Cell Cycle-Dependent Changes in Localization and Phosphorylation of the Plasma Membrane Kv2.1 K+ Channel Impact Endoplasmic Reticulum Membrane Contact Sites in COS-1 Cells [Cell Biology]

October 6th, 2015 by Cobb, M. M., Austin, D. C., Sack, J. T., Trimmer, J. S.

The plasma membrane (PM) comprises distinct subcellular domains with diverse functions that need to be dynamically coordinated with intracellular events, one of the most impactful being mitosis. The Kv2.1 voltage-gated potassium channel is conditionally localized to large PM clusters that represent specialized PM:endoplasmic reticulum membrane contact sites (PM:ER MCS), and overexpression of Kv2.1 induces more exuberant PM:ER MCS in neurons and in certain heterologous cell types. Localization of Kv2.1 at these contact sites is dynamically regulated by changes in phosphorylation at sites located on its large cytoplasmic C-terminus. Here, we show that Kv2.1 expressed in COS-1 cells undergoes dramatic cell cycle-dependent changes in its PM localization, having diffuse localization in interphase cells, and robust clustering during M phase. The mitosis-specific clusters of Kv2.1 are localized to PM:ER MCS, and M phase clustering of Kv2.1 induces more extensive PM:ER MCS. These cell cycle-dependent changes in Kv2.1 localization and the induction of PM:ER MCS are accompanied by increased mitotic Kv2.1 phosphorylation at several C-terminal phosphorylation sites. Phosphorylation of exogenously expressed Kv2.1 is significantly increased upon metaphase arrest in COS-1 and CHO cells, and in a pancreatic β cell line that express endogenous Kv2.1. The M phase clustering of Kv2.1 at PM:ER MCS in COS-1 cells requires the same C-terminal targeting motif needed for conditional Kv2.1 clustering in neurons. The cell cycle-dependent changes in localization and phosphorylation of Kv2.1 were not accompanied by changes in the electrophysiological properties of Kv2.1 expressed in CHO cells. Together, these results provide novel insights into the cell cycle-dependent changes in PM protein localization and phosphorylation.
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Dopamine Transporter Activity Is Modulated by {alpha}-Synuclein [Molecular Bases of Disease]

October 6th, 2015 by

The duration and strength of the dopaminergic signal is regulated by the dopamine transporter (DAT). Drug addiction, neurodegenerative and neuropsychiatric diseases have all been associated with altered DAT activity. The membrane localization and the activity of DAT are regulated by a number of intracellular proteins. α-Synuclein, a protein partner of DAT, is implicated in neurodegenerative disease and drug addiction. Little is known about the regulatory mechanisms of the interaction between DAT and α-synuclein, the cellular location of this interaction, and the functional consequences of this interaction on the basal, amphetamine (AMPH) induced DAT-meditated DA efflux and membrane microdomain distribution of the transporter. Here, we found that the majority of DAT/α-synuclein protein complexes are found at the plasma membrane of dopaminergic neurons or mammalian cells, and that AMPH-mediated increase in DAT activity enhances the association of these proteins at the plasma membrane. Further examination of the interaction of DAT and α-synuclein revealed a transient interaction between these two proteins at the plasma membrane. Additionally, we found DAT-induced membrane depolarization enhances plasma membrane localization of α-synuclein, which in turn increases DA efflux and enhances DAT localization in cholesterol rich membrane microdomains.

Assembly of a Tyr122-hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity-reduction and Release with Phosphoenzyme Isomerization [Bioenergetics]

October 6th, 2015 by Yamasaki, K., Daiho, T., Danko, S., Suzuki, H.

The mechanism whereby events in and around the catalytic site/head of Ca2+-ATPase effect Ca2+ release to the lumen from the transmembrane helices remains elusive. We have developed a method to determine deoccluded bound Ca2+ by taking advantage of its rapid occlusion upon formation of E1PCa2 and of stabilization afforded by a high concentration of Ca2+. The assay is applicable to minute amounts of Ca2+-ATPase expressed in COS-1 cells. It was validated by measuring the Ca2+ binding properties of unphosphorylated Ca2+-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca2+ release process E1PCa2 → E2PCa2 → E2P + 2Ca2+. In wild type, Ca2+ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa2 → E2PCa2) followed by very rapid Ca2+ release. In contrast, with alanine mutants of Leu119 and Tyr122 on cytoplasmic part of second transmembrane helix (M2) and Ile179 on the A domain, Ca2+ release in 10 μM Ca2+ lags EP isomerization indicating the presence of a transient E2P state with bound Ca2+. Results suggest that these residues function in Ca2+ affinity-reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and resulting association with the A and P domains, thus leading to Ca2+ release.
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Degradation of stop codon read-through mutant proteins via the ubiquitin-proteasome system causes hereditary disorders. [Protein Synthesis and Degradation]

October 6th, 2015 by

During translation, stop codon read-through occasionally happens when the stop codon is misread, skipped or mutated, resulting in the production of aberrant proteins with carboxy-terminal extension. These extended proteins are potentially deleterious, but their regulation is poorly understood. Here we show in vitro and in vivo evidence that mouse cFLIP-L with a 46 amino acid extension encoded by a read-through mutant gene is rapidly degraded by the ubiquitin-proteasome system (UPS), causing hepatocyte apoptosis during embryogenesis. The extended peptide interacts with an E3 ubiquitin ligase, TRIM21, to induce ubiquitylation of the mutant protein. In human, 20 read-through mutations are related to hereditary disorders, and extended peptides found in human PNPO and HSD3B2 similarly destabilize these proteins, involving TRIM21 for PNPO degradation. Our findings indicate that degradation of aberrant proteins with carboxy-terminal extension encoded by read-through mutant genes is a mechanism for loss-of-function resulting in hereditary disorders.
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The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy [Protein Synthesis and Degradation]

October 6th, 2015 by

Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses. Thus, we focused on a newly isolated thermotolerant yeast strain, Kluyveromyces marxianus DMKU3-1042, to utilize as a novel system elucidating autophagy. We developed experimental methods to monitor autophagy in K. marxianus cells, identified the complete set of K. marxianus Atg homologs, and confirmed that each Atg homolog is engaged in autophagosome formation. Biochemical and bioinformatic analyses revealed that recombinant K. marxianus Atg proteins have superior thermostability and solubility as compared with S. cerevisiae Atg proteins, probably due to the shorter primary sequences of KmAtg proteins. Furthermore, bioinformatic analyses showed that more than half of K. marxianus open reading frames are relatively short in length. These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, not only in autophagy field, but also in other fields.
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A Novel RB E3 Ubiquitin Ligase (NRBE3) Promotes RB Degradation and is Transcriptionally Regulated by E2F1 [Protein Synthesis and Degradation]

October 6th, 2015 by

Retinoblastoma protein (RB) plays critical roles in tumor suppression and is degraded through proteasomal way. However, E3 ubiquitin ligases responsible for proteasome-mediated degradation of RB are largely unknown. Here we characterize a novel RB E3 ubiquitin ligase NRBE3 that binds RB and promotes RB degradation. NRBE3 contains an LxCxE motif and binds RB in vitro. NRBE3 interacts with RB in cells when proteasome activity is inhibited. NRBE3 promotes RB ubiquitination and degradation via ubiquitin-proteasome pathway. Importantly, purified NRBE3 ubiquitinates recombinant RB in vitro and a U-box is identified as essential for its E3 activity. Surprisingly, NRBE3 is transcriptionally activated by E2F1/DP1. Consequently, NRBE3 affects cell cycle by promoting G1/S transition. Moreover, NRBE3 is up-regulated in breast cancer tissues. Taken together, we identify NRBE3 as a novel ubiquitin E3 ligase for RB, which might play as a potential oncoprotein in human cancers.
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Macrophage-specific de novo synthesis of ceramide is dispensable for inflammasome-driven inflammation and insulin-resistance in obesity [Metabolism]

October 5th, 2015 by

Dietary lipid overload and calorie excess during obesity is a low grade chronic inflammatory state with diminished ability to appropriately metabolize glucose or lipids. Macrophages are critical in maintaining adipose tissue homeostasis, in part by regulating lipid metabolism, energy homeostasis and tissue remodeling. During high fat diet-induced obesity, macrophages are activated by lipid derived "danger signals" such as ceramides and palmitate and promote the adipose tissue inflammation in an Nlrp3 inflammasome-dependent manner. Given that the metabolic fate of fatty acids in macrophages is not entirely elucidated, we have hypothesized that de novo synthesis of ceramide, through the rate-limiting enzyme serine palmitoyltransferase long chain (Sptlc)-2, is required for saturated fatty acid driven Nlrp3 inflammasome activation in macrophages. Here we report that mitochondrial targeted overexpression of catalase which is established to mitigate oxidative stress controls ceramide-induced Nlrp3 inflammasome activation but does not affect the ATP-mediated caspase-1 cleavage. Surprisingly, myeloid cell-specific deletion of Sptlc2 is not required for palmitate driven Nlrp3 inflammasome activation. Furthermore, the ablation of Sptlc2 in macrophages did not impact macrophage polarization or obesity-induced adipose tissue leukocytosis. Consistent with these data, investigation of insulin-resistance using hyperinsulinemic-euglycemic clamps revealed no significant differences in obese mice lacking ceramide de novo synthesis machinery in macrophages. These data suggest that alternate metabolic pathways control fatty acid derived ceramide synthesis in macrophage and the Nlrp3 inflammasome activation in obesity.
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Structural insights into the high-efficiency catalytic mechanism of the sterile {alpha}-motif/histidineaspartate domain-containing protein [Enzymology]

October 5th, 2015 by Li, Y., Kong, J., Peng, X., Hou, W., Qin, X., Yu, X.-F.

Sterile α-motif/histidine-aspartate domain-containing protein (SAMHD1), a homo-tetrameric GTP/dGTP-dependent dNTP triphosphohydrolase, catalyzes the conversion of dNTP into deoxynucleoside and triphosphate. As the only characterized dNTP triphosphohydrolase in human cells, SAMHD1 plays an important role in human innate immunity, autoimmunity, and cell cycle control. Previous biochemical studies and crystal structures have revealed that SAMHD1 interconverts between an inactive monomeric or dimeric form and a dGTP/GTP-induced active tetrameric form. Here, we describe a novel state of SAMHD1 (109-626 amino acids, SAMHD1C) that is characterized by a rapid initial hydrolysis rate. Interestingly, the crystal structure showed that this novel SAMHD1 tetramer contains only GTP and has structural features distinct from the GTP/dNTP-bound SAMHD1 tetramer. Our work thus reveals structural features of SAMHD1 that may represent one of its biological assembly states in cells. The biochemical and structural information generated by the present study not only provides an ordered pathway for the assembly and activation of SAMHD1 but also provides insights into the potential mechanisms of the high-efficiency catalytic activity of this enzyme family in vivo.
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PAK kinases mediate the phosphorylation of PREX2 to initiate feedback inhibition of Rac1 [Signal Transduction]

October 5th, 2015 by

Phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent Rac exchanger 2 (PREX2) is a guanine nucleotide exchange factor (GEF) for the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase, facilitating the exchange of GDP for GTP on Rac1. GTP bound Rac1 then activates its downstream effectors, including p21 activated kinases (PAK). PREX2 and Rac1 are frequently mutated in cancer, and have key roles within the insulin signaling pathway. Rac1 can be inactivated by multiple mechanisms; however, negative regulation by insulin is not well understood. Here, we show that in response to being activated after insulin stimulation, Rac1 initiates its own inactivation by decreasing PREX2 GEF activity. Following PREX2 mediated activation of Rac1 by the second messengers PIP3 or Gβγ, we found that PREX2 was phosphorylated through a PAK dependent mechanism. PAK mediated phosphorylation of PREX2 reduced GEF activity towards Rac1 by inhibiting PREX2 binding to PIP3 and Gβγ. Cell fractionation experiments also revealed that phosphorylation prevented PREX2 from localizing to the cellular membrane. Further, the onset of insulin induced phosphorylation of PREX2 was delayed compared to AKT. Altogether, we propose that second messengers activate the Rac1 signal, which sets in motion a cascade whereby PAK kinases phosphorylate and negatively regulate PREX2 to decrease Rac1 activation. This type of regulation would allow for transient activation of the PREX2-Rac1 signal, and may be relevant in multiple physiological processes, including diseases such as diabetes and cancer when insulin signaling is chronically activated.

High glucose-induced retinal pericyte apoptosis depends on association of GAPDH and Siah1 [Cell Biology]

October 5th, 2015 by Suarez, S., McCollum, G. W., Jayagopal, A., Penn, J. S.

Diabetic Retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive and are of limited effectiveness. Retinal pericyte death is an early pathologic
feature of DR. Though it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in ocular tissues. In this manuscript we examined the involvement of the GAPDH/Siah1 interaction in human retinal pericyte (hRP) apoptosis. HRP were cultured in 5mM normal glucose, 25mM L- or D-glucose for 48hrs (osmotic control and high glucose treatments, respectively). Siah1 siRNA was used to downregulate Siah1 expression. TAT-FLAG GAPDH and/or Siah1 directed peptides were used to block GAPDH and Siah1 interaction. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on the association of GAPDH and Siah1. Apoptosis was measured by Annexin V staining and caspase-3 enzymatic activity assay. High glucose increased Siah1 total protein levels, induced the association between GAPDH and Siah1, and led to GAPDH nuclear translocation. Our findings demonstrate that dissociation of the GAPDH/Siah1 pro-apoptotic complex can block high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Thus, the work presented in this manuscript can provide a foundation to identify novel targets for early treatment of DR.