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The importance of protein glycosylation in the interaction of pathogenic bacteria with their host is becoming increasingly clear. Neisseria meningitidis, the etiological agent of cerebrospinal meningitis, crosses cellular barriers after adhering to host cells through type IV pill. Pilin glycosylation genes (pgl) are responsible for the glycosylation of PilE, the major subunit of type IV pili, with the 2,4-diacetamido2,4,6-trideoxyhexose residue. Nearly half of the clinical isolates, however, display an insertion in the pglBCD operon, which is anticipated to lead to a different, unidentified glycosylation. Here the structure of pilin glycosylation was determined in such a strain by 'top-down' MS approaches. MALDI-TOF, nanoelectrospray ionization Fourier transform ion cyclotron resonance, and nanoelectrospray ionization quadrupole TOF MS analysis of purified pill preparations originating from IV. meningitidis strains, either wild type or deficient for pilin glycosylation, revealed a glycan mass inconsistent with 2,4-diacetamido-2,4,6-trideoxyhexose or any sugar in the databases. This unusual modification was determined by in-source dissociation of the sugar from the protein followed by tandem MS analysis with collision-induced fragmentation to be a hexose modified with a glyceramido and an acetamido group. We further show genetically that the nature of the sugar present on the pilin is determined by the carboxyl-terminal region of the pglB gene modified by the insertion in the pglBCD locus. We thus report a previously undiscovered monosaccharide involved in posttranslational modification of type IV pilin subunits by a MS-based approach and determine the molecular basis of its biosynthesis. mass spectrometry | pglB | Pili

Proinsulin is a key autoantigen in type 1 diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD[8.sup.+] T cell responses to the insulin B chain and adjacent C peptide, we selected 8- to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type 1 diabetic patients produced IFN-[gamma] in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted peptides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-A1 and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients. autoimmunity | beta cell | CD8 T cells | human autoantigens | proteasome

Exosomes, extracellular vesicles (EVs) of endosomal origin, emerge as master regulators of cell-to-cell signaling in physiology and disease. Exosomes are highly enriched in tetraspanins (TSPNs) and syndecans (SDCs), the latter occurring mainly in proteolytically cleaved form, as membrane-spanning C-terminal fragments of the proteins. While both protein families are membrane scaffolds appreciated for their role in exosome formation, composition, and activity, we currently ignore whether these work together to control exosome biology. Here we show that TSPN6, a poorly characterized tetraspanin, acts as a negative regulator of exosome release, supporting the lysosomal degradation of SDC4 and syntenin. We demonstrate that TSPN6 tightly associates with SDC4, the SDC4- TSPN6 association dictating the association of TSPN6 with syntenin and the TSPN6-dependent lysosomal degradation of SDC4-syntenin. TSPN6 also inhibits the shedding of the SDC4 ectodomain, mimicking the effects of matrix metalloproteinase inhibitors. Taken together, our data identify TSPN6 as a regulator of the trafficking and processing of SDC4 and highlight an important physical and functional interconnection between these membrane scaffolds for the production of exosomes. These findings clarify our understanding of the molecular determinants governing EV formation and have potentially broad impact for EV-related biomedicine. [ABSTRACT FROM AUTHOR]

Control of microtubule dynamics underlies several fundamental processes such as cell polarity, cell division, and cell motility. To gain insights into the mechanisms that control microtubule dynamics during cell motility, we investigated the interactome of the microtubule plus-end-binding protein end-binding 1 (EB1). Via molecular mapping and cross-linking mass spectrometry we identified and characterized a large complex associating a specific isoform of myomegalin termed "SMYLE" (for short myomegalin-like EB1 binding protein), the PKA scaffolding protein AKAP9, and the pericentrosomal protein CDK5RAP2. SMYLE was associated through an evolutionarily conserved N-terminal domain with AKAP9, which in turn was anchored at the centrosome via CDK5RAP2. SMYLE connected the pericentrosomal complex to the microtubule-nucleating complex (?-TuRC) via Galectin-3-binding protein. SMYLE associated with nascent centrosomal microtubules to promote microtubule assembly and acetylation. Disruption of SMYLE interaction with EB1 or AKAP9 prevented microtubule nucleation and their stabilization at the leading edge of migrating cells. In addition, SMYLE depletion led to defective astral microtubules and abnormal orientation of the mitotic spindle and triggered G1 cell-cycle arrest, which might be due to defective centrosome integrity. As a consequence, SMYLE loss of function had a profound impact on tumor cell motility and proliferation, suggesting that SMYLE might be an important player in tumor progression. [ABSTRACT FROM AUTHOR]

Proinsulin is a key autoantigen in type I diabetes. Evidence in the mouse has underscored the importance of the insulin B chain region in autoimmunity to pancreatic beta cells. In man, a majority of proteasome cleavage sites are predicted by proteasome cleavage algorithms within this region. To study CD8+ T cell responses to the insulin B chain and adjacent C peptide, we selected 8- to 11-mer peptides according to proteasome cleavage patterns obtained by digestion of two peptides covering proinsulin residues 28 to 64. We studied their binding to purified HLA class I molecules and their recognition by T cells from diabetic patients. Peripheral blood mononuclear cells from 17 of 19 recent-onset and 12 of 13 long-standing type I diabetic patients produced IFN-y in response to proinsulin peptides as shown by using an ELISPOT assay. In most patients, the response was against several class I-restricted pep- tides. Nine peptides were recognized within the proinsulin region covering residues 34 to 61. Four yielded a high frequency of recognition in HLA-Al and -B8 patients. Three peptides located in the proinsulin region 41-51 were shown to bind several HLA molecules and to be recognized in a high percentage of diabetic patients. [ABSTRACT FROM AUTHOR]