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.

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.

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.

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.

Transcriptional and Translational Modulation of Myo-Inositol Oxygenase (MIOX) by Fatty acids: Implications in Renal Tubular Injury Induced in Obesity and Diabetes [Molecular Bases of Disease]

November 17th, 2015 by

Kidney is one of the target organs for various metabolic diseases, including diabetes, metabolic syndrome and obesity. Most of the metabolic studies underscore glomerular pathobiology, while the tubulo-interstitial compartment has been under-emphasized. Current study highlights mechanisms concerning pathobiology of tubular injury in the context of myo-inositol oxygenase (MIOX), a tubular enzyme. Kidneys of mice fed with a high fat diet (HFD) had increased MIOX expression and activity, the latter was related to phosphorylation of serine/threonine residues. Also, expression of sterol regulatory element-binding protein1 (SREBP1) and markers of cellular/nuclear damage was increased along with accentuated apoptosis and loss of tubular brush border. Similar results were observed in cells treated with palmitate:BSA. Multiple sterol response elements and E-box motifs were found in the MIOX promoter, and its activity was modulated by palmitate:BSA. Electrophoretic mobility and Chip assays confirmed binding of SREBP to consensus sequences of MIOX promoter. Exposure of palmitate:BSA-treated cells to rapamycin normalized MIOX expression and prevented SREBP1 nuclear translocation. In addition, rapamycin treatment reduced p53 expression and apoptosis. Like rapamycin, SREBP siRNA reduced MIOX expression. Increased expression of MIOX was associated with generation of reactive oxygen species (ROS) in kidney tubules of mice fed HFD and cells exposed to palmitate:BSA. Both MIOX- and SREBP1-siRNAs reduced generation of ROS. Collectively, these findings suggest that HFD- or fatty acids modulate transcriptional, translational and post-translational regulation of MIOX expression/activity and underscore MIOX being a novel target of transcription factor SREBP1. Conceivably, activation of mTORC1/SREBP1/MIOX pathway leads to the generation of ROS culminating into tubulo-interstitial injury in states of obesity.
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Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity [Enzymology]

November 17th, 2015 by

Multiple isoforms of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) arise from the alternative splicing of its single gene-encoded pre-mRNA transcript. Among these, the longer Drp1 isoforms, expressed selectively in neurons, bear unique polypeptide sequences within their GTPase and variable (VD) domains, known as the A-insert and the B-insert, respectively. Their functions remain unresolved. A comparison of the various biochemical and biophysical properties of the neuronally expressed isoforms with that of the ubiquitously expressed, and shortest, Drp1 isoform (Drp1-short) has revealed the effect of these inserts on Drp1 function. Utilizing various biochemical, biophysical and cellular approaches, we find that the A- and B-inserts, distinctly alter the oligomerization propensity of Drp1 in solution as well as the preferred curvature of helical Drp1 self-assembly on membranes. Consequently, these sequences also suppress Drp1 cooperative GTPase activity. Mitochondrial fission factor (Mff), a tail-anchored membrane protein of the mitochondrial outer membrane that recruits cytosolic Drp1 to sites of ensuing fission, differentially stimulates the disparate Drp1 isoforms and alleviates the autoinhibitory effect imposed by these sequences on Drp1 function. Moreover, the differential stimulatory effects of Mff on Drp1 isoforms are dependent on the mitochondrial lipid, cardiolipin (CL). Whereas Mff stimulation of the intrinsically cooperative Drp1-short isoform is relatively modest, CL-independent, and is even counter-productive at high CL concentrations, Mff stimulation of the much-less cooperative longest Drp1 isoform (Drp-long) is robust and occurs synergistically with increasing CL content. Thus, membrane-anchored Mff differentially regulates various Drp1 isoforms by functioning as an allosteric effector of cooperative GTPase activity.
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Dynamin-Related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-Anchored Mitochondrial Fission Factor [Protein Structure and Folding]

November 17th, 2015 by

Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane (OMM) by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interactions between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ∆VD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil (CC) motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction.
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