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.

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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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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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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Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short [Cell Biology]

November 17th, 2015 by

Drosophila melanogaster Sarcomere Length Short (SALS) is a recently identified Wiskott - Aldrich syndrome protein homology 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcomeric actin filaments. Here we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2). Our results revealed that SALS-WH2 binds both monomeric and filamentous actin and shifts the monomer : filament equilibrium towards monomeric actin. In addition, SALS-WH2 can bind to but fails to depolymerize phalloidin-, or jasplakinolide-bound actin filaments. These interactions endow SALS-WH2 with two major activities in the regulation of actin dynamics: SALS-WH2 sequesters actin monomers into non-polymerizable complexes and enhances actin filament disassembly by severing, which is modulated by tropomyosin. We also show that profilin does not influence the activities of the WH2 domains of SALS in actin dynamics. In conclusion, the tandem WH2 domains of SALS are multifunctional regulators of actin dynamics. Our findings suggest that the activities of the WH2 domains do not reconstitute the presumed biological function of the full-length protein. Consequently, the interactions of the WH2 domains of SALS with actin must be tuned in the cellular context by other modules of the protein and/or sarcomeric components for its proper functioning.

Characterization of a Cyanobacterial Chloride-Pumping Rhodopsin and Its Conversion into a Proton Pump [Bioenergetics]

November 17th, 2015 by Hasemi, T., Kikukawa, T., Kamo, N., Demura, M.

Light-driven ion-pumping rhodopsins are widely distributed in microorganisms and are now classified into the categories of outward H+ and Na+ pumps and an inward Cl- pump. These different types share a common protein architecture and utilize the photoisomerization of the same chromophore, retinal, to evoke photoreactions. Despite these similarities, successful pump-to-pump conversion had been confined to only the H+ pump bacteriorhodopsin, which was converted to a Cl- pump in 1995 by a single amino acid replacement. In this study, we report the first success of the reverse conversion, from a Cl- pump to a H+ pump. A novel microbial rhodopsin (MrHR) from the cyanobacterium Mastigocladopsis repens functions as a Cl- pump and belongs to a cluster that is far distant from the known Cl- pumps. With a single amino acid replacement, MrHR is converted to a H+ pump in which dissociable residues function almost completely in the H+ relay reactions. MrHR most likely evolved from a H+ pump, but it has not yet been highly optimized into a mature Cl- pump.

Proteolytic processing of Neuregulin 1 type III by three intramembrane cleaving proteases [Neurobiology]

November 16th, 2015 by

Numerous membrane-bound proteins undergo regulated intramembrane proteolysis (RIP). RIP is initiated by shedding and the remaining stubs are further processed by intramembrane cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of β-secretase (β-site APP cleaving enzyme 1; BACE1). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Due to the hairpin nature of NRG1 type III two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1 oriented stub is further cleaved by γ-secretase at an ε-like site 5 amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ε-cleavage site is only 1 amino acid N-terminal to a V/L substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III β-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer's disease associated mutations within the Amyloid precursor protein. The type 2 oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with (Z-LL)2 ketone inhibits formation of a N-terminal ICD and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate, which is not only cleaved by multiple sheddases but also processed by three different I-CLiPs.

Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts Mitochondrial Membrane Potential and Induces Apoptosis [Microbiology]

November 16th, 2015 by Barnwal, B., Karlberg, H., Mirazimi, A., Tan, Y.-J.

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study for the first time suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid (aa) residues, in the CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by the caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase (PARP), in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93-140 aa residues. Using alanine scanning, we demonstrated that L127 and L135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that L127 and L135 are important residues for the disruption of mitochondrial membrane potential by NSs. Thus, these results indicate that the C-terminal of CCHFV NSs triggers mitochondrial membrane permeabilization leading to activation of caspases which ultimately leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells.
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