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BACKGROUND. Autoimmune diseases often have strong genetic associations with specific HLA-DR alleles. The synovial lesion in chronic inflammatory forms of arthritis shows marked upregulation of HLA-DR molecules, including in postinfectious Lyme arthritis (LA). However, the identity of HLA-DR-presented peptides, and therefore the reasons for these associations, has frequently remained elusive. METHODS. Using immunopeptidomics to detect HLA-DR-presented peptides from synovial tissue, we identified T cell epitopes from 3 extracellular matrix (ECM) proteins in patients with postinfectious LA, identified potential Borreliella burgdorferi-m\mk (Sb-mimic) epitopes, and characterized T and B cell responses to these peptides or proteins. RESULTS. Of 24 postinfectious LA patients, 58% had CD4*Tcell responses to at least 1 epitope of 3 ECM proteins, fibronectin-1, laminin B2, and/or collagen V[alpha]1, and 17% of 52 such patients had antibody responses to at least 1 of these proteins. Patients with autoreactive T cell responses had significantly increased frequencies of HLA-DRB1*04 or -DRB1*1501 alleles and more prolonged arthritis. When tetramer reagents were loaded with ECM or corresponding Sb-mimic peptides, binding was only with the autoreactive T cells. A high percentage of ECM-autoreactive [CD4.sup.+] T cells in synovial fluid were T-bet- expressing Th1 cells, a small percentage were RoR[gamma]t-expressingThl? cells, and a minimal percentage were FoxP3-expressingTregs. CONCLUSION. Autoreactive, proinflammatory CD4* T cells and autoantibodies develop to ECM proteins in a subgroup of postinfectious LA patients who have specific HLA-DR alleles. Ratherthan the traditional molecular mimicry model, we propose that epitope spreading provides the best explanation for this example of infection-induced autoimmunity. FUNDING. Supported by National Institute of Allergy and Infectious Diseases R01- AI101175, R01-AI144365, and F32- AI125764; National Institute of Arthritis and Musculoskeletal and Skin Diseases K01- AR062098 and T32-AR007258; NIH grants P41GM104603, R24-GM134210, S10-RR020946, S10-OD010724, S10-OD021651, and S10-OD021728; and the G. Harold and Leila Y. Mathers Foundation, the Eshe Fund, and the Lyme Disease and Arthritis Research Fund at Massachusetts General Hospital., Introduction Lyme arthritis (LA), which is caused by infection with the tick-borne spirochete Borreliella (Borrelia) burgdorferi (Bb), is typically manifested by swelling and pain in a few large joints, especially [...]

The cellular isoform of the prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and pathological functions of PrP depend on intramolecular interactions between these two domains, but the specific amino acid residues involved have proven challenging to define. Here, we employ a combination of chemical cross-linking, mass spectrometry, NMR, molecular dynamics simulations, and functional assays to identify residue-level contacts between the N- and C-terminal domains of PrPC. We also determine how these interdomain contacts are altered by binding of Cu2+ ions and by functionally relevant mutations. Our results provide a structural basis for interpreting both the normal and toxic activities of PrP.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Mitochondrial ribosomes of Trypanosoma brucei are composed of 9S and 12S rRNAs, eubacterial-type ribosomal proteins, polypeptides lacking discernible motifs and approximately 20 pentatricopeptide repeat (PPR) RNA binding proteins. Several PPRs also populate the polyadenylation complex; among these, KPAF1 and KPAF2 function as general mRNA 3' adenylation/uridylation factors. The A/U-tail enables mRNA binding to the small ribosomal subunit and is essential for translation. The presence of A/U-tail also correlates with requirement for translation of certain mRNAs in mammalian and insect parasite stages. Here, we inquired whether additional PPRs activate translation of individual mRNAs. Proteomic analysis identified KRIPP1 and KRIPP8 as components of the small ribosomal subunit in mammalian and insect forms, but also revealed their association with the polyadenylation complex in the latter. RNAi knockdowns demonstrated essential functions of KRIPP1 and KRIPP8 in the actively respiring insect stage, but not in the mammalian stage. In the KRIPP1 knockdown, A/U-tailed mRNA encoding cytochrome c oxidase subunit 1 declined concomitantly with the de novo synthesis of this subunit whereas polyadenylation and translation of cyb mRNA were unaffected. In contrast, the KRIPP8 knockdown inhibited A/U-tailing and translation of both CO1 and cyb mRNAs. Our findings indicate that ribosome-associated PPRs may selectively activate mRNAs for translation.

Small, noncoding RNA biogenesis typically involves cleavage of structured precursor by RNase III-like endonucleases. However, guide RNAs (gRNAs) that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5' triphosphate characteristic of the transcription initiation and possess a U-tail indicative of 3' processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase, DSS1 3'-5' exonuclease, and three additional subunits. This complex, termed mitochondrial 3' processome (MPsome), is responsible for primary uridylation of ∼800 nt gRNA precursors, their processive degradation to a mature size of 40-60 nt, and secondary U-tail addition. Both strands of the gRNA gene are transcribed into sense and antisense precursors of similar lengths. Head-to-head hybridization of these transcripts blocks symmetrical 3'-5' degradation at a fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5' extremity of a primary molecule by uridylation-induced, antisense transcription-controlled 3'-5' exonucleolytic degradation.

In rheumatoid arthritis (RA), immunological triggers at mucosal sites, such as the gut microbiota, may promote autoimmunity that affects joints. Here, we used discovery-based proteomics to detect HLA-DR-presented peptides in synovia or peripheral blood mononuclear cells and identified 2 autoantigens, N-acetylglucosamine-6-sulfatase (GNS) and filamin A (FLNA), as targets of T and B cell responses in 52% and 56% of RA patients, respectively. Both GNS and FLNA were highly expressed in synovia. GNS appeared to be citrullinated, and GNS antibody values correlated with anti -citrullinated protein antibody (ACPA) levels. FLNA did not show the same results. The HLA-DR-presented GNS peptide has marked sequence homology with epitopes from sulfatase proteins of the Prevotella sp. and Parabacteroides sp., whereas the HLA-DRpresented FLNA peptide has homology with epitopes from proteins of the Prevotella sp. and Butyricimonas sp., another gut commensal. Patients with T cell reactivity with each self-peptide also had responses to the corresponding microbial peptides, and the levels were directly correlated. Furthermore, HLA-DR molecules encoded by shared-epitope (SE) alleles were predicted to bind these self- and microbial peptides strongly, and these responses were more common in RA patients with SE alleles. Thus, sequence homology between T cell epitopes of 2 self-proteins and a related order of gut microbes may provide a link between mucosal and joint immunity in patients with RA., Introduction Rheumatoid arthritis (RA) is an HLA class II-associated autoimmune disease, in which arthritogenic T cells drive the progressive inflammation and destruction of synovial joints (1). Both genetic and environmental [...]

Objective: Terminal glycans on the Fc portion of IgG antibodies are critical for antibody‐triggered, proinflammatory or antiinflammatory responses. We undertook this study to compare glycan profiles of total IgG1 and Borrelia burgdorferi (Bb)–specific IgG1 antibodies in patients with oral antibiotic–responsive or antibiotic‐refractory Lyme arthritis (LA). Methods: Following affinity‐column processing, glycan profiles of IgG antibodies were determined in serum and synovial fluid (SF) samples of 21 LA patients using glycoblotting with hydrazide glycan enrichment and determination of glycan structure by matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry. Correlations between glycan profiles and treatment outcomes were analyzed. Results: Compared with patients with antibiotic‐refractory LA, those with antibiotic‐responsive LA had total and Bb‐specific IgG1 antibody glycans with less intense inflammatory profiles, containing lower percentages of N‐acetylglucosamine (GlcNAc) and bisecting GlcNAc and higher percentages of galactose and fucose. In contrast, patients with antibiotic‐refractory LA prior to receiving IV antibiotic therapy had total IgG1 and Bb IgG1 antibodies with maximal, minimally opposed, proinflammatory glycan profiles, containing high percentages of GlcNAc and bisecting GlcNAc, intermediate percentages with galactose and fucose, and low percentages with N‐acetylneuraminic acid (sialic acid). Patients with refractory LA who were first seen with synovitis after receiving IV antibiotic therapy still had Bb IgG1 antibodies with strongly inflammatory glycan profiles, but their inflammatory potential appeared to be waning. Conclusion: Patients with oral antibiotic–responsive LA had Bb IgG1 antibodies with more balanced proinflammatory/antiinflammatory glycan profiles, whereas patients with antibiotic‐refractory LA had Bb IgG1 antibodies with maximal, minimally opposed, proinflammatory glycan profiles. Among patients with antibiotic‐refractory LA, antibodies with this unbalanced inflammatory glycan profile may have a role in sustaining maladaptive joint inflammation. [ABSTRACT FROM AUTHOR]

Many peroxy-containing secondary metabolites (1,2) have been isolated and shown to provide beneficial effects to human health (3-5). Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although [...]

O -GlcNAcylation is a prominent modification of nuclear and cytoplasmic proteins in animals and plants and is mediated by a single O -GlcNAc transferase (OGT). Spindly (Spy), a paralog of OGT first discovered in higher plants, has an ortholog in the apicomplexan parasite Toxoplasma gondii , and both enzymes are now recognized as O -fucosyltransferases (OFTs). Here we investigate the evolution of spy -like genes and experimentally confirm OFT activity in the social amoeba Dictyostelium —a protist that is more related to fungi and metazoa. Immunofluorescence probing with the fucose-specific Aleuria aurantia lectin (AAL) and biochemical cell fractionation combined with western blotting suggested the occurrence of nucleocytoplasmic fucosylation. The absence of reactivity in mutants deleted in spy or gmd (unable to synthesize GDP-Fuc) suggested monofucosylation mediated by Spy. Genetic ablation of the modE locus, previously predicted to encode a GDP-fucose transporter, confirmed its necessity for fucosylation in the secretory pathway but not for the nucleocytoplasmic proteins. Affinity capture of these proteins combined with mass spectrometry confirmed monofucosylation of Ser and Thr residues of several known nucleocytoplasmic proteins. As in Toxoplasma , the Spy OFT was required for optimal proliferation of Dictyostelium under laboratory conditions. These findings support a new phylogenetic analysis of OGT and OFT evolution that indicates their occurrence in the last eukaryotic common ancestor but mostly complementary presence in its eukaryotic descendants with the notable exception that both occur in red algae and plants. Their generally exclusive expression, high degree of conservation, and shared monoglycosylation targets suggest overlapping roles in physiological regulation. [ABSTRACT FROM AUTHOR]