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Journal Cover Journal of Biological Chemistry
  [SJR: 3.151]   [H-I: 435]   [206 followers]  Follow
   Full-text available via subscription Subscription journal
   ISSN (Print) 0021-9258 - ISSN (Online) 1083-351X
   Published by ASBMB Homepage  [3 journals]
  • Checkpoint kinase 1-induced phosphorylation of O-linked
           {beta}-N-acetylglucosamine transferase regulates the intermediate filament
           network during cytokinesis [Cell Biology]
    • Authors: Zhe Li; Xueyan Li, Shanshan Nai, Qizhi Geng, Ji Liao, Xingzhi Xu, Jing Li
      Pages: 19548 - 19555
      Abstract: Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified O-linked β-N-acetylglucosamine (O-GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT–pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular O-GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an siOGT-based approach. Lastly, expression of plasmids resistant to siOGT efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1–OGT–vimentin pathway that regulates the intermediate filament network during cytokinesis.
      PubDate: 2017-12-01T00:06:01-08:00
      DOI: 10.1074/jbc.M117.811646
      Issue No: Vol. 292, No. 48 (2017)
  • Quantitative time-course metabolomics in human red blood cells reveal the
           temperature dependence of human metabolic networks [Computational Biology]
    • Authors: James T. Yurkovich; Daniel C. Zielinski, Laurence Yang, Giuseppe Paglia, Ottar Rolfsson, Olafur E. Sigurȷonsson, Jared T. Broddrick, Aarash Bordbar, Kristine Wichuk, Sigurthur Brynȷolfsson, Sirus Palsson, Sveinn Gudmundsson, Bernhard O. Palsson
      Pages: 19556 - 19564
      Abstract: The temperature dependence of biological processes has been studied at the levels of individual biochemical reactions and organism physiology (e.g. basal metabolic rates) but has not been examined at the metabolic network level. Here, we used a systems biology approach to characterize the temperature dependence of the human red blood cell (RBC) metabolic network between 4 and 37 °C through absolutely quantified exo- and endometabolomics data. We used an Arrhenius-type model (Q10) to describe how the rate of a biochemical process changes with every 10 °C change in temperature. Multivariate statistical analysis of the metabolomics data revealed that the same metabolic network-level trends previously reported for RBCs at 4 °C were conserved but accelerated with increasing temperature. We calculated a median Q10 coefficient of 2.89 ± 1.03, within the expected range of 2–3 for biological processes, for 48 individual metabolite concentrations. We then integrated these metabolomics measurements into a cell-scale metabolic model to study pathway usage, calculating a median Q10 coefficient of 2.73 ± 0.75 for 35 reaction fluxes. The relative fluxes through glycolysis and nucleotide metabolism pathways were consistent across the studied temperature range despite the non-uniform distributions of Q10 coefficients of individual metabolites and reaction fluxes. Together, these results indicate that the rate of change of network-level responses to temperature differences in RBC metabolism is consistent between 4 and 37 °C. More broadly, we provide a baseline characterization of a biochemical network given no transcriptional or translational regulation that can be used to explore the temperature dependence of metabolism.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.804914
      Issue No: Vol. 292, No. 48 (2017)
  • Phosphomimetic S3D cofilin binds but only weakly severs actin filaments
    • Authors: W. Austin Elam; Wenxiang Cao, Hyeran Kang, Andrew Huehn, Glen M. Hocky, Ewa Prochniewicz, Anthony C. Schramm, Karina Negron, Jean Garcia, Teresa T. Bonello, Peter W. Gunning, David D. Thomas, Gregory A. Voth, Charles V. Sindelar, Enrique M. De La Cruz
      Pages: 19565 - 19579
      Abstract: Many biological processes, including cell division, growth, and motility, rely on rapid remodeling of the actin cytoskeleton and on actin filament severing by the regulatory protein cofilin. Phosphorylation of vertebrate cofilin at Ser-3 regulates both actin binding and severing. Substitution of serine with aspartate at position 3 (S3D) is widely used to mimic cofilin phosphorylation in cells and in vitro. The S3D substitution weakens cofilin binding to filaments, and it is presumed that subsequent reduction in cofilin occupancy inhibits filament severing, but this hypothesis has remained untested. Here, using time-resolved phosphorescence anisotropy, electron cryomicroscopy, and all-atom molecular dynamics simulations, we show that S3D cofilin indeed binds filaments with lower affinity, but also with a higher cooperativity than wild-type cofilin, and severs actin weakly across a broad range of occupancies. We found that three factors contribute to the severing deficiency of S3D cofilin. First, the high cooperativity of S3D cofilin generates fewer boundaries between bare and decorated actin segments where severing occurs preferentially. Second, S3D cofilin only weakly alters filament bending and twisting dynamics and therefore does not introduce the mechanical discontinuities required for efficient filament severing at boundaries. Third, Ser-3 modification (i.e. substitution with Asp or phosphorylation) “undocks” and repositions the cofilin N terminus away from the filament axis, which compromises S3D cofilin's ability to weaken longitudinal filament subunit interactions. Collectively, our results demonstrate that, in addition to inhibiting actin binding, Ser-3 modification favors formation of a cofilin-binding mode that is unable to sufficiently alter filament mechanical properties and promote severing.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.808378
      Issue No: Vol. 292, No. 48 (2017)
  • A conserved tryptophan within the WRDPLVDID domain of yeast Pah1
           phosphatidate phosphatase is required for its in vivo function in lipid
           metabolism [Lipids]
    • Authors: Yeonhee Park; Gil-Soo Han, George M. Carman
      Pages: 19580 - 19589
      Abstract: PAH1-encoded phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol at the endoplasmic reticulum membrane, plays a major role in controlling the utilization of phosphatidate for the synthesis of triacylglycerol or membrane phospholipids. The conserved N-LIP and haloacid dehalogenase–like domains of Pah1 are required for phosphatidate phosphatase activity and the in vivo function of the enzyme. Its non-conserved regions, which are located between the conserved domains and at the C terminus, contain sites for phosphorylation by multiple protein kinases. Truncation analyses of the non-conserved regions showed that they are not essential for the catalytic activity of Pah1 and its physiological functions (e.g. triacylglycerol synthesis). This analysis also revealed that the C-terminal region contains a previously unrecognized WRDPLVDID domain (residues 637-645) that is conserved in yeast, mice, and humans. The deletion of this domain had no effect on the catalytic activity of Pah1 but caused the loss of its in vivo function. Site-specific mutational analyses of the conserved residues within WRDPLVDID indicated that Trp-637 plays a crucial role in Pah1 function. This work also demonstrated that the catalytic activity of Pah1 is required but is not sufficient for its in vivo functions.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.819375
      Issue No: Vol. 292, No. 48 (2017)
  • Transcriptome analysis reveals determinant stages controlling human
           embryonic stem cell commitment to neuronal cells [Developmental Biology]
    • Authors: Yuanyuan Li; Ran Wang, Nan Qiao, Guangdun Peng, Ke Zhang, Ke Tang, Jing-Dong J. Han, Naihe Jing
      Pages: 19590 - 19604
      Abstract: Proper neural commitment is essential for ensuring the appropriate development of the human brain and for preventing neurodevelopmental diseases such as autism spectrum disorders, schizophrenia, and intellectual disorders. However, the molecular mechanisms underlying the neural commitment in humans remain elusive. Here, we report the establishment of a neural differentiation system based on human embryonic stem cells (hESCs) and on comprehensive RNA sequencing analysis of transcriptome dynamics during early hESC differentiation. Using weighted gene co-expression network analysis, we reveal that the hESC neurodevelopmental trajectory has five stages: pluripotency (day 0); differentiation initiation (days 2, 4, and 6); neural commitment (days 8–10); neural progenitor cell proliferation (days 12, 14, and 16); and neuronal differentiation (days 18, 20, and 22). These stages were characterized by unique module genes, which may recapitulate the early human cortical development. Moreover, a comparison of our RNA-sequencing data with several other transcriptome profiling datasets from mice and humans indicated that Module 3 associated with the day 8–10 stage is a critical window of fate switch from the pluripotency to the neural lineage. Interestingly, at this stage, no key extrinsic signals were activated. In contrast, using CRISPR/Cas9–mediated gene knockouts, we also found that intrinsic hub transcription factors, including the schizophrenia-associated SIX3 gene and septo-optic dysplasia-related HESX1 gene, are required to program hESC neural determination. Our results improve the understanding of the mechanism of neural commitment in the human brain and may help elucidate the etiology of human mental disorders and advance therapies for managing these conditions.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.796383
      Issue No: Vol. 292, No. 48 (2017)
  • Stem cells take the stairs [Developmental Biology]
    • Authors: Caroline Vissers; Guo-li Ming, Hongjun Song
      Pages: 19605 - 19606
      Abstract: Human embryonic stem cells progress through multiple stages in their path to neural differentiation, but the steps taken along the way are difficult to distinguish, limiting our understanding of this important process. Jing and colleagues (2) now report comprehensive analyses of transcriptome dynamics during this process that reveal five discrete stages, defined in part by highly connected transcription factor networks that link progressive stages. Surprisingly, the third stage, which appears to be critical for neural fate commitment, depends almost entirely on intracellular signaling.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.H117.796383
      Issue No: Vol. 292, No. 48 (2017)
  • Reactivation of mutant p53 and induction of apoptosis in human tumor cells
           by maleimide analogs. [Additions and Corrections]
    • Authors: Vladimir J. N. Bykov; Natalia Issaeva, Nicole Zache, Alexandre Shilov, Monica Hultcrantz, Jan Bergman, Galina Selivanova, Klas G. Wiman
      Pages: 19607 - 19607
      Abstract: VOLUME 280 (2005) PAGES 30384–30391We would like to acknowledge that the controls shown in Fig. 5, B and C, and Fig. 6C have also been used in a previous publication (Bykov, V. J., Issaeva, N., Shilov, A., Hultcrantz, M., Pugacheva, E., Chumakov, P., Bergman, J., Wiman, K. G., and Selivanova, G. (2002) Restoration of the tumor suppressor function to mutant p53 by a low-molecular-weight compound. Nat. Med. 8, 282–288). In Fig. 5, B and C, the control lanes in the gel shift assays, i.e. no treatment and supershift with PAb421 or PAb1801 antibody in the absence of test compound, are the same as those shown in the previous paper. Likewise, in Fig. 6C, the p53 immunostaining and Hoechst staining of untreated control cells is the same as that shown in the previous paper. The reason for this mistake is that we examined the two novel mutant p53-targeting compounds PRIMA-1 and MIRA-1 side by side in several assays and then decided to publish our results in two separate papers. However, it should be noted that these controls can serve equally well as controls for both compounds. Therefore, this does not change the validity of the data nor the conclusions from the experiments.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.AAC117.000815
      Issue No: Vol. 292, No. 48 (2017)
  • Large-scale analysis of variation in the insulin-like growth factor family
           in humans reveals rare disease links and common polymorphisms. [Additions
           and Corrections]
    • Authors: Peter Rotwein
      Pages: 19608 - 19608
      Abstract: VOLUME 292 (2017) PAGES 9252–9261PAGE 9254:There was an error in the text on page 9254, right column, second paragraph. “… most of this could be attributed to a single non-coding C to T change located 5′ to the adjacent INS gene (see single nucleotide polymorphism (SNP): rs14948363)” should be changed as follows. “… most of this could be attributed to a single non-coding C to T change located 3′ to the adjacent INS gene (see single nucleotide polymorphism (SNP): rs149483638).”
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.AAC117.000854
      Issue No: Vol. 292, No. 48 (2017)
  • Cdh1, a substrate-recruiting component of anaphase-promoting
           complex/cyclosome (APC/C) ubiquitin E3 ligase, specifically interacts with
           phosphatase and tensin homolog (PTEN) and promotes its removal from
           chromatin. [Additions and Corrections]
    • Authors: Byeong Hyeok Choi; Michele Pagano, Chuanshu Huang, Wei Dai
      Pages: 19609 - 19609
      Abstract: VOLUME 289 (2014) PAGES 17951–17959PAGE 17955:Six lanes representing Flag-Cdh1 from the lysate input of Fig. 4C of the originally published version of this paper were mistakenly used and repeated in the IP:Flag lanes. This figure has now been corrected with the original experimental images. This correction does not affect the results or conclusions of this work.jbc;292/48/19609/FU1F1FU1
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.AAC117.000864
      Issue No: Vol. 292, No. 48 (2017)
  • Role for ribosome-associated complex and stress-seventy subfamily B
           (RAC-Ssb) in integral membrane protein translation [Protein Structure and
    • Authors: Ligia Acosta–Sampson; Kristina Doring, Yuping Lin, Vivian Y. Yu, Bernd Bukau, Gunter Kramer, Jamie H. D. Cate
      Pages: 19610 - 19627
      Abstract: Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex–stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.813857
      Issue No: Vol. 292, No. 48 (2017)
  • Defining the interaction of the protease CpaA with its type II secretion
           chaperone CpaB and its contribution to virulence in Acinetobacter species
    • Authors: Rachel L. Kinsella; Juvenal Lopez, Lauren D. Palmer, Nichole D. Salinas, Eric P. Skaar, Niraj H. Tolia, Mario F. Feldman
      Pages: 19628 - 19638
      Abstract: Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are a frequent cause of multidrug-resistant, healthcare-associated infections. Our previous work demonstrated that A. nosocomialis M2 possesses a functional type II secretion system (T2SS) that is required for full virulence. Further, we identified the metallo-endopeptidase CpaA, which has been shown previously to cleave human Factor V and deregulate blood coagulation, as the most abundant type II secreted effector protein. We also demonstrated that its secretion is dependent on CpaB, a membrane-bound chaperone. In this study, we show that CpaA expression and secretion are conserved across several medically relevant Acinetobacter species. Additionally, we demonstrate that deletion of cpaA results in attenuation of A. nosocomialis M2 virulence in moth and mouse models. The virulence defects resulting from the deletion of cpaA were comparable with those observed upon abrogation of T2SS activity. The virulence defects resulting from the deletion of cpaA are comparable with those observed upon abrogation of T2SS activity. We also show that CpaA and CpaB strongly interact, forming a complex in a 1:1 ratio. Interestingly, deletion of the N-terminal transmembrane domain of CpaB results in robust secretion of CpaA and CpaB, indicating that the transmembrane domain is dispensable for CpaA secretion and likely functions to retain CpaB inside the cell. Limited proteolysis of spheroplasts revealed that the C-terminal domain of CpaB is exposed to the periplasm, suggesting that this is the site where CpaA and CpaB interact in vivo. Last, we show that CpaB does not abolish the proteolytic activity of CpaA against human Factor V. We conclude that CpaA is, to the best of our knowledge, the first characterized, bona fide virulence factor secreted by Acinetobacter species.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.808394
      Issue No: Vol. 292, No. 48 (2017)
  • Molecular mechanisms for enhancement of stromal cell-derived factor
           1-induced chemotaxis by platelet endothelial cell adhesion molecule 1
           (PECAM-1) [Cell Biology]
    • Authors: Yoshihiro Umezawa; Hiroki Akiyama, Keigo Okada, Shinya Ishida, Ayako Nogami, Gaku Oshikawa, Tetsuya Kurosu, Osamu Miura
      Pages: 19639 - 19655
      Abstract: Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell adhesion protein involved in the regulation of cell adhesion and migration. Interestingly, several PECAM-1–deficient hematopoietic cells exhibit impaired chemotactic responses to stromal cell-derived factor 1 (SDF-1), a chemokine essential for B lymphopoiesis and bone marrow myelopoiesis. However, whether PECAM-1 is involved in SDF-1–regulated chemotaxis is unknown. We report here that SDF-1 induces tyrosine phosphorylation of PECAM-1 at its immunoreceptor tyrosine-based inhibition motifs in several hematopoietic cell lines via the Src family kinase Lyn, Bruton's tyrosine kinase, and JAK2 and that inhibition of these kinases reduced chemotaxis. Overexpression and knockdown of PECAM-1 enhanced and down-regulated, respectively, SDF-1–induced Gαi-dependent activation of the PI3K/Akt/mTORC1 pathway and small GTPase Rap1 in hematopoietic 32Dcl3 cells, and these changes in activation correlated with chemotaxis. Furthermore, pharmacological or genetic inhibition of the PI3K/Akt/mTORC1 pathway or Rap1, respectively, revealed that these pathways are independently activated and required for SDF-1–induced chemotaxis. When coexpressed in 293T cells, PECAM-1 physically associated with the SDF-1 receptor CXCR4. Moreover, PECAM-1 overexpression and knockdown reduced and enhanced SDF-1–induced endocytosis of CXCR4, respectively. Furthermore, when expressed in 32Dcl3 cells, an endocytosis-defective CXCR4 mutant, CXCR4-S324A/S325A, could activate the PI3K/Akt/mTORC1 pathway as well as Rap1 and induce chemotaxis in a manner similar to PECAM-1 overexpression. These findings suggest that PECAM-1 enhances SDF-1–induced chemotaxis by augmenting and prolonging activation of the PI3K/Akt/mTORC1 pathway and Rap1 and that PECAM-1, at least partly, exerts its activity by inhibiting SDF-1–induced internalization of CXCR4.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.779603
      Issue No: Vol. 292, No. 48 (2017)
  • Inhibition of the sarco/endoplasmic reticulum (ER) Ca2+-ATPase by
           thapsigargin analogs induces cell death via ER Ca2+ depletion and the
           unfolded protein response [Cell Biology]
    • Authors: Pankaȷ Sehgal; Paula Szalai, Claus Olesen, Helle A. Praetorius, Poul Nissen, Soren Brogger Christensen, Nikolai Engedal, Jesper V. Moller
      Pages: 19656 - 19673
      Abstract: Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 μm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca2+ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2–4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover, knockdown of two key store-operated Ca2+ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca2+ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca2+ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca2+ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca2+ levels and SOCE are not required.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.796920
      Issue No: Vol. 292, No. 48 (2017)
  • The hnRNP RALY regulates transcription and cell proliferation by
           modulating the expression of specific factors including the proliferation
           marker E2F1 [Gene Regulation]
    • Authors: Nicola Cornella; Toma Tebaldi, Lisa Gasperini, Jarnail Singh, Richard A. Padgett, Annalisa Rossi, Paolo Macchi
      Pages: 19674 - 19692
      Abstract: The heterogeneous nuclear ribonucleoproteins (hnRNP) form a large family of RNA-binding proteins that exert numerous functions in RNA metabolism. RALY is a member of the hnRNP family that binds poly-U–rich elements within several RNAs and regulates the expression of specific transcripts. RALY is up-regulated in different types of cancer, and its down-regulation impairs cell cycle progression. However, the RALY's role in regulating RNA levels remains elusive. Here, we show that numerous genes coding for factors involved in transcription and cell cycle regulation exhibit an altered expression in RALY–down-regulated HeLa cells, consequently causing impairments in transcription, cell proliferation, and cell cycle progression. Interestingly, by comparing the list of RALY targets with the list of genes affected by RALY down-regulation, we found an enrichment of RALY mRNA targets in the down-regulated genes upon RALY silencing. The affected genes include the E2F transcription factor family. Given its role as proliferation-promoting transcription factor, we focused on E2F1. We demonstrate that E2F1 mRNA stability and E2F1 protein levels are reduced in cells lacking RALY expression. Finally, we also show that RALY interacts with transcriptionally active chromatin in both an RNA-dependent and -independent manner and that this association is abolished in the absence of active transcription. Taken together, our results highlight the importance of RALY as an indirect regulator of transcription and cell cycle progression through the regulation of specific mRNA targets, thus strengthening the possibility of a direct gene expression regulation exerted by RALY.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.795591
      Issue No: Vol. 292, No. 48 (2017)
  • The SecA protein deeply penetrates into the SecYEG channel during
           insertion, contacting most channel transmembrane helices and periplasmic
           regions [Cell Biology]
    • Authors: Tithi Banerjee; Zeliang Zheng, Jane Abolafia, Shelby Harper, Donald Oliver
      Pages: 19693 - 19707
      Abstract: The bacterial Sec-dependent system is the major protein-biogenic pathway for protein secretion across the cytoplasmic membrane or insertion of integral membrane proteins into the phospholipid bilayer. The mechanism of SecA-driven protein transport across the SecYEG channel complex has remained controversial with conflicting claims from biochemical and structural studies regarding the depth and extent of SecA insertion into SecYEG during ongoing protein transport. Here we utilized site-specific in vivo photo-crosslinking to thoroughly map SecY regions that are in contact with SecA during its insertion cycle. An arabinose-inducible, rapidly folding OmpA-GFP chimera was utilized to jam the SecYEG channels with an arrested substrate protein to “freeze” them in their SecA-inserted state. Examination of 117 sites distributed throughout SecY indicated that SecA not only interacts extensively with the cytosolic regions of SecY as shown previously, but it also interacts with most of the transmembrane helices and periplasmic regions of SecY, with a clustering of interaction sights around the lateral gate and pore ring regions. Our observations support previous reports of SecA membrane insertion during in vitro protein transport as well as those documenting the membrane penetration properties of this protein. They suggest that one or more SecA regions transiently integrate into the heart of the SecY channel complex to span the membrane to promote the protein transport cycle. These findings indicate that high-resolution structural information about the membrane-inserted state of SecA is still lacking and will be critical for elucidating the bacterial protein transport mechanism.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.RA117.000130
      Issue No: Vol. 292, No. 48 (2017)
  • Activation and mechanism of a cryptic oviedomycin gene cluster via the
           disruption of a global regulatory gene, adpA, in Streptomyces
           ansochromogenes [Gene Regulation]
    • Authors: Jingjing Xu; Jihui Zhang, Jiming Zhuo, Yue Li, Yuqing Tian, Huarong Tan
      Pages: 19708 - 19720
      Abstract: Genome sequencing analysis has revealed at least 35 clusters of likely biosynthetic genes for secondary metabolites in Streptomyces ansochromogenes. Disruption of adpA encoding a global regulator (AdpA) resulted in the failure of nikkomycin production, whereas other antibacterial activities against Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis were observed with the fermentation broth of ΔadpA but not with that of the wild-type strain. Transcriptional analysis showed that a cryptic gene cluster (pks7), which shows high identity with an oviedomycin biosynthetic gene cluster (ovm), was activated in ΔadpA. The corresponding product of pks7 was characterized as oviedomycin by MS and NMR spectroscopy. To understand the molecular mechanism of ovm activation, the roles of six regulatory genes situated in the ovm cluster were investigated. Among them, proteins encoded by co-transcribed genes ovmZ and ovmW are positive regulators of ovm. AdpA directly represses the transcription of ovmZ and ovmW. Co-overexpression of ovmZ and ovmW can relieve the repression of AdpA on ovm transcription and effectively activate oviedomycin biosynthesis. The promoter of ovmOI–ovmH is identified as the direct target of OvmZ and OvmW. This is the first report that AdpA can simultaneously activate nikkomycin biosynthesis but repress oviedomycin biosynthesis in one strain. Our findings provide an effective strategy that is able to activate cryptic secondary metabolite gene clusters by genetic manipulation of global regulatory genes.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.809145
      Issue No: Vol. 292, No. 48 (2017)
  • Cystine uptake through the cystine/glutamate antiporter xCT triggers
           glioblastoma cell death under glucose deprivation [Molecular Bases of
    • Authors: Takeo Goji; Kazuhiko Takahara, Manabu Negishi, Hironori Katoh
      Pages: 19721 - 19732
      Abstract: Oncogenic signaling in cancer cells alters glucose uptake and utilization to supply sufficient energy and biosynthetic intermediates for survival and sustained proliferation. Oncogenic signaling also prevents oxidative stress and cell death caused by increased production of reactive oxygen species. However, elevated glucose metabolism in cancer cells, especially in glioblastoma, results in the cells becoming sensitive to glucose deprivation (i.e. in high glucose dependence), which rapidly induces cell death. However, the precise mechanism of this type of cell death remains unknown. Here, we report that glucose deprivation alone does not trigger glioblastoma cell death. We found that, for cell death to occur in glucose-deprived glioblastoma cells, cystine and glutamine also need to be present in culture media. We observed that cystine uptake through the cystine/glutamate antiporter xCT under glucose deprivation rapidly induces NADPH depletion, reactive oxygen species accumulation, and cell death. We conclude that although cystine uptake is crucial for production of antioxidant glutathione in cancer cells its transport through xCT also induces oxidative stress and cell death in glucose-deprived glioblastoma cells. Combining inhibitors targeting cancer-specific glucose metabolism with cystine and glutamine treatment may offer a therapeutic approach for glioblastoma tumors exhibiting high xCT expression.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.814392
      Issue No: Vol. 292, No. 48 (2017)
  • Pyruvate dehydrogenase complex (PDC) subunits moonlight as interaction
           partners of phosphorylated STAT5 in adipocytes and adipose tissue [Gene
    • Authors: Allison J. Richard; Hardy Hang, Jacqueline M. Stephens
      Pages: 19733 - 19742
      Abstract: STAT5 proteins play a role in adipocyte development and function, but their specific functions are largely unknown. To this end, we used an unbiased MS-based approach to identify novel STAT5-interacting proteins. We observed that STAT5A bound the E1β and E2 subunits of the pyruvate dehydrogenase complex (PDC). Whereas STAT5A typically localizes to the cytosol or nucleus, PDC normally resides within the mitochondrial matrix where it converts pyruvate to acetyl-CoA. We employed affinity purification and immunoblotting to validate the interaction between STAT5A and PDC subunits in murine and human cultured adipocytes, as well as in adipose tissue. We found that multiple PDC subunits interact with hormone-activated STAT5A in a dose- and time-dependent manner that coincides with tyrosine phosphorylation of STAT5. Using subcellular fractionation and immunofluorescence microscopy, we observed that PDC-E2 is present within the adipocyte nucleus where it associates with STAT5A. Because STAT5A is a transcription factor, we used chromatin immunoprecipitation (ChIP) to assess PDC's ability to interact with STAT5 DNA-binding sites. These analyses revealed that PDC-E2 is bound to a STAT5-binding site in the promoter of the STAT5 target gene cytokine-inducible SH2-containing protein (cish). We have demonstrated a compelling interaction between STAT5A and PDC subunits in adipocytes under physiological conditions. There is previous evidence that PDC localizes to cancer cell nuclei where it plays a role in histone acetylation. On the basis of our ChIP data and these previous findings, we hypothesize that PDC may modulate STAT5's ability to regulate gene expression by controlling histone or STAT5 acetylation.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.811794
      Issue No: Vol. 292, No. 48 (2017)
  • S-Nitrosylation inhibits the kinase activity of tomato
           phosphoinositide-dependent kinase 1 (PDK1) [Plant Biology]
    • Authors: Jian-Zhong Liu; Jicheng Duan, Min Ni, Zhen Liu, Wen-Li Qiu, Steven A. Whitham, Wei-Jun Qian
      Pages: 19743 - 19751
      Abstract: It is well known that the reactive oxygen species NO can trigger cell death in plants and other organisms, but the underlying molecular mechanisms are not well understood. Here we provide evidence that NO may trigger cell death in tomato (Solanum lycopersicum) by inhibiting the activity of phosphoinositide-dependent kinase 1 (SlPDK1), a conserved negative regulator of cell death in yeasts, mammals, and plants, via S-nitrosylation. Biotin-switch assays indicated that SlPDK1 is a target of S-nitrosylation. Moreover, the kinase activity of SlPDK1 was inhibited by S-nitrosoglutathione in a concentration-dependent manner, indicating that SlPDK1 activity is abrogated by S-nitrosylation. The S-nitrosoglutathione–induced inhibition was reversible in the presence of a reducing agent but additively enhanced by hydrogen peroxide (H2O2). Our LC-MS/MS analyses further indicated that SlPDK1 is primarily S-nitrosylated on a cysteine residue at position 128 (Cys128), and substitution of Cys128 with serine completely abolished SlPDK1 kinase activity, suggesting that S-nitrosylation of Cys128 is responsible for SlPDK1 inhibition. In summary, our results establish a potential link between NO-triggered cell death and inhibition of the kinase activity of tomato PDK1.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.803882
      Issue No: Vol. 292, No. 48 (2017)
  • Structural analysis of the STAT1:STAT2 heterodimer revealed the mechanism
           of Sendai virus C protein-mediated blockade of type 1 interferon signaling
           [Protein Structure and Folding]
    • Authors: Kosuke Oda; Takashi Oda, Yasuyuki Matoba, Mamoru Sato, Takashi Irie, Takemasa Sakaguchi
      Pages: 19752 - 19766
      Abstract: Sendai virus (SeV), which causes respiratory diseases in rodents, possesses the C protein that blocks the signal transduction of interferon (IFN), thereby escaping from host innate immunity. We previously demonstrated by using protein crystallography that two molecules of Y3 (the C-terminal half of the C protein) can bind to the homodimer of the N-terminal domain of STAT1 (STAT1ND), elucidating the mechanism of inhibition of IFN-γ signal transduction. SeV C protein also blocks the signal transduction of IFN-α/β by inhibiting the phosphorylation of STAT1 and STAT2, although the mechanism for the inhibition is unclear. Therefore, we sought to elucidate the mechanism of inhibition of the IFN signal transduction via STAT1 and STAT2. Small angle X-ray scattering analysis indicated that STAT1ND associates with the N-terminal domain of STAT2 (STAT2ND) with the help of a Gly-rich linker. We generated a linker-less recombinant protein possessing a STAT1ND:STAT2ND heterodimeric structure via an artificial disulfide bond. Analytical size-exclusion chromatography and surface plasmon resonance revealed that one molecule of Y3 can associate with a linker-less recombinant protein. We propose that one molecule of C protein associates with the STAT1:STAT2 heterodimer, inducing a conformational change to an antiparallel form, which is easily dephosphorylated. This suggests that association of C protein with the STAT1ND:STAT2ND heterodimer is an important factor to block the IFN-α/β signal transduction.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.786285
      Issue No: Vol. 292, No. 48 (2017)
  • Sirtuin 5 is required for mouse survival in response to cardiac pressure
           overload [Molecular Bases of Disease]
    • Authors: Kathleen A. Hershberger; Dennis M. Abraham, Angelical S. Martin, Lan Mao, Juan Liu, Hongbo Gu, Jason W. Locasale, Matthew D. Hirschey
      Pages: 19767 - 19781
      Abstract: In mitochondria, the sirtuin SIRT5 is an NAD+-dependent protein deacylase that controls several metabolic pathways. Although a wide range of SIRT5 targets have been identified, the overall function of SIRT5 in organismal metabolic homeostasis remains unclear. Given that SIRT5 expression is highest in the heart and that sirtuins are commonly stress-response proteins, we used an established model of pressure overload–induced heart muscle hypertrophy caused by transverse aortic constriction (TAC) to determine SIRT5's role in cardiac stress responses. Remarkably, SIRT5KO mice had reduced survival upon TAC compared with wild-type mice but exhibited no mortality when undergoing a sham control operation. The increased mortality with TAC was associated with increased pathological hypertrophy and with key abnormalities in both cardiac performance and ventricular compliance. By combining high-resolution MS-based metabolomic and proteomic analyses of cardiac tissues from wild-type and SIRT5KO mice, we found several biochemical abnormalities exacerbated in the SIRT5KO mice, including apparent decreases in fatty acid oxidation and glucose oxidation as well as an overall decrease in mitochondrial NAD+/NADH. Together, these abnormalities suggest that SIRT5 deacylates protein substrates involved in cellular oxidative metabolism to maintain mitochondrial energy production. Overall, the functional and metabolic results presented here suggest an accelerated development of cardiac dysfunction in SIRT5KO mice in response to TAC, explaining increased mortality upon cardiac stress. Our findings reveal a key role for SIRT5 in maintaining cardiac oxidative metabolism under pressure overload to ensure survival.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.809897
      Issue No: Vol. 292, No. 48 (2017)
  • A lower isoelectric point increases signal sequence-mediated secretion of
           recombinant proteins through a bacterial ABC transporter [Molecular
    • Authors: Hyunjong Byun; Jiyeon Park, Sun Chang Kim, Jung Hoon Ahn
      Pages: 19782 - 19791
      Abstract: Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter–mediated secretions.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.786749
      Issue No: Vol. 292, No. 48 (2017)
  • 3-Sulfogalactosyl-dependent adhesion of Escherichia coli HS multivalent
           adhesion molecule is attenuated by sulfatase activity [Molecular Bases of
    • Authors: Fitua Al-Saedi; Diana Pereira Vaz, Daniel H. Stones, Anne Marie Krachler
      Pages: 19792 - 19803
      Abstract: Bacterial adhesion to host receptors is an early and essential step in bacterial colonization, and the nature of adhesin–receptor interactions determines bacterial localization and thus the outcome of these interactions. Here, we determined the host receptors for the multivalent adhesion molecule (MAM) from the gut commensal Escherichia coli HS (MAMHS), which contains an array of seven mammalian cell entry domains. The MAMHS adhesin interacted with a range of host receptors, through recognition of a shared 3-O-sulfogalactosyl moiety. This functional group is also found in mucin, a component of the intestinal mucus layer and thus one of the prime adherence targets for commensal E. coli. Mucin gels impeded the motility of E. coli by acting as a physical barrier, and the barrier effect was enhanced by specific interactions between mucin and MAMHS in a sulfation-dependent manner. Desulfation of mucin by pure sulfatase or the sulfatase-producing commensal Bacteroides thetaiotaomicron decreased binding of E. coli to mucin and increased the attachment of bacteria to the epithelial surface via interactions with surface-localized sulfated lipid and protein receptors. Together, our results demonstrate that the E. coli adhesin MAMHS facilitates retention of a gut commensal by attachment to mucin. They further suggest that the amount of sulfatase secreted by mucin-foraging bacteria such as B. thetaiotaomicron, inhabiting the same niche, may affect the capacity of the mucus barrier to retain commensal E. coli.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.817908
      Issue No: Vol. 292, No. 48 (2017)
  • AMP-activated protein kinase-mediated feedback phosphorylation controls
           the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase
           kinase {beta} [Enzymology]
    • Authors: Akihiro Nakanishi; Naoya Hatano, Yuya Fujiwara, Arian Sha'ri, Shota Takabatake, Hiroki Akano, Naoki Kanayama, Masaki Magari, Naohito Nozaki, Hiroshi Tokumitsu
      Pages: 19804 - 19813
      Abstract: The Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5′-AMP–activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr144 phosphorylation by activated AMPK converts CaMKKβ into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471–587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr144 phosphorylation–dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr144 antibody revealed phosphorylation of Thr144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKKβ.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.805085
      Issue No: Vol. 292, No. 48 (2017)
  • Resistance to pyridine-based inhibitor KF116 reveals an unexpected role of
           integrase in HIV-1 Gag-Pol polyprotein proteolytic processing [Molecular
           Bases of Disease]
    • Authors: Ashley C. Hoyte; Augusta V. Jamin, Pratibha C. Koneru, Matthew J. Kobe, Ross C. Larue, James R. Fuchs, Alan N. Engelman, Mamuka Kvaratskhelia
      Pages: 19814 - 19825
      Abstract: The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.816645
      Issue No: Vol. 292, No. 48 (2017)
  • Quantitative phosphoproteomics reveals involvement of multiple signaling
           pathways in early phagocytosis by the retinal pigmented epithelium
           [Protein Synthesis and Degradation]
    • Authors: Cheng-Kang Chiang; Aleksander Tworak, Brian M. Kevany, Bo Xu, Janice Mayne, Zhibin Ning, Daniel Figeys, Krzysztof Palczewski
      Pages: 19826 - 19839
      Abstract: One of the major biological functions of the retinal pigmented epithelium (RPE) is the clearance of shed photoreceptor outer segments (POS) through a multistep process resembling phagocytosis. RPE phagocytosis helps maintain the viability of photoreceptors that otherwise could succumb to the high metabolic flux and photo-oxidative stress associated with visual processing. The regulatory mechanisms underlying phagocytosis in the RPE are not fully understood, although dysfunction of this process contributes to the pathogenesis of multiple human retinal degenerative disorders, including age-related macular degeneration. Here, we present an integrated transcriptomic, proteomic, and phosphoproteomic analysis of phagocytosing RPE cells, utilizing three different experimental models: the human-derived RPE-like cell line ARPE-19, cultured murine primary RPE cells, and RPE samples from live mice. Our combined results indicated that early stages of phagocytosis in the RPE are mainly characterized by pronounced changes in the protein phosphorylation level. Global phosphoprotein enrichment analysis revealed involvement of PI3K/Akt, mechanistic target of rapamycin (mTOR), and MEK/ERK pathways in the regulation of RPE phagocytosis, confirmed by immunoblot analyses and in vitro phagocytosis assays. Most strikingly, phagocytosis of POS by cultured RPE cells was almost completely blocked by pharmacological inhibition of phosphorylation of Akt. Our findings, along with those of previous studies, indicate that these phosphorylation events allow the RPE to integrate multiple signals instigated by shed POS at different stages of the phagocytic process.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.812677
      Issue No: Vol. 292, No. 48 (2017)
  • Molecular characterization and verification of
           azido-3,8-dideoxy-d-manno-oct-2-ulosonic acid incorporation into bacterial
           lipopolysaccharide [Membrane Biology]
    • Authors: Inga Nilsson; Kerri Grove, Dustin Dovala, Tsuyoshi Uehara, Guillaume Lapointe, David A. Six
      Pages: 19840 - 19848
      Abstract: 3-Deoxy-d-manno-oct-2-ulosonic acid (Kdo) is an essential component of LPS in the outer leaflet of the Gram-negative bacterial outer membrane. Although labeling of Escherichia coli with the chemical reporter 8-azido-3,8-dideoxy-d-manno-oct-2-ulosonic acid (Kdo-N3) has been reported, its incorporation into LPS has not been directly shown. We have now verified Kdo-N3 incorporation into E. coli LPS at the molecular level. Using microscopy and PAGE analysis, we show that Kdo-N3 is localized to the outer membrane and specifically incorporates into rough and deep-rough LPS. In an E. coli strain lacking endogenous Kdo biosynthesis, supplementation with exogenous Kdo restored full-length core-LPS, which suggests that the Kdo biosynthetic pathways might not be essential in vivo in the presence of sufficient exogenous Kdo. In contrast, exogenous Kdo-N3 only restored a small fraction of core LPS with the majority incorporated into truncated LPS. The truncated LPS were identified as Kdo-N3–lipid IVA and (Kdo-N3)2–lipid IVA by MS analysis. The low level of Kdo-N3 incorporation could be partly explained by a 6-fold reduction in the specificity constant of the CMP-Kdo synthetase KdsB with Kdo-N3 compared with Kdo. These results indicate that the azido moiety in Kdo-N3 interferes with its utilization and may limit its utility as a tracer of LPS biosynthesis and transport in E. coli. We propose that our findings will be helpful for researchers using Kdo and its chemical derivatives for investigating LPS biosynthesis, transport, and assembly in Gram-negative bacteria.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.814962
      Issue No: Vol. 292, No. 48 (2017)
  • Isomer activation controls stereospecificity of class I
           fructose-1,6-bisphosphate aldolases [Enzymology]
    • Authors: Paul W. Heron; Jurgen Sygusch
      Pages: 19849 - 19860
      Abstract: Fructose-1,6-bisphosphate (FBP) aldolase, a glycolytic enzyme, catalyzes the reversible and stereospecific aldol addition of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (d-G3P) by an unresolved mechanism. To afford insight into the molecular determinants of FBP aldolase stereospecificity during aldol addition, a key ternary complex formed by DHAP and d-G3P, comprising 2% of the equilibrium population at physiological pH, was cryotrapped in the active site of Toxoplasma gondii aldolase crystals to high resolution. The growth of T. gondii aldolase crystals in acidic conditions enabled trapping of the ternary complex as a dominant population. The obligate 3(S)-4(R) stereochemistry at the nascent C3–C4 bond of FBP requires a si-face attack by the covalent DHAP nucleophile on the d-G3P aldehyde si-face in the active site. The cis-isomer of the d-G3P aldehyde, representing the dominant population trapped in the ternary complex, would lead to re-face attack on the aldehyde and yield tagatose 1,6-bisphosphate, a competitive inhibitor of the enzyme. We propose that unhindered rotational isomerization by the d-G3P aldehyde moiety in the ternary complex generates the active trans-isomer competent for carbonyl bond activation by active-site residues, thereby enabling si-face attack by the DHAP enamine. C–C bond formation by the cis-isomer is suppressed by hydrogen bonding of the cis-aldehyde carbonyl with the DHAP enamine phosphate dianion through a tetrahedrally coordinated water molecule. The active site geometry further suppresses C–C bond formation with the l-G3P enantiomer of d-G3P. Understanding C–C formation is of fundamental importance in biological reactions and has considerable relevance to biosynthetic reactions in organic chemistry.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.811034
      Issue No: Vol. 292, No. 48 (2017)
  • Electrostatic lipid-protein interactions sequester the curli amyloid fold
           on the lipopolysaccharide membrane surface [Protein Structure and Folding]
    • Authors: Hema M. Swasthi; Samrat Mukhopadhyay
      Pages: 19861 - 19872
      Abstract: Curli is a functional amyloid protein in the extracellular matrix of enteric Gram-negative bacteria. Curli is assembled at the cell surface and consists of CsgA, the major subunit of curli, and a membrane-associated nucleator protein, CsgB. Oligomeric intermediates that accumulate during the lag phase of amyloidogenesis are generally toxic, but the underlying mechanism by which bacterial cells overcome this toxicity during curli assembly at the surface remains elusive. Here, we elucidated the mechanism of curli amyloidogenesis and provide molecular insights into the strategy by which bacteria can potentially bypass the detrimental consequences of toxic amyloid intermediates. Using a diverse range of biochemical and biophysical tools involving circular dichroism, fluorescence, Raman spectroscopy, and atomic force microscopy imaging, we characterized the molecular basis of the interaction of CsgB with a membrane-mimetic anionic surfactant as well as with lipopolysaccharide (LPS) constituting the outer leaflet of Gram-negative bacteria. Aggregation studies revealed that the electrostatic interaction of the positively charged C-terminal region of the protein with a negatively charged head group of surfactant/LPS promotes a protein–protein interaction that results in facile amyloid formation without a detectable lag phase. We also show that CsgB, in the presence of surfactant/LPS, accelerates the fibrillation rate of CsgA by circumventing the lag phase during nucleation. Our findings suggest that the electrostatic interactions between lipid and protein molecules play a pivotal role in efficiently sequestering the amyloid fold of curli on the membrane surface without significant accumulation of toxic oligomeric intermediates.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.815522
      Issue No: Vol. 292, No. 48 (2017)
  • Disruption of amyloid precursor protein ubiquitination selectively
           increases amyloid {beta} (A{beta}) 40 levels via presenilin 2-mediated
           cleavage [Neurobiology]
    • Authors: Rebecca L. Williamson; Karine Laulagnier, Andre M. Miranda, Marty A. Fernandez, Michael S. Wolfe, Remy Sadoul, Gilbert Di Paolo
      Pages: 19873 - 19889
      Abstract: Amyloid plaques, a neuropathological hallmark of Alzheimer's disease, are largely composed of amyloid β (Aβ) peptide, derived from cleavage of amyloid precursor protein (APP) by β- and γ-secretases. The endosome is increasingly recognized as an important crossroad for APP and these secretases, with major implications for APP processing and amyloidogenesis. Among various post-translational modifications affecting APP accumulation, ubiquitination of cytodomain lysines may represent a key signal controlling APP endosomal sorting. Here, we show that substitution of APP C-terminal lysines with arginine disrupts APP ubiquitination and that an increase in the number of substituted lysines tends to increase APP metabolism. An APP mutant lacking all C-terminal lysines underwent the most pronounced increase in processing, leading to accumulation of both secreted and intracellular Aβ40. Artificial APP ubiquitination with rapalog-mediated proximity inducers reduced Aβ40 generation. A lack of APP C-terminal lysines caused APP redistribution from endosomal intraluminal vesicles (ILVs) to the endosomal limiting membrane, with a subsequent decrease in APP C-terminal fragment (CTF) content in secreted exosomes, but had minimal effects on APP lysosomal degradation. Both the increases in secreted and intracellular Aβ40 were abolished by depletion of presenilin 2 (PSEN2), recently shown to be enriched on the endosomal limiting membrane compared with PSEN1. Our findings demonstrate that ubiquitin can act as a signal at five cytodomain-located lysines for endosomal sorting of APP. They further suggest that disruption of APP endosomal sorting reduces its sequestration in ILVs and results in PSEN2-mediated processing of a larger pool of APP-CTF on the endosomal membrane.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.818138
      Issue No: Vol. 292, No. 48 (2017)
  • SUMOylation regulates nuclear accumulation and signaling activity of the
           soluble intracellular domain of the ErbB4 receptor tyrosine kinase
           [Molecular Bases of Disease]
    • Authors: Anna M. Knittle; Maria Helkkula, Mark S. Johnson, Maria Sundvall, Klaus Elenius
      Pages: 19890 - 19904
      Abstract: Erb-B2 receptor tyrosine kinase 4 (ErbB4) is a kinase that can signal via a proteolytically released intracellular domain (ICD) in addition to classical receptor tyrosine kinase–activated signaling cascades. Previously, we have demonstrated that ErbB4 ICD is posttranslationally modified by the small ubiquitin-like modifier (SUMO) and functionally interacts with the PIAS3 SUMO E3 ligase. However, direct evidence of SUMO modification in ErbB4 signaling has remained elusive. Here, we report that the conserved lysine residue 714 in the ErbB4 ICD undergoes SUMO modification, which was reversed by sentrin-specific proteases (SENPs) 1, 2, and 5. Although ErbB4 kinase activity was not necessary for the SUMOylation, the SUMOylated ErbB4 ICD was tyrosine phosphorylated to a higher extent than unmodified ErbB4 ICD. Mutation of the SUMOylation site compromised neither ErbB4-induced phosphorylation of the canonical signaling pathway effectors Erk1/2, Akt, or STAT5 nor ErbB4 stability. In contrast, SUMOylation was required for nuclear accumulation of the ErbB4 ICD. We also found that Lys-714 was located within a leucine-rich stretch, which resembles a nuclear export signal, and could be inactivated by site-directed mutagenesis. Furthermore, SUMOylation modulated the interaction of ErbB4 with chromosomal region maintenance 1 (CRM1), the major nuclear export receptor for proteins. Finally, the SUMO acceptor lysine was functionally required for ErbB4 ICD-mediated inhibition of mammary epithelial cell differentiation in a three-dimensional cell culture model. Our findings indicate that a SUMOylation-mediated mechanism regulates nuclear localization and function of the ICD of ErbB4 receptor tyrosine kinase.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.794271
      Issue No: Vol. 292, No. 48 (2017)
  • Autophagy induction under carbon starvation conditions is negatively
           regulated by carbon catabolite repression [Metabolism]
    • Authors: Atsuhiro Adachi; Michiko Koizumi, Yoshinori Ohsumi
      Pages: 19905 - 19918
      Abstract: Autophagy is a conserved process in which cytoplasmic components are sequestered for degradation in the vacuole/lysosomes in eukaryotic cells. Autophagy is induced under a variety of starvation conditions, such as the depletion of nitrogen, carbon, phosphorus, zinc, and others. However, apart from nitrogen starvation, it remains unclear how these stimuli induce autophagy. In yeast, for example, it remains contentious whether autophagy is induced under carbon starvation conditions, with reports variously suggesting both induction and lack of induction upon depletion of carbon. We therefore undertook an analysis to account for these inconsistencies, concluding that autophagy is induced in response to abrupt carbon starvation when cells are grown with glycerol but not glucose as the carbon source. We found that autophagy under these conditions is mediated by nonselective degradation that is highly dependent on the autophagosome-associated scaffold proteins Atg11 and Atg17. We also found that the extent of carbon starvation–induced autophagy is positively correlated with cells' oxygen consumption rate, drawing a link between autophagy induction and respiratory metabolism. Further biochemical analyses indicated that maintenance of intracellular ATP levels is also required for carbon starvation–induced autophagy and that autophagy plays an important role in cell viability during prolonged carbon starvation. Our findings suggest that carbon starvation–induced autophagy is negatively regulated by carbon catabolite repression.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.817510
      Issue No: Vol. 292, No. 48 (2017)
  • Structural insights into marine carbohydrate degradation by family GH16
           {kappa}-carrageenases [Protein Structure and Folding]
    • Authors: Maria Matard–Mann; Thomas Bernard, Cedric Leroux, Tristan Barbeyron, Robert Larocque, Aurelie Prechoux, Alexandra Jeudy, Murielle Jam, Pi Nyvall Collen, Gurvan Michel, Mirȷam Czȷzek
      Pages: 19919 - 19934
      Abstract: Carrageenans are sulfated α-1,3-β-1,4-galactans found in the cell wall of some red algae that are practically valuable for their gelation and biomimetic properties but also serve as a potential carbon source for marine bacteria. Carbohydrate degradation has been studied extensively for terrestrial plant/bacterial systems, but sulfation is not present in these cases, meaning the marine enzymes used to degrade carrageenans must possess unique features to recognize these modifications. To gain insights into these features, we have focused on κ-carrageenases from two distant bacterial phyla, which belong to glycoside hydrolase family 16 and cleave the β-1,4 linkage of κ-carrageenan. We have solved the crystal structure of the catalytic module of ZgCgkA from Zobellia galactanivorans at 1.66 Å resolution and compared it with the only other structure available, that of PcCgkA from Pseudoalteromonas carrageenovora 9T (ATCC 43555T). We also describe the first substrate complex in the inactivated mutant form of PcCgkA at 1.7 Å resolution. The structural and biochemical comparison of these enzymes suggests key determinants that underlie the functional properties of this subfamily. In particular, we identified several arginine residues that interact with the polyanionic substrate, and confirmed the functional relevance of these amino acids using a targeted mutagenesis strategy. These results give new insight into the diversity of the κ-carrageenase subfamily. The phylogenetic analyses show the presence of several distinct clades of enzymes that relate to differences in modes of action or subtle differences within the same substrate specificity, matching the hybrid character of the κ-carrageenan polymer.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.808279
      Issue No: Vol. 292, No. 48 (2017)
  • Photorhabdus luminescens lectin A (PllA): A new probe for detecting
           {alpha}-galactoside-terminating glycoconČ·ugates [Protein Structure and
    • Authors: Ghamdan Beshr; Asfandyar Sikandar, Eva-Maria Jemiller, Nikolai Klymiuk, Dirk Hauck, Stefanie Wagner, Eckhard Wolf, Jesko Koehnke, Alexander Titz
      Pages: 19935 - 19951
      Abstract: Lectins play important roles in infections by pathogenic bacteria, for example, in host colonization, persistence, and biofilm formation. The Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically lives in insect-infecting Heterorhabditis nematodes and kills the insect host upon invasion by the nematode. The P. luminescens genome harbors the gene plu2096, coding for a novel lectin that we named PllA. We analyzed the binding properties of purified PllA with a glycan array and a binding assay in solution. Both assays revealed a strict specificity of PllA for α-galactoside–terminating glycoconjugates. The crystal structures of apo PllA and complexes with three different ligands revealed the molecular basis for the strict specificity of this lectin. Furthermore, we found that a 90° twist in subunit orientation leads to a peculiar quaternary structure compared with that of its ortholog LecA from Pseudomonas aeruginosa. We also investigated the utility of PllA as a probe for detecting α-galactosides. The α-Gal epitope is present on wild-type pig cells and is the main reason for hyperacute organ rejection in pig to primate xenotransplantation. We noted that PllA specifically recognizes this epitope on the glycan array and demonstrated that PllA can be used as a fluorescent probe to detect this epitope on primary porcine cells in vitro. In summary, our biochemical and structural analyses of the P. luminescens lectin PllA have disclosed the structural basis for PllA's high specificity for α-galactoside–containing ligands, and we show that PllA can be used to visualize the α-Gal epitope on porcine tissues.
      PubDate: 2017-12-01T00:06:02-08:00
      DOI: 10.1074/jbc.M117.812792
      Issue No: Vol. 292, No. 48 (2017)
School of Mathematical and Computer Sciences
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