITGB1 étaient impliquées dans l’activité antifongique des cellules. Les cellules épithéliales bronchiques reconnaissent la lectine FleA des spores d’A. fumigatus ce qui entraine une réponse antifongique. Cette réponse est altérée lorsque l’expression des intégrines α3β1 est inhibée. L’interaction directe de ces intégrines avec la lectine du champignon reste à confirmer.

To investigate changes in the tear proteome of patients with Sjögren's syndrome (SSp) using a high-resolution trapped ion mobility spectrometry (TIMS) powered with parallel accumulation–serial fragmentation (PASEF). Tear samples were collected using Schirmer strips from healthy controls (HC) and SSp, then extracted in ammonium bicarbonate. The protein content of each sample was normalized to 250 ng. Proteomics analysis was performed by using TIMS coupled quadrupole time-of-flight (timsTOF Pro) followed by data processing using MaxQuant software for protein identification. Differentially expressed proteins were detected by a Limma statistical test with a false discovery rate (FDR)˂1%. Protein Gene Ontology classification was performed by using Panther. The proteins identified decreased by 23.9% in SSp compared to HC. Off the proteins identified in SSp, 90% were common with HC while only 68% of the proteins identified in HC were detected in SSp. A total of 175 proteins were significantly modulated in SSp with a fold-change ≥ 1.5 (p-value ≤0.05). None of the proteins detected only in SSp was among significantly modulated proteins but 25 proteins identified only in HC were significantly modulated. Catalytic activity (46.9%) and binding proteins (38.5%) formed the major molecular function groups among significantly modulated proteins. Proteins that exhibit binding activity were mainly down-regulated in SSp while proteins involved in apoptosis such as caspase-3 and proteasome subunits were up-regulated. Several oxidoreductase enzymes (18) such as sulfhydryl oxidase-1, Glutathione peroxidase-3 and lactoperoxidase have been down-regulated. Actins, actin-binding proteins, tubulins, microtubule-binding proteins were decreased in SSp. These results highlight an alteration in cellular process and antioxidase activity. A large amount of proteins expressed differently in SSp that involve important biological processes and signalling pathways have been revealed. The improved spatial resolution, sensitivity, speed, and specificity of timsTOF Pro can dramatically increase the ability to fully investigate the molecular factors behind the pathophysiology of SS.

AstractPurposeTo analyze and compare protein composition in different parts of the Schirmer strips (called the bulb and the rest of the strip) by using a comprehensive proteomics approach based on highly sensitive trapped ion‐mobility spectrometry coupled quadrupole time‐of‐flight (timsTOF Pro).MethodsSchirmer strips were collected from healthy subjects on 4 different visits over two consecutive days to rationally prepare three groups: 4 whole strips, 4 bulbs and 4 rests of the strips. Each group was extracted in ammonium bicarbonate before analysis with nanoElute UHPLC (ultra‐high‐pressure liquid chromatography) coupled timsTOF Pro. Protein Gene Ontology classification was performed by using Panther. Mass spectrometry data were processed using MaxQuant for protein identification.ResultsThe number of identified proteins increased by 49.6% when the bulbs are separated from the rests of the strips, each group being processed independently. Most of the identified proteins (%50.5) were common in the bulbs and the rests, 26.3% identified only in the bulbs and 23.2% only in the rests. Among all, 1650 proteins were identified from three groups, binding (44%) and catalytic activities (38.3%) constituted the main groups of molecular functions. The cellular (31.3%), metabolic (20.9%), biological regulation processes (13%) formed the main classes in the biological processes of identified proteins. A high number of enzymes (480), formed of hydrolases (47.5%), oxidoreductases (22.1%), transferases (16.7%), ligases (10%), isomerases, (2.1%) and lyases (1.7%) were identified in tear proteome.ConclusionsThe separate study of the bulbs and the rests identified several proteins that are not found in the entire strip alone. Probably, treating bulbs and rests apart allowed to elute more proteins from each part. A decrease in the concentration of abundant tear proteins might have enabled the identification of less abundant ones too. This methodology could improve the pre‐analytical steps before MS analysis. The dataset created can help to model and compare multiple signaling pathways associated with ocular surface pathologies. Moreover, the TimsTOF Pro could also add a technical improvement for the investigation of biomarkers in ocular diseases.

CD4+ T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC‐II molecules. The pathways leading to endogenous MHC‐II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy‐dependent and ‐independent endogenous presentation of HIV‐ and HCMV‐derived peptides. By studying the immunopeptidome of MHC‐II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC‐II‐loading compartments and rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC‐II molecules. Defining the interactome of T6BP, we identify calnexin as a T6BP partner. We show that the calnexin cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4+ T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC‐II‐restricted endogenous presentation pathway, and we propose one potential mechanism of action. The autophagy receptor T6BP (TAX1BP1) stabilizes the invariant chain (CD74) and regulates MHC‐II trafficking, thereby favoring the presentation of high‐affinity peptides to virus‐specific CD4+ T cells. The autophagy receptor T6BP (TAX1BP1) stabilises the invariant chain (CD74) and regulates MHC‐II trafficking, thereby favouring the presentation of high affinity peptides to virus‐specific CD4+ T cells.

CD4(+) T lymphocytes play a major role in the establishment and maintenance of immunity. They are activated by antigenic peptides derived from extracellular or newly synthesized (endogenous) proteins presented by the MHC-II molecules. The pathways leading to endogenous MHC-II presentation remain poorly characterized. We demonstrate here that the autophagy receptor, T6BP, influences both autophagy-dependent and -independent endogenous presentation of HIV- and HCMV-derived peptides. By studying the immunopeptidome of MHC-II molecules, we show that T6BP affects both the quantity and quality of peptides presented. T6BP silencing induces the mislocalization of the MHC-II-loading compartments and rapid degradation of the invariant chain (CD74) without altering the expression and internalization kinetics of MHC-II molecules. Defining the interactome of T6BP, we identify calnexin as a T6BP partner. We show that the calnexin cytosolic tail is required for this interaction. Remarkably, calnexin silencing replicates the functional consequences of T6BP silencing: decreased CD4(+) T cell activation and exacerbated CD74 degradation. Altogether, we unravel T6BP as a key player of the MHC-II-restricted endogenous presentation pathway, and we propose one potential mechanism of action.

This study aimed to investigate the human proteome profile of samples collected from whole (W) Schirmer strips (ScS) and their two parts—the bulb (B) and the rest of the strip (R)—with a comprehensive proteomic approach using a trapped ion mobility mass spectrometer, the timsTOF Pro. Eight ScS were collected from two healthy subjects at four different visits to be separated into three batches, i.e., 4W, 4B, and 4R. In total, 1582 proteins were identified in the W, B, and R batches. Among all identified proteins, binding proteins (43.4%) and those with catalytic activity (42.2%) constituted more than 80% of the molecular functions. The most represented biological processes were cellular processes (31.2%), metabolic processes (20.8%), and biological regulation (13.1%). Enzymes were the most represented protein class (41%), consisting mainly of hydrolases (47.5%), oxidoreductases (22.1%), and transferases (16.7%). The bulb (B), which is in contact with the conjunctiva, might collect both tear and cell proteins and therefore promote the identification of more proteins. Processing B and R separately before mass spectrometry (MS) analysis, combined with the high data acquisition speed and the addition of ion-mobility-based separation in the timsTOF Pro, can bring a new dimension to biomarker investigations of a limited sample such as tear fluid.