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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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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Zn2+-dependent activation of the Trk signaling pathway induces phosphorylation of the brain-enriched tyrosine phosphatase STEP: molecular basis for Zn2+-induced ERK MAPK activation [Neurobiology]

November 16th, 2015 by Poddar, R., Rajagopal, S., Shuttleworth, C. W., Paul, S.

Excessive release of Zn2+ in the brain is implicated in the progression of acute brain injuries. Although several signaling cascades have been reported to be involved in Zn2+-induced neurotoxicity, a potential contribution of tyrosine phosphatases in this process has not been well explored. Here we show that exposure to high concentrations of Zn2+ led to a progressive increase in phosphorylation of the striatal-enriched phosphatase (STEP), a component of the excitotoxic-signaling pathway that plays a role in neuroprotection. Zn2+ mediated phosphorylation of STEP61 at multiple sites (hyperphosphorylation) was induced by the up-regulation of brain-derived neurotropic factor (BDNF), tropomyosin receptor kinase (Trk) signaling and activation of cAMP-dependent PKA (protein kinase A). Mutational studies further showed that differential phosphorylation of STEP61 at the PKA sites, ser160 and ser221 regulates the affinity of STEP61 towards its substrates. Consistent with these findings we also show that BDNF/Trk/PKA mediated signaling is required for Zn2+-induced phosphorylation of extracellular regulated kinase 2 (ERK2), a substrate of STEP that is involved in Zn2+-dependent neurotoxicity. The strong correlation between the temporal profile of STEP61 hyperphosphorylation and ERK2 phosphorylation indicates that loss of function of STEP61 through phosphorylation is necessary for maintaining sustained ERK2 phosphorylation. This interpretation is further supported by the findings that deletion of the STEP gene led to a rapid and sustained increase in ERK2 phosphorylation within minutes of exposure to Zn2+. The study provides further insight into the mechanisms of regulation of STEP61 and also offers a molecular basis for the Zn2+-induced sustained activation of ERK2.
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Structural plasticity of the protein plug that traps newly packaged genomes in podoviridae virions [Microbiology]

November 16th, 2015 by

Bacterial viruses of the P22-like family encode a specialized tail needle essential for genome stabilization after DNA-packaging and implicated in Gram-negative cell envelope penetration. The atomic structure of P22 tail needle (gp26) crystallized at acidic pH reveals a slender fiber containing an N-terminal trimer-of-hairpins tip. Though the length and composition of tail needles vary significantly in Podoviridae, unexpectedly, the amino acid sequence of the N-terminal tip is exceptionally conserved in more than two hundred genomes of P22-like phages and prophages. In this paper, we used X-ray crystallography and EM to investigate the neutral pH structure of three tail needles from bacteriophage P22, HK620 and Sf6. In all cases, we found the N-terminal tip is poorly structured, in stark contrast to the compact trimer-of-hairpins seen in gp26 crystallized at acidic pH. Hydrogen/deuterium exchange mass spectrometry, limited proteolysis, circular dichroism spectroscopy and gel filtration chromatography revealed that the N-terminal tip is highly dynamic in solution and unlikely to adopt a stable trimeric conformation at physiological pH. This is supported by the cryo-EM reconstruction of P22 mature virion tail, where the density of gp26 N-terminal tip is incompatible with a trimer-of-hairpins. We propose the tail needle N-terminal tip exists in two conformations: a pre-ejection extended conformation, which seals the portal vertex after genome-packaging and a post-ejection trimer-of-hairpins that form upon its release from the virion. The conformational plasticity of the tail needle N-terminal tip is built in the amino acid sequence, explaining its extraordinary conservation in nature.

Ankyrin-G inhibits endocytosis of cadherin dimers [Membrane Biology]

November 16th, 2015 by Cadwell, C. M., Jenkins, P. M., Bennett, V., Kowalczyk, A. P.

Dynamic regulation of endothelial cell adhesion is central to vascular development and maintenance. Furthermore, altered endothelial adhesion is implicated in numerous diseases. Thus, normal vascular patterning and maintenance require tight regulation of endothelial cell adhesion dynamics. Yet, the mechanisms that control junctional plasticity are not fully understood. VE-cadherin is an adhesive protein found in adherens junctions of endothelial cells. VE-cadherin mediates adhesion through trans interactions formed by its extracellular domain. Trans binding is followed by cis interactions that laterally cluster the cadherin in junctions. VE-cadherin is linked to the actin cytoskeleton through cytoplasmic interactions with β and α-catenin, which serve to increase adhesive strength. Furthermore, p120-catenin binds to the cytoplasmic tail of the cadherin and stabilizes it at the plasma membrane. Here, we report that induced cis-dimerization of VE-cadherin inhibits endocytosis independent of both p120 binding and trans interactions. However, we find that ankyrin-G, a protein that links membrane proteins to the spectrin-actin cytoskeleton, associates with VE-cadherin and inhibits its endocytosis. Ankyrin-G inhibits VE-cadherin endocytosis independent of p120 binding. We propose a model in which ankyrin-G associates with and inhibits the endocytosis of VE-cadherin cis-dimers. Our findings support a novel mechanism for regulation of VE-cadherin endocytosis through ankyrin association with cadherin engaged in lateral interactions.

Downregulation of miRs 203, 887, 3619 and 182 prevent vimentin-triggered, phospholipase D (PLD)-mediated cancer cell invasion [Signal Transduction]

November 15th, 2015 by Fite, K., Gomez-Cambronero, J.

Breast cancer is a leading cause of morbidity and mortality among women. Metastasis is initiated after epithelial-mesenchymal-transition (EMT). We have found a connection between EMT markers and the expression of 4 microRNAs (miRs), mediated by the signaling enzyme phospholipase D (PLD). Low aggressive MCF-7 and BT-474 breast cancer cells have low endogenous PLD enzymatic activity and cell invasion, concomitantly with high expression of miR-203, 887 and 3619 (that decrease PLD2 translation and a luciferase reporter) and miR-182 (targeting PLD1) that are therefore tumor-suppresor like miRs. The combination miR-887+miR-3619 abolished >90% PLD enzymatic activity. Conversely, post-EMT MDA-MB-231 and BT-549 cells, have low miR expression, high levels of PLD1/2 and high aggressiveness. The latter was reversed by ectopically transfecting the miRs, which was negated by silencing miRs with specific siRNAs. We uncovered that the molecular mechanism is that E-cadherin triggers expression of the miRs in pre-EMT cells, whereas Vimentin dampens expression of the miRs in post-EMT, invasive cells. This novel work identifies for the first time a set of miRs that are activated by a major pre-EMT marker and deactivated by a post-EMT marker, boosting the transition from low invasion to high invasion, as mediated by the key phospholipid metabolism enzyme PLD.
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