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Adam M Benham

Publications and source records attributed to Adam M Benham.

8 recordsLinked to original sources
PubMed2006

The DMalpha and DMbeta chain cooperate in the oxidation and folding of HLA-DM.

HLA-DM (DM) is a heterodimeric MHC molecule that catalyzes the peptide loading of classical MHC class II molecules in the endosomal/lysosomal compartments of APCs. Although the function of DM is well-established, little is known about how DMalpha and beta-chains fold, oxidize, and form a complex in the endoplasmic reticulum (ER). In this study, we show that glycosylation promotes, but is not essential for, DMalphabeta ER exit. However, glycosylation of DMalpha N15 is required for oxidation of the alpha-chain. The DMalpha and beta-chains direct each others fate: single DMalpha chains cannot fully oxidize without DMbeta, while DMbeta forms disulfide-linked homodimers without DMalpha. Correct oxidation and subsequent ER egress depend on the unique DMbeta C25 and C35 residues. This suggests that the C25-C35 disulfide bond in the peptide-binding domain overcomes the need for stabilizing peptides required by other MHC molecules.

Antigen-Presenting Cells↗
PubMed2006

Mutations in the FAD binding domain cause stress-induced misoxidation of the endoplasmic reticulum oxidoreductase Ero1beta.

Disulfide bond catalysis is an essential component of protein biogenesis in the secretory pathway, from yeast through to man. In the endoplasmic reticulum (ER), protein-disulfide isomerase (PDI) catalyzes the oxidation and isomerization of disulfide bonds and is re-oxidized by an endoplasmic reticulum oxidoreductase (ERO). The elucidation of ERO function was greatly aided by the genetic analysis of two ero mutants, whose impairment results from point mutations in the FAD binding domain of the Ero protein. The ero1-1 and ero1-2 yeast strains have conditional and dithiothreitol-sensitive phenotypes, but the effects of the mutations on the behavior of Ero proteins has not been reported. Here, we show that these Gly to Ser and His to Tyr mutations do not prevent the dimerization of Ero1beta or the non-covalent interaction of Ero1beta with PDI. However, the Gly to Ser mutation abolishes disulfide-dependent PDI-Ero1beta heterodimers. Both the Gly to Ser and His to Tyr mutations make Ero1beta susceptible to misoxidation and aggregation, particularly during a temperature or redox stress. We conclude that the Ero FAD binding domain is critical for conformational stability, allowing Ero proteins to withstand stress conditions that cause client proteins to misfold.

Dimerization↗
PubMed2006

An introduction to thiol redox proteins in the endoplasmic reticulum and a review of current electrochemical methods of detection of thiols.

This aim of this paper is to expound the complexity of thiol redox systems in the endoplasmic reticulum of eukaryotic cells to the electroanalytical community. A summary of the state of the art in electrochemical methods for detection of thiols gives an insight into the challenges that need to be addressed to bridge the disparity between current analytical techniques and applications in a 'real' biological scenario.

Animals↗
PubMed2005

Tissue-specific expression and dimerization of the endoplasmic reticulum oxidoreductase Ero1beta.

Endoplasmic reticulum oxidoreductases (Eros) are essential for the formation of disulfide bonds. Understanding disulfide bond catalysis in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. Mammals express two related Ero proteins, Ero1alpha and Ero1beta. Ero1beta is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1beta can form homodimers and mixed heterodimers with Ero1alpha, in addition to Ero-PDI dimers. Ero-Ero dimers require the Ero active site, occur in vivo, and can be modeled onto the Ero1p crystal structure. Our data indicate that the Ero1beta protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1beta occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1beta expression is high, but is inversely correlated with PDI and PDIp levels, demonstrating that cell-specific differences exist in the regulation of oxidative protein folding in vivo.

Binding Sites↗
PubMed2004

PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum.

Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.

Amino Acid Motifs↗
PubMedDate not supplied

Differential oxidation of HLA-B2704 and HLA-B2705 in lymphoblastoid and transfected adherent cells.

MHC class I molecules are predominantly involved in the presentation of antigens from viral proteins to CD8+ T cells of the immune system. However, MHC proteins can also be linked to autoimmune diseases, and the HLA-B27 allele is expressed by 95% of people with the rheumatic condition ankylosing spondylitis (AS). A precise molecular explanation for the association between HLA-B27 and AS is still lacking, although it is known that inappropriately disulfide bonded HLA-B27 heavy chains can be found at both the cell surface and in the endoplasmic reticulum (ER) of HLA-B27 expressing cells. This papers shows that HLA-B27 heavy chain misfolding does not depend on any unpaired cysteine residue per se when HLA-B27 is highly expressed. Also shown is that major differences exist in the disulfide-dependent conformations of two HLA-B27 subtypes, HLA-B2704 and HLA-B2705. The results imply that residues 77, 152, and/or 211 influence the redox potential of the MHC class I heavy chain and suggest that manipulating the redox environment can alter the conformational state of HLA-B27 subtypes.

CD8-Positive T-Lymphocytes↗