The human resource implications of improving financial risk protection for mothers and newborns in Zimbabwe

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Rhesus macaque MHC class I molecules show differential subcellular localizations

The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding α1 and α2 domains, suggesting failure of peptide binding is responsible for retaining ‘intracellular’ Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes

Proteomic Analysis of S-Acylated Proteins in Human B Cells Reveals Palmitoylation of the Immune Regulators CD20 and CD23

S-palmitoylation is a reversible post-translational modification important for controlling the membrane targeting and function of numerous membrane proteins with diverse roles in signalling, scaffolding, and trafficking. We sought to identify novel palmitoylated proteins in B lymphocytes using acyl-biotin exchange chemistry, coupled with differential analysis by liquid-chromatography tandem mass spectrometry. In total, we identified 57 novel palmitoylated protein candidates from human EBV-transformed lymphoid cells. Two of them, namely CD20 and CD23 (low affinity immunoglobulin epsilon Fc receptor), are immune regulators that are effective/potential therapeutic targets for haematological malignancies, autoimmune diseases and allergic disorders. Palmitoylation of CD20 and CD23 was confirmed by heterologous expression of alanine mutants coupled with bioorthogonal metabolic labeling. This study demonstrates a new subset of palmitoylated proteins in B cells, illustrating the ubiquitous role of protein palmitoylation in immune regulation

Specificity of Transmembrane Protein Palmitoylation in Yeast

Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called “Asp-His-His-Cys- Cysteine Rich Domain” (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether

The activity of TRAF RING homo- and heterodimers is regulated by zinc finger 1

Ubiquitin chains linked through lysine63 (K63) play a critical role in inflammatory signalling. Following ligand engagement of immune receptors, the RING E3 ligase TRAF6 builds K63-linked chains together with the heterodimeric E2 enzyme Ubc13-Uev1A. Dimerisation of the TRAF6 RING domain is essential for the assembly of K63-linked ubiquitin chains. Here, we show that TRAF6 RING dimers form a catalytic complex where one RING interacts with a Ubc13~Ubiquitin conjugate, while the zinc finger 1 (ZF1) domain and linker-helix of the opposing monomer contact ubiquitin. The RING dimer interface is conserved across TRAFs and we also show that TRAF5–TRAF6 heterodimers form. Importantly, TRAF5 can provide ZF1, enabling ubiquitin transfer from a TRAF6-bound Ubc13 conjugate. Our study explains the dependence of activity on TRAF RING dimers, and suggests that both homo- and heterodimers mediated by TRAF RING domains have the capacity to synthesise ubiquitin chains

Retroviral mRNA nuclear export elements regulate protein function and virion assembly

Rodent cells are notable for their inability to support normal assembly of HIV particles. In this report, we address possible causes for this defect by considering the hypothesis that mRNA-associated events occurring in the nucleus can regulate the activity of their encoded proteins in the cytoplasm. We show that altering the RNA nuclear export element used by HIV gag-pol mRNA from the Rev response element to the constitutive transport element restores both the trafficking of Gag to cellular membranes and efficient HIV assembly in murine cells. These results suggest that two phases of the HIV life cycle, RNA export and capsid assembly, that have hitherto been regarded as distinct are, in fact, linked. Thus, protein function and fate may depend upon the full and precise history of its encoding mRNA

Stoichiometry of the T-cell receptor–CD3 complex and key intermediates assembled in the endoplasmic reticulum

The T-cell receptor (TCR)–CD3 complex is critical for T-cell development and function, and represents one of the most complex transmembrane receptors. Models of different stoichiometry and valency have been proposed based on cellular experiments and these have important implications for the mechanisms of receptor triggering. Since determination of receptor stoichiometry in T-cells is not possible due to the presence of previously synthesized, unlabeled receptor components with different half-lives, we examined the stoichiometry of the receptor assembled in endoplasmic reticulum (ER) microsomes of B-cell origin. The stoichiometric relationship among all subunits was directly determined using intact radiolabeled TCR–CD3 complexes that were isolated with a sequential, non-denaturing immunoprecipitation method, and identical results were obtained with two detergents belonging to different structural classes. The results firmly establish that the αβ TCR–CD3 complex assembled in the ER is monovalent and composed of one copy of the TCRαβ, CD3δɛ, CD3γɛ and ζ−ζ dimers

Structure of a trimeric domain of the MHC class II-associated chaperonin and targeting protein Ii.

The invariant chain (Ii) plays a critical role in MHC class II antigen processing by stabilizing peptide-free class II alphabeta heterodimers in a nonameric (alphabetaIi)3 complex soon after their synthesis and directing transport of the complex from the endoplasmic reticulum to compartments where peptide loading of class II takes place. Loading progresses following Ii proteolysis and via an intermediate complex of MHC class II with an Ii-derived peptide, CLIP. CLIP is substituted by exogenous peptidic fragments in an exchange reaction catalyzed by HLA-DM. The CLIP region of Ii, roughly residues 81-104, is one of two segments shown to interact with class II HLA-DR molecules. The other segment, Ii 118-216, is C-terminal to CLIP, mediates trimerization of the ectodomain of Ii and interferes with DM/class II binding. Here we report the three-dimensional structure of this trimeric domain, determined by nuclear magnetic resonance (NMR) studies of a 27 kDa trimer of human Ii 118-192. The cylindrical shape of the molecule and the mapping of conserved residues delimit surfaces which may be important for interactions between Ii and class II molecules

Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation

Protein palmitoylation is a post-translational modification that affects a great number of proteins. In most cases, the enzymes responsible for this modification have not been identified. Some proteins use palmitoylation to attach themselves to membranes; however, palmitoylation also occurs in transmembrane proteins, and the function of this palmitoylation is less clear. Here we identify Swf1, a member of the DHHC-CDR family of palmitoyltransferases, as the protein responsible for modifying the yeast SNAREs Snc1, Syn8 and Tlg1, at cysteine residues close to the cytoplasmic end of their single transmembrane domains (TMDs). In an swf1Δ mutant, Tlg1 is mis-sorted to the vacuole. This occurs because unpalmitoylated Tlg1 is recognised by the ubiquitin ligase Tul1, resulting in its targeting to the multivesicular body pathway. Our results suggest that one role of palmitoylation is to protect TMDs from the cellular quality control machinery, and that Swf1 may be the enzyme responsible for most, if not all, TMD-associated palmitoylation in yeast