Chaperone-Assisted Post-Translational Transport of Plastidic Type I Signal Peptidase 1 [Membrane Biology]

October 7th, 2015 by Endow, J. K., Singhal, R., Fernandez, D. E., Inoue, K.

Type I signal peptidase (SPase I) is an integral membrane Ser/Lys-protease with one or two transmembrane domains (TMDs), cleaving transport signals off translocated precursor proteins. The catalytic domain of SPase I folds to form a hydrophobic surface and inserts into the lipid bilayers at the trans-side of the membrane. In bacteria, SPase I is targeted co-translationally and the catalytic domain remains unfolded until it reaches the periplasm. By contrast, SPases I in eukaryotes are targeted post-translationally, requiring an alternative strategy to prevent premature folding. Here we demonstrate that two distinct stromal components are involved in posttranslational transport of plastidic SPase I (Plsp1) from Arabidopsis thaliana which contains a single TMD. During import into isolated chloroplasts, Plsp1 was targeted to the membrane via a soluble intermediate in an ATP-hydrolysis dependent manner. Insertion of Plsp1 into isolated chloroplast membranes, by contrast, was found to occur by two distinct mechanisms. The first mechanism requires ATP hydrolysis and the protein conducting channel cpSecY1, and was strongly enhanced by exogenously-added cpSecA1. The second mechanism was independent of nucleoside triphosphates and proteinaceous components but with a high frequency of mis-orientation. This unassisted insertion was inhibited by urea and stroma extract. During import-chase assays using intact chloroplasts, Plsp1 was incorporated into a soluble 700-kD complex that co-migrated with the CPN60 complex before inserting into the membrane. The TMD within Plsp1 was required for the cpSecA1-dependent insertion but was dispensable for association with the 700-kD complex and also for unassisted membrane insertion. These results indicate cooperation of Cpn60 and cpSecA1 for proper membrane insertion of Plsp1 by cpSecY1.

Porphyrin Binding to Gun4 protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway [Computational Biology]

October 7th, 2015 by

In oxygenic phototrophs, chlorophylls, hemes and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated and an important regulatory role is attributed to Mg-chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the Mg-chelatase activity but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for Mg-protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding and the magnesium ion held within the porphyrin is coordinated by Asn211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in Mg-chelatase assay showing that tight porphyrin-binding in Gun4 facilitates its interaction with the Mg-chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.
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MAPK Activated by Prostaglandin E2 Phosphorylates Connexin 43 and Closes Osteocytic Hemichannels in Response to Continuous Flow Shear Stress [Cell Biology]

October 6th, 2015 by Riquelme, M. A., Burra, S., Kar, R., Lampe, P. D., Jiang, J. X.

Cx43 hemichannels serve as a portal for the release of prostaglandins, a critical process in mediating biological responses of mechanical loading on bone formation and remodeling. We have previously observed that fluid flow shear stress (FFSS) opens hemichannels; however sustained FFSS results in hemichannel closure, as continuous opening of hemichannels is detrimental to cell viability and bone remodeling. However, the mechanism that regulates the closure of the hemichannels is unknown. Here, we show that activation of p44/42 ERK upon continuous FFSS leads to Cx43 phosphorylation at S279/282, sites known to be phosphorylated sites by p44/42 MAPK. Incubation of osteocytic MLO-Y4 cells with conditioned media (CM) collected after continuous FFSS increased MAPK-dependent phosphorylation of Cx43. CM treatment inhibited hemichannel opening and this inhibition was reversed when cells were pre-treated with MAPK pathway inhibitor. We found that PGE2 accumulates in the CM in a time-dependent manner. Treatment with PGE2 increased phospho-p44/42 ERK levels and also Cx43 phosphorylation at S279/282 sites. Depletion of PGE2 from CM and pre-treatment with a p44/42 ERK pathway specific inhibitor, resulted in a complete inhibition of ERK-dependent Cx43 phosphorylation and attenuated the inhibition of hemichannels by CM and PGE2. Consistently, the opening of hemichannels by FFSS was blocked by PGE2 and CM and this blockage was reversed by U0126 and the CM depleted of PGE2. Similar observation was also obtained in isolated primary osteocytes. Together, results from this study suggest that extracellular PGE2 accumulated after continuous FFSS is responsible for activation of p44/42 ERK signaling and subsequently, direct Cx43 phosphorylation by activated ERK leads to hemichannel closure.
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Tumor suppressive function of p21-activated kinase 6 in hepatocellular carcinoma [Gene Regulation]

October 6th, 2015 by Liu, W., Liu, Y., Liu, H., Zhang, W., Fu, Q., Xu, J., Gu, J.

Our previous studies identified the oncogenic role of p21-activated kinase 1 (PAK1) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Contrarily, PAK6 was found to predict favorable prognosis in RCC patients. Nevertheless, the ambiguous tumor suppressive function of PAK6 in hepatocarcinogenesis remains obscure. Herein, decreased PAK6 expression was found to be associated with Tumor Node Metastasis (TNM) stage progression and unfavorable overall survival (OS) in HCC patients. Additionally, overexpression and silence of PAK6 experiments showed that PAK6 inhibited xenografted tumor growth in vivo, and restricted cell proliferation, colony formation, migration and invasion and promoted cell apoptosis and anoikis in vitro. Moreover, overexpression of kinase dead and nuclear localization signal (NLS) deletion mutants of PAK6 experiments indicated tumor suppressive function of PAK6 was dependent on its kinase activity and nuclear translocation partially. Furthermore, gain or loss of function in polycomb repressive complex 2 (PRC2) components including EZH2, SUZ12 and EED elucidated that epigenetic control of H3K27me3 arbitrated PAK6 downregulation in hepatoma cells. More importantly, negative correlation between PAK6 and EZH2 expression was observed in hepatoma tissues from HCC patients. These data identified the tumor suppressive role and potential underlying mechanism of PAK6 in hepatocarcinogenesis.

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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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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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.

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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