Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic {alpha}{beta}T cell Receptor Chains [Protein Structure and Folding]

September 23rd, 2015 by Feige, M. J., Behnke, J., Mittag, T., Hendershot, L. M.

In eukaryotic cells, secretory pathway proteins must pass stringent quality control checkpoints before exiting the endoplasmic reticulum (ER). Acquisition of native structure is generally considered to be the most important prerequisite for ER exit. However, structurally detailed protein folding studies in the ER are few. Furthermore, aberrant ER quality control (ERQC) decisions are associated with a large and increasing number of human diseases, highlighting the need for more detailed studies on the molecular determinants that result in proteins being either secreted or retained. Here, we used the clonotypic αβ chains of the T cell receptor (TCR) as a model to analyze lumenal determinants of ERQC with a particular emphasis on how proper assembly of oligomeric proteins can be monitored in the ER. A combination of in vitro and in vivo approaches allowed us to provide a detailed model for αβTCR assembly control in the cell. We found that folding of the TCR α-chain constant domain Cα is dependent on αβ-heterodimerization. Furthermore, our data show that some variable regions associated with either chain can remain incompletely folded until chain pairing occurs. Together, these data argue for template-assisted folding at more than one point in the TCR α/β assembly process that allows specific recognition of unassembled clonotypic chains by the ER chaperone machinery and thus reliable quality control of this important immune receptor. Additionally, it highlights an unreported possible limitation in the α- and β-chain combinations that will comprise the T cell repertoire.
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Cystathionine {beta}-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria [Signal Transduction]

September 23rd, 2015 by

Among numerous proteins containing pairs of regulatory cystathionine β-synthase (CBS) domains, Family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP, ADP) or activation (ATP). Here we show that linear P1,Pn-diadenosine 5′-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi and Clostridium perfringens, and increase their activity up to 30-, 5- and 7-fold, respectively. Ap4A, Ap5A and Ap6A bound non-cooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were approximately 10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A, but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.
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Antigen-B Cell Receptor Complexes Associate with Intracellular MHC Class II Molecules [Cell Biology]

September 23rd, 2015 by Barroso, M., Tucker, H., Drake, L., Nichol, K., Drake, J. R.

Antigen processing and MHC class II-restricted antigen presentation by antigen presenting cells (APC) such as dendritic cells and B cells allows for activation of naive CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted APC in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging / FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, previously shown to be critical for CD4 T cell activation, is selectively incorporated into these complexes and selectively loaded with peptide derived from BCR-internalized cognate antigen. These results demonstrate that in B cells internalized Ag-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms that B cells use to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.

Insights into the determination of the templating nucleotide at the initiation of ϕ29 DNA replication [Enzymology]

September 23rd, 2015 by

Bacteriophage φ29 from Bacillus subtilis starts replication of its terminal protein (TP)-DNA by a protein-priming mechanism. To start replication, the DNA polymerase forms a heterodimer with a free TP that recognises the replication origins, placed at both 5' ends of the linear chromosome, and initiates replication using as primer the OH-group of Ser232 of the TP. The initiation of φ29 TP-DNA replication mainly occurs opposite the second nucleotide at the 3' end of the template. Earlier analyses of the template position that directs the initiation reaction were performed using single-stranded and double-stranded oligonucleotides containing the replication origin sequence without the parental TP. Here, we show that the parental TP has no influence in the determination of the nucleotide used as template in the initiation reaction. Previous studies showed that the priming domain of the primer TP determines the template position used for initiation. The results obtained here using mutant TPs at the priming loop where Ser232 is located, indicate that the aromatic residue Phe230 is one of the determinants that allows the positioning of the penultimate nucleotide at the polymerisation active site to direct insertion of the initiator dAMP during the initiation reaction. The role of Phe230 in limiting the internalisation of the template strand in the polymerisation active site is discussed.

Correlations between photodegradation of bisretinoid constituents of retina and dicarbonyl-adduct deposition [Glycobiology and Extracellular Matrices]

September 22nd, 2015 by Zhou, J., Ueda, K., Zhao, J., Sparrow, J. R.

Non-enzymatic collagen cross-linking and carbonyl-adduct deposition are features of Bruch's membrane aging in the eye and disturbances in extracellular matrix turnover are considered to contribute to Bruch′s membrane thickening. Since bisretinoid constitutents of the lipofuscin of retinal pigment epithelial cells (RPE) are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal, GO and methylglyoxal, MG) we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side−chains. Abca4-/- and Rdh8-/-/Abca4-/- mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice while carbonylation of an RPE specific protein was observed in Abca4-/- but not in wild-type mice under the same conditions. Immunolabeling of cryostat sectioned eyes harvested from Abca4-/- mice revealed that carbonyl adduct deposition in Bruch′s membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, receptor for advanced glycation end-products (RAGE) that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO, were upregulated in Abca4-/- mice. Additionally, in acellular assays, peptides were cross−linked in the presence of A2E photodegradation products and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch′s membrane that can confer risk of age-related macular degeneration.
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Feedback Mechanisms Regulate Ets variant 2 (Etv2) Gene Expression and Hematoendothelial Lineages [Developmental Biology]

September 22nd, 2015 by

Etv2 is an essential transcriptional regulator of hematoendothelial lineages during embryogenesis. While Etv2 downstream targets have been identified, little is known regarding the upstream transcriptional regulation of Etv2 gene expression. In the present study, we have established a novel methodology that utilizes the differentiating embryonic stem cell and differentiating embryoid body (ES/EB) system, to define the modules and enhancers embedded within the Etv2 promoter. Using this system, we defined an autoactivating role for Etv2, which is mediated by two adjacent Ets motifs in the proximal promoter. In addition, we have defined the role of VEGF/Flk1-Calcineurin-NFAT signaling cascade in the transcriptional regulation of Etv2. Furthermore, we have defined an Etv2-Flt1-Flk1 cascade, which serves as a negative feedback mechanism to regulate Etv2 gene expression. To complement and extend these studies, we have demonstrated that the Flt1 null embryonic phenotype was partially rescued in the Etv2 conditional knockout background. In summary, these studies define upstream and downstream networks that serve as a transcriptional rheostat to regulate Etv2 gene expression.

Structural Basis for the Regulation of the MmpL Transporters of Mycobacterium tuberculosis [Gene Regulation]

September 22nd, 2015 by

The mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complex regulatory network, including the TetR-family transcriptional regulators Rv3249c and Rv1816. Here we report the crystal structures of these two regulators, revealing dimeric, two-domain molecules with architecture consistent with the TetR family of regulators. Buried extensively within the C-terminal regulatory domains of Rv3249c and Rv1816 we found fortuitous bound ligands, which were identified as palmitic acid (a fatty acid) and isopropyl laurate (a fatty acid ester), respectively. Our results suggest that fatty acids may be the natural ligands of these regulatory proteins. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by these proteins. Binding of palmitic acid renders these regulators incapable of interacting with their respective operator DNAs, which will result in derepression of the corresponding mmpL genes. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

Natural polymorphisms and oligomerization of human APOBEC3H contribute to single-stranded DNA scanning ability [Immunology]

September 22nd, 2015 by

APOBEC3H is a deoxycytidine deaminase that can restrict the replication of HIV-1 in the absence of the viral protein Vif that induces APOBEC3H degradation in cells. APOBEC3H exists in humans as seven haplotypes (I-VII) with different cellular stabilities. Of the three stable APOBEC3H haplotypes (II, V, and VII), haplotypes II and V occur most frequently in the population. Despite APOBEC3H being a bona fide restriction factor, there has been no comparative biochemical characterization of APOBEC3H haplotypes. We characterized the single-stranded (ss)DNA scanning mechanisms that haplotypes II and V use to search their ssDNA substrate for cytosine containing deamination motifs. APOBEC3H haplotype II was able to processively deaminate multiple cytosines in a single enzyme-substrate encounter by using sliding, jumping, and intersegmental transfer movements. In contrast, APOBEC3H haplotype V exhibited diminished sliding and intersegmental transfer abilities, but was able to jump along ssDNA. Due to an Asp or Glu at amino acid 178 differentiating these APOBEC3H haplotypes, the data indicated that this amino acid on helix 6 contributes to processivity. The diminished processivity of APOBEC3H haplotype V did not result in a reduced efficiency to restrict HIV-1 replication in single-cycle infectivity assays, suggesting a redundancy in the contributions of jumping and intersegmental transfer to mutagenic efficiency. Optimal processivity on ssDNA also required dimerization of APOBEC3H through the β2 strands. The findings support a model in which jumping can compensate for deficiencies in intersegmental transfer and suggest that APOBEC3H haplotypes II and V induce HIV-1 mutagenesis efficiently, but by different mechanisms.

Small Molecules Detected by Second-Harmonic Generation Modulate the Conformation of Monomeric {alpha}-Synuclein and Reduce its Aggregation in Cells [Molecular Bases of Disease]

September 22nd, 2015 by

Proteins are structurally dynamic molecules that perform specialized functions through unique conformational changes accessible in physiological environments. An ability to specifically and selectively control protein function via conformational modulation is an important goal for development of novel therapeutics and studies of protein mechanism in biological networks and disease. Here we applied a second-harmonic generation (SHG)-based technique for studying protein conformation in solution and in real time to the intrinsically disordered, Parkinson's disease related protein α-synuclein. From a fragment library, we identified small molecule modulators that bind to monomeric α-synuclein in vitro and significantly reduce α-synuclein aggregation in a neuronal cell culture model. Our results indicate that the conformation of α-synuclein is linked to the protein's aggregation in cells. They also provide support for a therapeutic strategy of targeting specific conformations of the protein to suppress or control its aggregation.
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Discovery of immunodominant B-cell epitopes within surface pneumococcal virulence proteins in paediatric patients with invasive pneumococcal disease [Immunology]

September 22nd, 2015 by

The identification of immunodominant B-cell epitopes within surface pneumococcal virulence proteins (PnVPs) in paediatric patients with invasive pneumococcal disease (IPD) is a valuable approach to define novel vaccine candidates. To this aim, we evaluated sera from children with IPD and age-matched controls against 141 twenty-mer synthetic peptides covering the entire sequence of major antigenic fragments within PnVPs, namely the choline-binding protein D (CbpD), the pneumococcal histidine-triad proteins (PhtD, PhtE), the pneumococcal surface protein A (PspA), the plasminogen and fibronectin binding protein B (PfbB) and the zinc metalloproteinase B (ZmpB). Ten immunodominant B-cell epitopes were identified: CbpD-pep4 [aa291-310], PhtD-pep11 [aa88-107], PhtD-pep17 [aa172-191], PhtD-pep19 [aa200-219], PhtE-pep32 [aa300-319], PhtE-pep40 [aa79-98], PfbB-pep76 [aa180-199], PfbB-pep79 [aa222-241], PfbB-pep90 [aa484-503] and ZmpB-pep125 [aa431-450]. All epitopes were highly conserved among different pneumococcal serotypes and 4 of them were located within the functional zinc-binding domain of histidine triad proteins phtD and PhtE. Peptides CbpD-pep4, PhtD-pep19 and PhtE-pep40 were broadly recognized by IPD patients` sera with prevalence 96.4%, 92.9% and 71.4% respectively, while control sera exhibited only minor reactivities(<10.7%). Their specificity for IPD was 93.3%, 95% and 96.7%, their sensitivity was 96.4%, 92.9% and 71.4% and their positivity likelihood ratio for IPD was 14.5, 18.6 and 21.4 respectively. Furthermore, purified antibodies against CbpD-pep4, PhtD-pep19 and PhtE-pep40 readily bound on the surface of different pneumococcal serotypes, as assessed by FACS and immunofluorescence analysis. The identified immunodominant B-cell epitopes provide a better understanding of immune response in IPD and are worth evaluation in additional studies as potential vaccine candidates.
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