Phosphorylation of the amyloid precursor protein (APP) at Ser-675 promotes APP processing involving meprin {beta} [Molecular Bases of Disease]
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by abnormal deposition of β-amyloid (Aβ) peptides. Aβ is a cleavage product of the amyloid precursor protein (APP), and aberrant posttranslational modifications of APP can alter APP processing and increase Aβ generation. In the AD brain, seven different residues, including Ser-675 (APP695 numbering) in the APP cytoplasmic domain has been found to be phosphorylated. Here, we show that expression of a phosphomimetic variant of Ser-675 in APP (APP-S675E), in human neuroblastoma SK-N-AS cells, reduces secretion of the soluble APP ectodom...
Source: Journal of Biological Chemistry - November 22, 2019 Category: Chemistry Authors: Preeti Kumaran Menon, Niina Anneli Koistinen, Kerstin Iverfeldt, Anna–Lena Strom Tags: Neurobiology Source Type: research

Structural and functional contributions of lipids to the stability and activity of the photosynthetic cytochrome b6f lipoprotein complex [Membrane Biology]
The photosynthetic cytochrome b6f complex, a homodimer containing eight distinct subunits and 26 transmembrane helices per monomer, catalyzes proton-coupled electron transfer across the thylakoid membrane. The 2.5-Å-resolution structure of the complex from the cyanobacterium Nostoc sp. revealed the presence of 23 lipid-binding sites per monomer. Although the crystal structure of the cytochrome b6f from a plant source has not yet been solved, the identities of the lipids present in a plant b6f complex have previously been determined, indicating that the predominant lipid species are monogalactosyldiacylglycerol (MGDG)...
Source: Journal of Biological Chemistry - November 22, 2019 Category: Chemistry Authors: Satarupa Bhaduri, Huamin Zhang, Satchal Erramilli, William A. Cramer Tags: Bioenergetics Source Type: research

Neuronal ER-plasma membrane ȷunctions organized by Kv2-VAP pairing recruit Nir proteins and affect phosphoinositide homeostasis [Membrane Biology]
The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER–PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER–PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir...
Source: Journal of Biological Chemistry - November 22, 2019 Category: Chemistry Authors: Michael Kirmiz, Taryn E. Gillies, Eamonn J. Dickson, James S. Trimmer Tags: Neurobiology Source Type: research

Glucose starvation induces mitochondrial fragmentation depending on the dynamin GTPase Dnm1/Drp1 in fission yeast [Cell Biology]
Mitochondria undergo morphological and dynamic changes in response to environmental stresses. Few studies have focused on addressing mitochondrial remodeling under stress. Using the fission yeast Schizosaccharomyces pombe as a model organism, here we investigated mitochondrial remodeling under glucose starvation. We employed live-cell microscopy to monitor mitochondrial morphology and dynamics of cells in profusion chambers under glucose starvation. Our results revealed that mitochondria fragment within minutes after glucose starvation and that the dynamin GTPase Dnm1 is required for promoting mitochondrial fragmentation. ...
Source: Journal of Biological Chemistry - November 22, 2019 Category: Chemistry Authors: Fan Zheng, Bowen Jia, Fenfen Dong, Ling Liu, Faiz Rasul, Jiajia He, Chuanhai Fu Tags: Cell Biology Source Type: research

From the wings to the center stage of chromosomes [Gene Regulation]
Telomere-binding protein TRF2 protects the linear chromosome ends, telomeres, from being recognized as damaged DNA. TRF2 also regulates gene expression outside telomeres, but the detailed mechanism has not been fully understood. Mukherjee and colleagues have employed ChIP-Seq and biochemical analyses to identify G-quadruplexes at gene promoters across the genome as nontelomeric TRF2-binding sites. TRF2 occupancy on such target sites leads to epigenetic gene repression, implicating TRF2–G-quadruplex interaction as a sophisticated regulator of gene expression. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 22, 2019 Category: Chemistry Authors: Keiji Okamoto, Hiroyuki Seimiya Tags: Editors ' Picks Highlights Source Type: research

Telomere repeat-binding factor 2 binds extensively to extra-telomeric G-quadruplexes and regulates the epigenetic status of several gene promoters [Genomics and Proteomics]
The role of the telomere repeat-binding factor 2 (TRF2) in telomere maintenance is well-established. However, recent findings suggest that TRF2 also functions outside telomeres, but relatively little is known about this function. Herein, using genome-wide ChIP-Seq assays of TRF2-bound chromatin from HT1080 fibrosarcoma cells, we identified thousands of TRF2-binding sites within the extra-telomeric genome. In light of this observation, we asked how TRF2 occupancy is organized within the genome. Interestingly, we found that extra-telomeric TRF2 sites throughout the genome are enriched in potential G-quadruplex–forming ...
Source: Journal of Biological Chemistry - November 22, 2019 Category: Chemistry Authors: Ananda Kishore Mukherjee, Shalu Sharma, Sulochana Bagri, Rintu Kutum, Pankaj Kumar, Asgar Hussain, Prateek Singh, Dhurjhoti Saha, Anirban Kar, Debasis Dash, Shantanu Chowdhury Tags: Editors ' Picks Source Type: research

Expression of Concern: Tumor suppressor SMAR1 activates and stabilizes p53 through its arginine-serine-rich motif. [Expressions of Concern]
VOLUME 280 (2005) PAGES 16019–16029The publisher of the Journal of Biological Chemistry is issuing an Expression of Concern to inform readers that credible concerns have been raised regarding some of the data and conclusions in the article listed above. The Journal of Biological Chemistry will provide additional information as it becomes available. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Tags: Expressions of Concern Source Type: research

Correction: Engineered protein disaggregases mitigate toxicity of aberrant prion-like fusion proteins underlying sarcoma. [Additions and Corrections]
VOLUME 294 (2019) PAGES 11286–11296Throughout the text, the abbreviations “EWS-FLI1” and “EWS-FLI3” refer to EWS-FLI1, type 1 and EWS-FLI1, type 3, respectively. These are EWS-FLI1 fusion proteins that arise from slightly different chromosomal translocations that are found naturally in human sarcomas (1). Domain maps for these distinct fusion proteins are shown in Fig. 2A. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Jeremy J. Ryan, Macy L. Sprunger, Kayla Holthaus, James Shorter, Meredith E. Jackrel Tags: Additions and Corrections Source Type: research

Control of cellular responses to mechanical cues through YAP/TAZ regulation [Cell Biology]
To perceive their three-dimensional environment, cells and tissues must be able to sense and interpret various physical forces like shear, tensile, and compression stress. These forces can be generated both internally and externally in response to physical properties, like substrate stiffness, cell contractility, and forces generated by adjacent cells. Mechanical cues have important roles in cell fate decisions regarding proliferation, survival, and differentiation as well as the processes of tissue regeneration and wound repair. Aberrant remodeling of the extracellular space and/or defects in properly responding to mechan...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Ishani Dasgupta, Dannel McCollum Tags: JBC Reviews Source Type: research

The biosynthetic diversity of the animal world [Enzymology]
Secondary metabolites are often considered within the remit of bacterial or plant research, but animals also contain a plethora of these molecules with important functional roles. Classical feeding studies demonstrate that, whereas some are derived from diet, many of these compounds are made within the animals. In the past 15 years, the genetic and biochemical origin of several animal natural products has been traced to partnerships with symbiotic bacteria. More recently, a number of animal genome-encoded pathways to microbe-like natural products have come to light. These pathways are sometimes horizontally acquired from b...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Joshua P. Torres, Eric W. Schmidt Tags: JBC Reviews Source Type: research

How organic chemistry became one of UCLA's most popular classes [ASBMB Award Articles]
Organic chemistry has a bad reputation, despite having a tremendous impact on our everyday lives. It has remained a notorious “weed-out” class for decades—striking fear in the hearts of students—and has long been viewed as a gatekeeper course for those interested in pursuing a career in medicine or other health-related professions. This personal account examines the underlying teaching philosophies that transformed organic chemistry into one of the most popular classes on the UCLA campus. Special emphasis is placed on ways to increase engagement and help students feel connected. Educational initiati...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Neil K. Garg Tags: ASBMB Award Articles Source Type: research

Hypoxia-inducible gene domain 1 proteins in yeast mitochondria protect against proton leak through complex IV [Membrane Biology]
Hypoxia-inducible gene domain 1 (HIGD1) proteins are small integral membrane proteins, conserved from bacteria to humans, that associate with oxidative phosphorylation supercomplexes. Using yeast as a model organism, we have shown previously that its two HIGD1 proteins, Rcf1 and Rcf2, are required for the generation and maintenance of a normal membrane potential (ΔΨ) across the inner mitochondrial membrane (IMM). We postulated that the lower ΔΨ observed in the absence of the HIGD1 proteins may be due to decreased proton pumping by complex IV (CIV) or enhanced leak of protons across the IMM. Here we meas...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Ngoc H. Hoang, Vera Strogolova, Jaramys J. Mosley, Rosemary A. Stuart, Jonathan Hosler Tags: Bioenergetics Source Type: research

Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens [Enzymology]
WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another majo...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Victor Clausse, Dingyin Tao, Subrata Debnath, Yuhong Fang, Harichandra D. Tagad, Yuhong Wang, Hongmao Sun, Christopher A. LeClair, Sharlyn J. Mazur, Kelly Lane, Zhen-Dan Shi, Olga Vasalatiy, Rebecca Eells, Lynn K. Baker, Mark J. Henderson, Martin R. Webb, Tags: Methods and Resources Source Type: research

Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition [Microbiology]
Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m7G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m7G1405) methyltransferases. W...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Meisam Nosrati, Debayan Dey, Atousa Mehrani, Sarah E. Strassler, Natalia Zelinskaya, Eric D. Hoffer, Scott M. Stagg, Christine M. Dunham, Graeme L. Conn Tags: RNA Source Type: research

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions [Plant Biology]
We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) con...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Munkhtsetseg Tsednee, Madeli Castruita, Patrice A. Salome, Aȷay Sharma, Brianne E. Lewis, Stefan R. Schmollinger, Daniela Strenkert, Kristen Holbrook, Marisa S. Otegui, Kaustav Khatua, Sayani Das, Ankona Datta, Si Chen, Christina Ramon, Martina Ral Tags: Cell Biology Source Type: research

A dual-chain assembly pathway generates the high structural diversity of cell-wall polysaccharides in Lactococcus lactis [Glycobiology and Extracellular Matrices]
In this study, using a CRISPR/Cas-based method, we performed a mutational analysis of the cwps genes. MALDI-TOF MS-based structural analysis of the mutant CWPS combined with sequence homology, transmission EM, and phage sensitivity analyses enabled us to infer a role for each protein encoded by the cwps cluster. We propose a comprehensive CWPS biosynthesis scheme in which the rhamnan and PSP chains are independently synthesized from two distinct lipid-sugar precursors and are joined at the extracellular side of the cytoplasmic membrane by a mechanism involving a membrane-embedded glycosyltransferase with a GT-C fold. The p...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Ilias Theodorou, Pascal Courtin, Simon Palussiere, Saulius Kulakauskas, Elena Bidnenko, Christine Pechoux, Francois Fenaille, Christophe Penno, Jennifer Mahony, Douwe van Sinderen, Marie–Pierre Chapot–Chartier Tags: Microbiology Source Type: research

Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH [Cell Biology]
In this study, we investigated Avastin's capacity to modulate VEGF165 binding to porcine aortic endothelial cells and to heparin and fibronectin (FN) across a range of pH values (pH 5–8). We observed that Avastin slightly enhanced VEGF binding to heparin and that heparin increased VEGF binding to Avastin. In contrast, Avastin inhibited VEGF binding to cells and FN, yet Avastin could still bind to VEGF that was bound to FN, indicating that these binding events are not mutually exclusive. Avastin binding to VEGF was dramatically reduced at acidic pH values (pH 5.0–6.5), whereas VEGF binding to FN and nonreceptor ...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Divyabharathy Tsiros, Casey E. Sheehy, Surenna Pecchia, Matthew A. Nugent Tags: Glycobiology and Extracellular Matrices Source Type: research

Mitochondrial amidoxime-reducing component 2 (MARC2) has a significant role in N-reductive activity and energy metabolism [Enzymology]
In this study, we characterized an existing KO mouse model lacking the functional MARC2 gene and fed a high-fat diet and also performed in vivo and in vitro experiments to characterize reductase activity toward known MARC substrates. MARC2 KO significantly decreased reductase activity toward several N-oxygenated substrates, and for typical MARC substrates, only small residual reductive activity was still detectable in MARC2 KO mice. The residual detected reductase activity in MARC2 KO mice could be explained by MARC1 expression that was hardly unaffected by KO, and we found no evidence of significant activity of other redu...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Sophia Rixen, Antje Havemeyer, Anita Tyl-Bielicka, Kazimiera Pysniak, Marta Gajewska, Maria Kulecka, Jerzy Ostrowski, Michal Mikula, Bernd Clement Tags: Metabolism Source Type: research

Proteomic analysis of affinity-purified 26S proteasomes identifies a suite of assembly chaperones in Arabidopsis [Plant Biology]
The 26S proteasome is an essential protease that selectively eliminates dysfunctional and short-lived regulatory proteins in eukaryotes. To define the composition of this proteolytic machine in plants, we tagged either the core protease (CP) or the regulatory particle (RP) sub-complexes in Arabidopsis to enable rapid affinity purification followed by mass spectrometric analysis. Studies on proteasomes enriched from whole seedlings, with or without ATP needed to maintain the holo-proteasome complex, identified all known proteasome subunits but failed to detect isoform preferences, suggesting that Arabidopsis does not constr...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: David C. Gemperline, Richard S. Marshall, Kwang-Hee Lee, Qingzhen Zhao, Weiming Hu, Fionn McLoughlin, Mark Scalf, Lloyd M. Smith, Richard D. Vierstra Tags: Protein Synthesis and Degradation Source Type: research

FOXO1 transcription factor regulates chondrogenic differentiation through transforming growth factor {beta}1 signaling [Developmental Biology]
The forkhead box O (FOXO) proteins are transcription factors involved in the differentiation of many cell types. Type II collagen (Col2) Cre-Foxo1-knockout and Col2-Cre-Foxo1,3,4 triple-knockout mice exhibit growth plate malformation. Moreover, recent studies have reported that in some cells, the expressions and activities of FOXOs are promoted by transforming growth factor β1 (TGFβ1), a growth factor playing a key role in chondrogenic differentiation. Here, using a murine chondrogenic cell line (ATDC5), mouse embryos, and human mesenchymal stem cells, we report the mechanisms by which FOXOs affect chondrogenic d...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Ichiro Kurakazu, Yukio Akasaki, Mitsumasa Hayashida, Hidetoshi Tsushima, Norio Goto, Takuya Sueishi, Masakazu Toya, Masanari Kuwahara, Ken Okazaki, Tomas Duffy, Martin K. Lotz, Yasuharu Nakashima Tags: Cell Biology Source Type: research

Calmodulin is involved in the dual subcellular location of two chloroplast proteins [Cell Biology]
This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Lucas Moyet, Daniel Salvi, Imen Bouchnak, Stephane Miras, Laura Perrot, Daphne Seigneurin–Berny, Marcel Kuntz, Norbert Rolland Tags: Plant Biology Source Type: research

{beta}-Catenin mutations as determinants of hepatoblastoma phenotypes in mice [Metabolism]
Hepatoblastoma (HB) is the most common pediatric liver cancer. Although long-term survival of HB is generally favorable, it depends on clinical stage, tumor histology, and a variety of biochemical and molecular features. HB appears almost exclusively before the age of 3 years, is represented by seven histological subtypes, and is usually associated with highly heterogeneous somatic mutations in the catenin β1 (CTNNB1) gene, which encodes β-catenin, a Wnt ligand–responsive transcriptional co-factor. Numerous recurring β-catenin mutations, not previously documented in HB, have also been identified in var...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Weiqi Zhang, Jennifer Meyfeldt, Huabo Wang, Sucheta Kulkarni, Jie Lu, Jordan A. Mandel, Brady Marburger, Ying Liu, Joanna E. Gorka, Sarangarajan Ranganathan, Edward V. Prochownik Tags: Molecular Bases of Disease Source Type: research

A piperidinol-containing molecule is active against Mycobacterium tuberculosis by inhibiting the mycolic acid flippase activity of MmpL3 [Microbiology]
In conclusion, our findings have uncovered bactericidal activity of a new chemical scaffold. Its anti-tubercular activity is mediated by direct inhibition of the flippase activity of MmpL3 rather than by inhibition of the inner membrane proton motive force, significantly advancing our understanding of MmpL3-targeted inhibition in mycobacteria. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Christian Dupont, Yushu Chen, Zhuȷun Xu, Francoise Roquet–Baneres, Mickael Blaise, Anne–Kathrin Witt, Faustine Dubar, Christophe Biot, Yann Guerardel, Florian P. Maurer, Shu–Sin Chng, Laurent Kremer Tags: Microbiology Source Type: research

Xenogeneic modulation of the ClpCP protease of Bacillus subtilis by a phage-encoded adaptor-like protein [Protein Synthesis and Degradation]
Like eukaryotic and archaeal viruses, which coopt the host's cellular pathways for their replication, bacteriophages have evolved strategies to alter the metabolism of their bacterial host. SPO1 bacteriophage infection of Bacillus subtilis results in comprehensive remodeling of cellular processes, leading to conversion of the bacterial cell into a factory for phage progeny production. A cluster of 26 genes in the SPO1 genome, called the host takeover module, encodes for potentially cytotoxic proteins that specifically shut down various processes in the bacterial host, including transcription, DNA synthesis, and cell divisi...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Nancy Mulvenna, Ingo Hantke, Lynn Burchell, Sophie Nicod, David Bell, Kurşad Turgay, Sivaramesh Wigneshweraraȷ Tags: Microbiology Source Type: research

The transcription factor PU.1 mediates enhancer-promoter looping that is required for IL-1{beta} eRNA and mRNA transcription in mouse melanoma and macrophage cell lines [Immunology]
The DNA-binding protein PU.1 is a myeloid lineage–determining and pioneering transcription factor due to its ability to bind “closed” genomic sites and maintain “open” chromatin state for myeloid lineage–specific genes. The precise mechanism of PU.1 in cell type–specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1β ...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Soon-Duck Ha, Woohyun Cho, Rodney P. DeKoter, Sung Ouk Kim Tags: Gene Regulation Source Type: research

A thiazole-derived oridonin analogue exhibits antitumor activity by directly and allosterically inhibiting STAT3 [Gene Regulation]
Constitutive activation of signal transducer and activator of transcription 3 (STAT3) occurs in ∼70% of human cancers, and STAT3 is regarded as one of the most promising targets for cancer therapy. However, specific direct STAT3 inhibitors remain to be developed. Oridonin is an ent-kaurane plant-derived diterpenoid with anti-cancer and anti-inflammatory activities. Here, using an array of cell-based and biochemical approaches, including cell proliferation and apoptosis assays, pulldown and reporter gene assays, site-directed mutagenesis, and molecular dynamics analyses, we report that a thiazole-derived oridonin analog...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Xiaofei Shen, Lin Zhao, Peihao Chen, Yanqiu Gong, Dingdong Liu, Xia Zhang, Lunzhi Dai, Qingxiang Sun, Jizhong Lou, Zhong Jin, Baohua Zhang, Dawen Niu, Ceshi Chen, Xiangbing Qi, Da Jia Tags: Signal Transduction Source Type: research

Kinetic and structural evidence that Asp-678 plays multiple roles in catalysis by the quinoprotein glycine oxidase [Bioenergetics]
In this study, mutation of Asp-678 in PlGoxA did not abolish CTQ formation. This allowed, for the first time, studying the role of this residue in catalysis. D678A and D678N substitutions yielded enzyme variants with CTQ, which did not react with glycine, although glycine was present in the crystal structures in the active site. D678E PlGoxA was active but exhibited a much slower kcat. This mutation altered the kinetic mechanism of the reductive half-reaction such that one could observe a previously undetected reactive intermediate, an initial substrate-oxidized CTQ adduct, which converted to the product-reduced CTQ adduct...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Kyle J. Mamounis, Dante Avalos, Erik T. Yukl, Victor L. Davidson Tags: Enzymology Source Type: research

Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties [Molecular Biophysics]
Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β-cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result i...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Carlos D. Vera, Chloe A. Johnson, Jonathan Walklate, Arjun Adhikari, Marina Svicevic, Srboljub M. Mijailovich, Ariana C. Combs, Stephen J. Langer, Kathleen M. Ruppel, James A. Spudich, Michael A. Geeves, Leslie A. Leinwand Tags: Molecular Bases of Disease Source Type: research

Impact of key residues within chloroplast thioredoxin-f on recognition for reduction and oxidation of target proteins [Bioenergetics]
Thioredoxin (Trx) is a redox-responsive protein that modulates the activities of its target proteins mostly by reducing their disulfide bonds. In chloroplasts, five Trx isoforms (Trx-f, Trx-m, Trx-x, Trx-y, and Trx-z) regulate various photosynthesis-related enzymes with distinct target selectivity. To elucidate the determinants of the target selectivity of each Trx isoform, here we investigated the residues responsible for target recognition by Trx-f, the most well-studied chloroplast-resident Trx. As reported previously, we found that positively-charged residues on the Trx-f surface are involved in the interactions with i...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Yuichi Yokochi, Kazunori Sugiura, Kazuhiro Takemura, Keisuke Yoshida, Satoshi Hara, Ken-ichi Wakabayashi, Akio Kitao, Toru Hisabori Tags: Plant Biology Source Type: research

Comparative analysis of the catalytic regulation of NEDD4-1 and WWP2 ubiquitin ligases [Protein Synthesis and Degradation]
NEDD4-1 E3 ubiquitin protein ligase (NEDD4-1) and WW domain-containing E3 ubiquitin ligase (WWP2) are HECT family ubiquitin E3 ligases. They catalyze Lys ubiquitination of themselves and other proteins and are important in cell growth and differentiation. Regulation of NEDD4-1 and WWP2 catalytic activities is important for controlling cellular protein homeostasis, and their dysregulation may lead to cancer and other diseases. Previous work has implicated noncatalytic regions, including the C2 domain and/or WW domain linkers in NEDD4-1 and WWP2, in contributing to autoinhibition of the catalytic HECT domains by intramolecul...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Hanjie Jiang, Stefani N. Thomas, Zan Chen, Claire Y. Chiang, Philip A. Cole Tags: Enzymology Source Type: research

Genetic code expansion and photocross-linking identify different {beta}-arrestin binding modes to the angiotensin II type 1 receptor [Signal Transduction]
The angiotensin II (AngII) type 1 receptor (AT1R) is a member of the G protein–coupled receptor (GPCR) family and binds β-arrestins (β-arrs), which regulate AT1R signaling and trafficking. These processes can be biased by different ligands or mutations in the AGTR1 gene. As for many GPCRs, the exact details for AT1R–β-arr interactions driven by AngII or β-arr–biased ligands remain largely unknown. Here, we used the amber-suppression technology to site-specifically introduce the unnatural amino acid (UAA) p-azido-l-phenylalanine (azF) into the intracellular loops (ICLs) and the C-tail o...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Laurence Gagnon, Yubo Cao, Aaron Cho, Dana Sedki, Thomas Huber, Thomas P. Sakmar, Stephane A. Laporte Tags: Methods and Resources Source Type: research

Mechanically activated Piezo1 channels of cardiac fibroblasts stimulate p38 mitogen-activated protein kinase activity and interleukin-6 secretion [Signal Transduction]
Piezo1 is a mechanosensitive cation channel with widespread physiological importance; however, its role in the heart is poorly understood. Cardiac fibroblasts help preserve myocardial integrity and play a key role in regulating its repair and remodeling following stress or injury. Here we investigated Piezo1 expression and function in cultured human and mouse cardiac fibroblasts. RT-PCR experiments confirmed that Piezo1 mRNA in cardiac fibroblasts is expressed at levels similar to those in endothelial cells. The results of a Fura-2 intracellular Ca2+ assay validated Piezo1 as a functional ion channel that is activated by i...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Nicola M. Blythe, Katsuhiko Muraki, Melanie J. Ludlow, Vasili Stylianidis, Hamish T. J. Gilbert, Elizabeth L. Evans, Kevin Cuthbertson, Richard Foster, Joe Swift, Jing Li, Mark J. Drinkhill, Frans A. van Nieuwenhoven, Karen E. Porter, David J. Beech, Neil Tags: Cell Biology Source Type: research

Intrinsic disorder and amino acid specificity modulate binding of the WW2 domain in kidney and brain protein (KIBRA) to synaptopodin [Molecular Biophysics]
The second WW domain (WW2) of the kidney and brain scaffolding protein, KIBRA, has an isoleucine (Ile-81) rather than a second conserved tryptophan and is primarily unstructured. However, it adopts the canonical triple-stranded antiparallel β-sheet structure of WW domains when bound to a two-PPXY motif peptide of the synaptic protein Dendrin. Here, using a series of biophysical experiments, we demonstrate that the WW2 domain remains largely disordered when bound to a 69-residue two-PPXY motif polypeptide of the synaptic and podocyte protein synaptopodin (SYNPO). Isothermal titration calorimetry and CD experiments reve...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Ethiene Kwok, Diego J. Rodriguez, Joachim Kremerskothen, Afua Nyarko Tags: Molecular Biophysics Source Type: research

Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses [Computational Biology]
Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in ...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Kateřina Machačkova, Květoslava Mlčochova, Pavlo Potalitsyn, Kateřina Hankova, Ondřeȷ Socha, Miloš Buděšinsky, Anȷa Muždalo, Martin Lepšik, Michaela Černekov Tags: Protein Structure and Folding Source Type: research

Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate [Signal Transduction]
Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 contro...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Neeladri Sekhar Roy, Xiaoying Jian, Olivier Soubias, Peng Zhai, Jessica R. Hall, Jessica N. Dagher, Nathan P. Coussens, Lisa M. Jenkins, Ruibai Luo, Itoro O. Akpan, Matthew D. Hall, R. Andrew Byrd, Marielle E. Yohe, Paul A. Randazzo Tags: Enzymology Source Type: research

A carbohydrate-binding family 48 module enables feruloyl esterase action on polymeric arabinoxylan [Protein Structure and Folding]
Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan an...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Jesper Holck, Folmer Fredslund, Marie S. Moller, Jesper Brask, Kristian B. R. M. Krogh, Lene Lange, Ditte H. Welner, Birte Svensson, Anne S. Meyer, Casper Wilkens Tags: Enzymology Source Type: research

Glucoselysine is derived from fructose and accumulates in the eye lens of diabetic rats [Glycobiology and Extracellular Matrices]
Prolonged hyperglycemia generates advanced glycation end-products (AGEs), which are believed to be involved in the pathogenesis of diabetic complications. In the present study, we developed a polyclonal antibody against fructose-modified proteins (Fru-P antibody) and identified its epitope as glucoselysine (GL) by NMR and LC-electrospray ionization (ESI)- quadrupole TOF (QTOF) analyses and evaluated its potential role in diabetes sequelae. Although the molecular weight of GL was identical to that of fructoselysine (FL), GL was distinguishable from FL because GL was resistant to acid hydrolysis, which converted all of the F...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Rei-ichi Ohno, Kenta Ichimaru, Seitaro Tanaka, Hikari Sugawa, Nana Katsuta, Shiori Sakake, Yu-ki Tominaga, Ikuho Ban, Jun-ichi Shirakawa, Yoshiki Yamaguchi, Emi Ito, Naoyuki Taniguchi, Ryoji Nagai Tags: Molecular Bases of Disease Source Type: research

Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil [Molecular Biophysics]
We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human β-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absen...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Wanjian Tang, William C. Unrath, Rohini Desetty, Christopher M. Yengo Tags: Enzymology Source Type: research

Regulation of human trophoblast syncytialization by histone demethylase LSD1 [Developmental Biology]
A successful pregnancy is critically dependent upon proper placental development and function. During human placentation, villous cytotrophoblast (CTB) progenitors differentiate to form syncytiotrophoblasts (SynTBs), which provide the exchange surface between the mother and fetus and secrete hormones to ensure proper progression of pregnancy. However, epigenetic mechanisms that regulate SynTB differentiation from CTB progenitors are incompletely understood. Here, we show that lysine-specific demethylase 1 (LSD1; also known as KDM1A), a histone demethylase, is essential to this process. LSD1 is expressed both in CTB progeni...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Jessica Milano-Foster, Soma Ray, Pratik Home, Avishek Ganguly, Bhaswati Bhattacharya, Shilpika Bajpai, Aratrika Pal, Clifford W. Mason, Soumen Paul Tags: Gene Regulation Source Type: research

Ostreolysin A and anthrolysin O use different mechanisms to control movement of cholesterol from the plasma membrane to the endoplasmic reticulum [Methods and Resources]
Recent studies using two cholesterol-binding bacterial toxin proteins, perfringolysin O (PFO) and domain 4 of anthrolysin O (ALOD4), have shown that cholesterol in the plasma membranes (PMs) of animal cells resides in three distinct pools. The first pool comprises mobile cholesterol, accessible to both PFO and ALOD4, that is rapidly transported to the endoplasmic reticulum (ER) to signal cholesterol excess and maintain cholesterol homeostasis. The second is a sphingomyelin (SM)-sequestered pool inaccessible to PFO and ALOD4 but that becomes accessible by treatment with SM-degrading sphingomyelinase (SMase). The third is an...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Kristen A. Johnson, Shreya Endapally, Danya C. Vazquez, Rodney E. Infante, Arun Radhakrishnan Tags: Lipids Source Type: research

The ACT domain in chloroplast precursor-phosphorylating STY kinases binds metabolites and allosterically regulates kinase activity [Cell Biology]
Protein import of nucleus-encoded proteins into plant chloroplasts is a highly regulated process, requiring fine-tuning mechanisms especially during chloroplast differentiation. One way of altering import efficiency is phosphorylation of chloroplast transit peptides in the cytosol. We recently investigated the role of three serine/threonine/tyrosine (STY) kinases, STY8, STY17, and STY46, in precursor phosphorylation. These three kinases have a high degree of similarity and harbor a conserved aspartate kinase–chorismate mutase–tyrA (prephenate dehydrogenase) (ACT) domain upstream of the kinase domain. The ACT do...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Ahmed Eisa, Bettina Bolter, Serena Schwenkert Tags: Plant Biology Source Type: research

The phosphorylation status of Ser-637 in dynamin-related protein 1 (Drp1) does not determine Drp1 recruitment to mitochondria [Membrane Biology]
In conclusion, the Ser-637 phosphorylation status in Drp1 is not a determinant that controls Drp1 recruitment to mitochondria. (Source: Journal of Biological Chemistry)
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Rong Yu, Tong Liu, Chenfei Ning, Fei Tan, Shao–Bo Jin, Urban Lendahl, Jian Zhao, Monica Nister Tags: Cell Biology Source Type: research

The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting complex/cyclosome by combining high-affinity activator binding with disruption of Doc1/Apc10 function [Cell Biology]
The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their c...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Liang Qin, Arda Mizrak, Dimitrius Santiago P. S. F. Guimaraes, Hana M. Tambrin, David O. Morgan, Mark C. Hall Tags: Protein Synthesis and Degradation Source Type: research

Differential regulation of AMP-activated protein kinase in healthy and cancer cells explains why V-ATPase inhibition selectively kills cancer cells [Metabolism]
The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic hub regulating various pathways involved in tumor metabolism. Here we report that vacuolar H+-ATPase (V-ATPase) inhibition differentially affects regulation of AMPK in tumor and nontumor cells and that this differential regulation contributes to the selectivity of V-ATPase inhibitors for tumor cells. In nonmalignant cells, the V-ATPase inhibitor archazolid increased phosphorylation and lysosomal localization of AMPK. We noted that AMPK localization has a prosurvival role, as AMPK silencing decreased cellular growth rates. In contrast, in cancer c...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Karin Bartel, Rolf Muller, Karin von Schwarzenberg Tags: Molecular Bases of Disease Source Type: research

Carbon source regulates polysaccharide capsule biosynthesis in Streptococcus pneumoniae [Glycobiology and Extracellular Matrices]
The exopolysaccharide capsule of Streptococcus pneumoniae is an important virulence factor, but the mechanisms that regulate capsule thickness are not fully understood. Here, we investigated the effects of various exogenously supplied carbohydrates on capsule production and gene expression in several pneumococcal serotypes. Microscopy analyses indicated a near absence of the capsular polysaccharide (CPS) when S. pneumoniae was grown on fructose. Moreover, serotype 7F pneumococci produced much less CPS than strains of other serotypes (6B, 6C, 9V, 15, and 23F) when grown on glucose or sucrose. RNA-sequencing revealed carbon ...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Lukas J. Troxler, Joel P. Werren, Thierry O. Schaffner, Nadezda Mostacci, Peter Vermathen, Martina Vermathen, Daniel Wuthrich, Cedric Simillion, Silvio D. Brugger, Remy Bruggmann, Lucy J. Hathaway, Julien Furrer, Markus Hilty Tags: Microbiology Source Type: research

The E3 ubiquitin ligase Pib1 regulates effective gluconeogenic shutdown upon glucose availability [Protein Synthesis and Degradation]
Cells use multiple mechanisms to regulate their metabolic states in response to changes in their nutrient environment. One example is the response of cells to glucose. In Saccharomyces cerevisiae growing in glucose-depleted medium, the re-availability of glucose leads to the down-regulation of gluconeogenesis and the activation of glycolysis, leading to “glucose repression.” However, our knowledge of the mechanisms mediating the glucose-dependent down-regulation of the gluconeogenic transcription factors is limited. Using the major gluconeogenic transcription factor Rds2 as a candidate, we identify here a novel...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Vineeth Vengayil, Zeenat Rashida, Sunil Laxman Tags: Signal Transduction Source Type: research

Molecular analysis of an enigmatic Streptococcus pneumoniae virulence factor: The raffinose-family oligosaccharide utilization system [Protein Structure and Folding]
Streptococcus pneumoniae is an opportunistic respiratory pathogen that can spread to other body sites, including the ears, brain, and blood. The ability of this bacterium to break down, import, and metabolize a wide range of glycans is key to its virulence. Intriguingly, S. pneumoniae can utilize several plant oligosaccharides for growth in vitro, including raffinose-family oligosaccharides (RFOs, which are α-(1→6)-galactosyl extensions of sucrose). An RFO utilization locus has been identified in the pneumococcal genome; however, none of the proteins encoded by this locus have been biochemically characterized. T...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Joanne K. Hobbs, Edward P. W. Meier, Benjamin Pluvinage, Mackenzie A. Mey, Alisdair B. Boraston Tags: Glycobiology and Extracellular Matrices Source Type: research

The RNA-binding protein SART3 promotes miR-34a biogenesis and G1 cell cycle arrest in lung cancer cells [RNA]
MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of most biological processes. Global miRNA biogenesis is altered in many cancers, and RNA-binding proteins play a role in miRNA biogenesis, presenting a promising avenue for targeting miRNA dysregulation in diseases. miR-34a exhibits tumor-suppressive activities by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor BCL-2, among other regulatory pathways such as Wnt, TGF-β, and Notch signaling. Many cancers exhibit down-regulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tu...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Emily J. Sherman, Dylan C. Mitchell, Amanda L. Garner Tags: Accelerated Communications Source Type: research

New gadget in the membrane trafficking toolbox: A novel inhibitor of SNARE priming [Membrane Biology]
NSF (N-ethylmaleimide sensitive factor) and its yeast counterpart Sec18 are highly conserved homohexameric proteins that play vital roles in eukaryotic membrane trafficking. Sec18 functions by disrupting SNARE complexes formed in cis, on the same membrane. However, the molecular mechanisms of this process are poorly understood, in large part due to the lack of selective, reversible inhibitors. A new study by Sparks et al. now reports a small molecule that appears to selectively inhibit Sec18 action in an in vitro assay. Their finding now paves the way to elucidate further details of Sec18-mediated SNARE priming. (Source: J...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Hagai Abeliovich Tags: Editors ' Picks Highlights Source Type: research

A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion [Cell Biology]
The homeostasis of most organelles requires membrane fusion mediated by soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual c...
Source: Journal of Biological Chemistry - November 15, 2019 Category: Chemistry Authors: Robert P. Sparks, Andres S. Arango, Matthew L. Starr, Zachary L. Aboff, Logan R. Hurst, David A. Rivera-Kohr, Chi Zhang, Kevin A. Harnden, Jermaine L. Jenkins, Wayne C. Guida, Emad Tajkhorshid, Rutilio A. Fratti Tags: Editors ' Picks Source Type: research