The Activation Mechanism of Glycoprotein Hormone Receptors with Implications in the Cause and Therapy of Endocrine Diseases [Molecular Bases of Disease]

November 18th, 2015 by

Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain triggering G-protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves' disease, hypo- and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves' disease and other dysfunctions of GPHRs.
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A Single glycan at the 99-loop of human kallikrein-related peptidase 2 regulates activation and enzymatic activity [Glycobiology and Extracellular Matrices]

November 18th, 2015 by

Human kallikrein-related peptidase 2 (KLK26) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/PSA. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells (LEXSY) and compared it with its non-glycosylated counterpart from E. coli expression. Mass spectrometry revealed that Asn95 carries a core glycan, consisting of two GlcNAc and three hexoses. Auto-catalytic activation was retarded in glyco-pro-KLK2, while the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. While glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 towards peptidic substrates with P2-Phe, the situation was reverted towards protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates, i.e., by a reduction of KM. Also, cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of KLK biology and pathology.
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Two Na+ Sites Control Conformational Change in a Neurotransmitter Transporter Homolog [Neurobiology]

November 18th, 2015 by

In LeuT, a prokaryotic homolog of neurotransmitter transporters, Na+ stabilizes outward-open conformational states. We examined how each of the two LeuT Na+ binding sites contributes to Na +-dependent closure of the cytoplasmic pathway using biochemical and biophysical assays of conformation. Mutating either of two residues that contribute to the Na2 site completely prevented cytoplasmic closure in response to Na+, suggesting that Na2 is essential for this conformational change, whereas Na1 mutants retained Na+ responsiveness. However, mutation of Na1 residues also influenced the Na+-dependent conformational change in ways that varied depending on the position mutated. Computational analyses suggest those mutants influence the ability of Na1 binding to hydrate the substrate pathway and perturb an interaction network leading to the extracellular gate. Overall, the results demonstrate that occupation of Na2 stabilizes outward-facing conformations presumably through a direct interaction between Na+ and transmembrane helices 1 and 8 whereas Na+ binding at Na1 influences conformational change through a network of intermediary interactions. The results also provide evidence that N-terminus release and helix motions represent distinct steps in cytoplasmic pathway opening.

Fc{gamma}RIIIa-Syk Co-signal Modulates CD4+ T-cell Response and Up-regulate TLR expression [Cell Biology]

November 18th, 2015 by Chauhan, A. K., Moore, T. L., Bi, Y., Chen, C.

CD4+ T-cells in SLE patients show altered TCR signaling, which utilize FcRγ-Syk. A role for FcγRIIIa activation from immune complex (IC) ligation and sublytic terminal complement complex (C5b-9) in CD4+ T-cell responses is not investigated. In this study, we show that the ICs present in SLE patients by ligating to FcγRIIIa on CD4+ T-cells phosphorylate Syk and provide a co-stimulatory signal to CD4+ T-cells in the absence of CD28 signal. This led to the development of pathogenic IL-17A+ and IFN-γhigh CD4+ T-cells in vitro. Cytokines IL-1β, IL-6, TGF-β1, and IL-23 were the only requirement for the development of both populations. SLE patients CD4+ T-cells that expressed CD25, CD69, and CD98 bound to ICs, showed pSyk, and produced IFN-γ and IL-17A. This FcγRIIIa mediated co-signal differentially up-regulated the expression of IFN pathway genes compared to CD28 co-signal. FcγRIIIa-pSyk up-regulated several TLR genes as well as the HMGB1 and MyD88 gene transcripts. ICs co-localized with these TLR pathway proteins. These results suggest a role for the FcγRIIIa-pSyk signal in modulating adaptive immune responses.

Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis [Gene Regulation]

November 17th, 2015 by Gallie, D. R.

The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5′‐leader (called Ω) of tobacco mosaic virus (TMV) preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation.
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Hypoxia Potentiates Palmitate-induced Pro-inflammatory Activation of Primary Human Macrophages [Immunology]

November 17th, 2015 by

Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids (FFAs) within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid, palmitate. Compared to normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Whereas palmitate-induced endoplasmic reticulum stress and nuclear factor-κB pathway activation were not enhanced by hypoxia, hypoxia increased activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors (HIF) HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein-1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with FFA exacerbates macrophage-mediated inflammation.

Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset in Type 1 Diabetes Mellitus [Immunology]

November 17th, 2015 by

Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-EdU incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of approximately 80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta-cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans.
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Mutations in Replicative Stress Response Pathways Are Associated with S Phase-Specific Defects in Nucleotide Excision Repair [Cell Biology]

November 17th, 2015 by

Nucleotide excision repair (NER) is a highly-conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens including UV light. Our laboratory previously demonstrated that human cells deficient in either ataxia telangiectasia and rad3-related (ATR) kinase or translesion DNA polymerase η (polη), i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress, are characterized by profound inhibition of NER exclusively during S phase. Towards elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism S. cerevisiae. Using this assay we demonstrate that in yeast, deficiency of the ATR homologue Mec1, or of any among several other proteins involved in the cellular response to replicative stress, significantly abrogates NER uniquely during S. Moreover initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover ectopic RPA1-3 overexpression rescues such deficiency in either ATR- or polη-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.

Cell Migration and Invadopodia Formation Require a Membrane-binding Domain of CARMIL2 [Cell Biology]

November 17th, 2015 by Lanier, M. H., McConnell, P., Cooper, J. A.

CARMILs regulate capping protein (CP), a critical determinant of actin assembly and actin-based cell motility. Vertebrates have three conserved CARMIL genes with distinct functions. In migrating cells, CARMIL2 is important for cell polarity, lamellipodial assembly, ruffling, and macropinocytosis. In cells, CARMIL2 localizes with a distinctive dual pattern to vimentin intermediate filaments and to membranes at leading edges and macropinosomes. The mechanism by which CARMIL2 localizes to membranes has not been defined. Here, we report that CARMIL2 has a conserved membrane-binding domain composed of basic and hydrophobic residues, which is necessary and sufficient for membrane localization, based on expression studies in cells and on direct binding of purified protein to lipids. Most important, we find that the membrane-binding domain is necessary for CARMIL2 to function in cells, based on rescue expression with a set of biochemically-defined mutants. CARMIL1 and CARMIL3 contain similar membrane-binding domains, based on sequence analysis and on experiments, but other CPI-motif proteins, such as CD2AP, do not. Based on these results, we propose a model in which the membrane-binding domain of CARMIL2 tethers this multidomain protein to the membrane, where it links dynamic vimentin filaments with regulation of actin assembly via CP.

Annexin A6 and late endosomal cholesterol modulates integrin recycling and cell migration [Molecular Bases of Disease]

November 17th, 2015 by

Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated Annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix (ECM). We recently showed that elevated AnxA6 levels significantly reduced secretion of the ECM protein fibronectin (FN). Since FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Upregulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking as well as transwell and 3D-migration/invasion assays. The reduced ability of AnxA6 expressing cells to migrate was associated with decreased cell surface expression of αVβ3 and α5β1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes (RE) while siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LE) inhibits Stx6-dependent integrin recycling, and that elevated AnxA6 levels cause LE-cholesterol accumulation, we propose that AnxA6 and blockage of LE-cholesterol transport is critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration.