Biology subjects

Barra, L.

Publications and source records attributed to Barra, L..

5 recordsLinked to original sources

bioRxiv2026

S-Adenosyl-D-methionine as a Non-Physiological Substrate for a Wide Range of SAM-Dependent Enzymes

The ability of SAM-dependent enzymes to accept S-adenosyl-D-methionine [D-SAM, (SS,RC)-SAM] instead of the native cofactor S-adenosyl-L-methionine [L-SAM, (SS,SC)-SAM] remains largely unexplored. Challenging the stereochemical preference of SAM-dependent enzymes, we investigated the ability of different enzyme classes to accept D-SAM. Contrary to common assumptions, the tested N- and O-methyl transferases (MTs), as well as one of the examined C-MTs accepted D-SAM. Docking studies suggest that acceptance of D-SAM by C-MTs may be influenced by the angle between the transferable methyl group of SAM and the nucleophilic carbon of the substrate, along with enzyme and substrate flexibility. In addition to conventional MTs, the radical SAM glutamine C-MT QCMT showed low but detectable methylation activity with D-SAM. Furthermore, the azetidine-2-carboxylic acid synthase AzeJ not only uses D-SAM but also incorporates the stereocentre of D-methionine into the cyclic amino acid product. The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropyl-1-carboxylic acid synthase (ACCS) also showed detectable turnover with D-SAM. These findings broaden the understanding of enzyme stereoselectivity, provide an overview of D-SAM-utilising enzymes, and identify first enzyme systems that may serve as starting points for engineering efforts aimed at shifting cofactor preference towards D-SAM.

biochemistry↗

bioRxiv2025

From genomic decay to functional advantage: Trait-based evidence for long-term host residency of a next-generation coral probiotic

A key bottleneck in microbiome engineering is ensuring the long-term host association of introduced microbes. Selecting probiotic candidates based on evolutionary genomic decay signatures of emerging host dependency offers a potential solution. The Ruegeria strain B4 of the population MC10, identified by such signatures, has shown persistent colonization in corals in a companion study. Whether this persistence translates into a measurable host benefit compared to other coral-associated Ruegeria strains, and which mechanisms underlie such benefit, remained unknown. Here we directly compare the probiotic efficacy of MC10-B4 against two sympatric Ruegeria strains isolated from the same coral colony and mucus compartment, controlling for host genotype and microenvironment. MC10-B4 inoculation significantly increased heat stress tolerance in the coral model cnidarian Aiptasia (Exaiptasia diaphana strain H2), outperforming both sympatric control strains. To understand the mechanistic basis of this probiotic efficacy, we characterized the functional profile of MC10 using an integrated multi-omics approach. The genome of MC10 is enriched in genes associated with host-interaction, including siderophore-mediated iron acquisition and exopolysaccharide biosynthesis, which were phenotypically confirmed by detectable iron scavenging and enhanced biofilm formation. Following exposure to coral tissue extract, MC10-B4 underwent a coordinated motility-to-sessility proteomic reprogramming, downregulating flagellar motor components while upregulating flagellin and biofilm regulators. This response was distinct from its sympatric relatives, which instead mounted broad upregulation of nutrient acquisition systems. MC10-B4s functional profile, particularly its sensitivity to oxidative stress, contrasts with the traits typically favored in conventional probiotic screens. Our results move beyond the descriptive identification of a coral probiotic by providing initial mechanistic insight into traits associated with long-term host association and improved thermal performance. These findings validate an evolution-guided approach that prioritizes innate colonization potential over pre-defined laboratory functionalities, informing the rational design of durable probiotics.

microbiology↗

bioRxiv2025

Homocitrullinated Peptides Drive Pro-Inflammatory T-Cell Responses in a Humanized Mouse Model of Rheumatoid Arthritis

ObjectiveAnti-homocitrullinated protein/peptide antibodies (AHCPA) are specific to rheumatoid arthritis (RA) and predictive of worse prognosis, suggesting a pathogenic role for autoreactivity to homocitrullinated antigens. However, T-cell responses to homocitrullinated peptides remain largely unexplored. We investigated these responses in a humanized HLA-DR4-transgenic (DR4tg) mouse model of RA, which expresses the strongest genetic risk factor for this disease. MethodsDR4tg mice were injected subcutaneously with a homocitrullinated peptide called HomoCitJED while control mice received phosphate-buffered saline. After 10 days, T-cells were analyzed for their phenotypic characteristics, cytokine production, and proliferative capacities in the draining lymph nodes (dLNs) and spleens by flow cytometry, enzyme-linked immunosorbent assays, and ProQuantum immunoassays. ResultsHomoCitJED immunization drove robust expansion of T helper (Th) 1, Th17 and hybrid Th1/Th17 CD4+ T cells in dLNs, alongside elevated CD25 activation marker within these subsets. Intracellular cytokine staining confirmed effector activity, revealing higher frequencies of IL-17A+, TNF-+IL-17A+, and IFN-{gamma}+IL-17A+ CD4+ T cells. CD4+ T cells from dLNs also up-regulated the exhaustion markers LAG-3 and Tim-3. CD8+ T cells (Tc) mirrored these findings, as HomoCitJED immunization augmented CD25 and KLRG1 expression, Tc1/Tc17-type IL-17A and IFN-{gamma}/IL-17A production, and antigen-specific proliferation. Additionally, Tim-3, LAG-3 and PD-1 expression on these subsets was augmented. ConclusionsA homocitrullinated peptide elicited Th1/Th17 and Tc1/Tc17 responses marked by concurrent activation and exhaustion signatures, pointing to a dysregulated T-cell state. These findings position homocitrulline-driven T-cell imbalance across both CD4+ and CD8+ T-cell compartments as a potential mechanistic contributor to RA pathogenesis and a target for future immunomodulatory therapies.

immunology↗

bioRxiv2025

Immune Responses Targeting Homocitrullinated Peptides Drive Rheumatoid Arthritis-like Pathology in HLA-DR4 Transgenic Mice

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by persistent synovial inflammation and progressive joint destruction. A hallmark feature of RA is the presence of anti-citrullinated protein autoantibodies (ACPAs), which have been extensively studied in disease pathogenesis. The development of these autoantibodies is strongly associated with the expression of HLA-DR4, containing the shared epitope. More recently, anti-homocitrullinated protein autoantibodies (AHCPAs) have also been implicated in RA and demonstrate a similar genetic association. Therefore, this study aimed to evaluate the role of homocitrulline immune responses in RA pathogenesis. HLA-DR4 transgenic (DR4tg) and B6 mice were immunized with a synthetic homocitrullinated peptide (HomoCitJED) or PBS followed by a booster 21 days later. Beginning on day 75 post-primary immunization mice received knee intra-articular (i.a.) injections of HomoCitJED once a week for three consecutive weeks. Swelling post i.a. injection was measured using digital calipers. IgG antibodies against homocitrullinated peptides/proteins were measured using enzyme-linked immunosorbent assay (ELISA). Knees were sectioned and analyzed for histopathological damage and citrullinated/homocitrullinated protein levels. HomoCitJED immunized DR4tg mice exhibited significantly greater swelling and incidence of anti-HomoCitJED and -homocitrullinated fibrinogen IgG autoantibodies. Additionally, 30% had responses against CitJED, a synthetic citrullinated peptide. HomoCitJED immunized DR4tg mice also experienced greater total histopathological damage; however, the immunizations/injections did not affect citrullinated/homocitrullinated protein levels. In conclusion, homocitrulline-specific immune responses can induce RA-like disease in DR4tg mice, supporting the pathogenic role of AHCPAs in RA.

immunology↗

bioRxiv2019

GATA2 Expression by Intima-Infiltrating Macrophages Drives Early Atheroma Formation

Aberrant macrophage polarization is a major contributor to the onset and progression of atherosclerosis. Despite this, macrophage polarization during in early stages of human atherosclerotic disease is poorly understood. Using transcriptomic analysis of macrophages recovered from early-stage human atherosclerotic lesions, we have identified a unique gene expression profile dissimilar to that observed in later stages of disease that is characterized by upregulation of the hematopoietic transcription factor GATA2. GATA2 overexpression in vitro recapitulated defects observed in patient macrophages, including deficiencies in the uptake and processing of apoptotic cells, and in the catalysis of atherogenic protein modifications, with GATA2 knockdown abrogating these defects. Our data describe a previously unreported macrophage differentiation state present in early atheroma formation and identifies GATA2 as a driver of macrophage functional defects during the early stages of atherosclerosis in humans.

immunology↗