CGA-N9, an antimicrobial peptide derived from chromogranin A: direct cell penetration of and endocytosis by Candida tropicalis
CGA-N9 is a peptide derived from the N-terminus of human chromogranin A comprising amino acids 47–55. Minimum inhibitory concentration (MIC) assays showed that CGA-N9 had antimicrobial activity and exhibited time-dependent inhibition activity against Candida tropicalis, with high safety in human red blood cells (HRBCs) and mouse brain microvascular endothelial cells (bEnd.3). According to the results of transmission electron microscopy (TEM), flow cytometry and confocal microscopy, CGA-N9 accumulated in cells without destroying the integrity of the cell membrane; the peptide was initially localized to the cell membra...
Source: Biochemical Journal - February 5, 2019 Category: Biochemistry Authors: Li, R., Chen, C., Zhu, S., Wang, X., Yang, Y., Shi, W., Chen, S., Wang, C., Yan, L., Shi, J. Tags: Research Articles Source Type: research

MOV10 sequesters the RNP of influenza A virus in the cytoplasm and is antagonized by viral NS1 protein
MOV10 has emerged as an important host antiviral factor. MOV10 not only inhibits various viruses, including human immunodeficiency virus type 1, hepatitis C virus and vesicular stomatitis virus, but also restricts the activity of retroelements long interspersed nucleotide element-1, Alu, SVA and intracisternal A particles. Here, we report that MOV10 suppresses influenza A virus infection through interacting with viral nucleoprotein (NP), sequestering viral RNP in the cytoplasm and causing the degradation of viral vRNA. The antiviral activity of MOV10 depends on the integrity of P-bodies. We also found that the antiviral ac...
Source: Biochemical Journal - February 5, 2019 Category: Biochemistry Authors: Li, J., Hu, S., Xu, F., Mei, S., Liu, X., Yin, L., Zhao, F., Zhao, X., Sun, H., Xiong, Z., Zhang, D., Cen, S., Wang, J., Liang, C., Guo, F. Tags: Research Articles Source Type: research

The transmembrane autophagy cargo receptors ATI1 and ATI2 interact with ATG8 through intrinsically disordered regions with distinct biophysical properties
Selective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8-interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8-interacting protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs)...
Source: Biochemical Journal - February 5, 2019 Category: Biochemistry Authors: Sjogaard, I. M. Z., Bressendorff, S., Prestel, A., Kausika, S., Oksbjerg, E., Kragelund, B. B., Brodersen, P. Tags: Research Articles Source Type: research

Antibody specificity and promiscuity
The immune system is capable of making antibodies against anything that is foreign, yet it does not react against components of self. In that sense, a fundamental requirement of the body's immune defense is specificity. Remarkably, this ability to specifically attack foreign antigens is directed even against antigens that have not been encountered a priori by the immune system. The specificity of an antibody for the foreign antigen evolves through an iterative process of somatic mutations followed by selection. There is, however, accumulating evidence that the antibodies are often functionally promiscuous or multi-specific...
Source: Biochemical Journal - February 5, 2019 Category: Biochemistry Authors: Jain, D., Salunke, D. M. Tags: Review Articles Source Type: research

Solution structure of TbTFIIS2-2 PWWP domain from Trypanosoma brucei and its binding to H4K17me3 and H3K32me3
Posttranslational modifications (PTMs) of core histones, such as histone methylation, play critical roles in a variety of biological processes including transcription regulation, chromatin condensation and DNA repair. In T. brucei, no domain recognizing methylated histone has been identified so far. TbTFIIS2-2, as a potential transcription elongation factors in T. brucei, contains a PWWP domain in the N-terminus which shares low sequence similarity compared with other PWWP domains and is absent from other TFIIS factors. In the present study, the solution structure of TbTFIIS2-2 PWWP domain was determined by NMR spectroscop...
Source: Biochemical Journal - January 31, 2019 Category: Biochemistry Authors: Wang, R., Gao, J., Zhang, J., Zhang, X., Xu, C., Liao, S., Tu, X. Tags: Research Articles Source Type: research

Silencing of type II phosphatidylinositol 4-kinase {beta} stabilizes prostate apoptosis response-4 and induces apoptosis in cancer cells
Type II phosphatidylinositol 4-kinase β (PtdIns 4-kinase II β) is an enigma among the phosphatidylinositol 4-kinase family. The role of PtdIns 4-kinase II β in MCF-7 cells was addressed with the help of short hairpin RNA (shRNA). PtdIns 4-kinase II β shRNA transfection increased pan-caspase activity and induced apoptosis in cancerous MCF-7 cells. Non-cancerous MCF-10A cells were resistant to PtdIns 4-kinase II β shRNA-induced apoptosis. Caspase 8 and 9 inhibitors rescued MCF-7 cells from apoptosis. Shotgun proteomic studies with Flag-tagged PtdIns 4-kinase II β immunoprecipitates showed tumor ...
Source: Biochemical Journal - January 31, 2019 Category: Biochemistry Authors: Chaudhry, S., Joshi, V., Bojjireddy, N., Thoh, M., Sandur, S. K., Subrahmanyam, G. Tags: Research Articles Source Type: research

ZNF300 stimulates fatty acid oxidation and alleviates hepatosteatosis through regulating PPAR{alpha}
ZNF300 plays an important role in the regulation of HBV-related hepatocellular carcinoma. However, little is known about the role of ZNF300 in lipid metabolism and NAFLD. In the present study, we observed that ZNF300 expression was markedly decreased in free fatty acid (FFA)-induced fatty liver. Overexpressed ZNF300 alleviated hepatic lipid accumulation, whereas knockdown of ZNF300 enhanced the FFA-induced lipid accumulation. Investigations of the underlying mechanisms revealed that ZNF300 directly binds to and regulates the PPARα expression, thus promoting fatty acid oxidation. Furthermore, bisulfite pyrosequencing ...
Source: Biochemical Journal - January 31, 2019 Category: Biochemistry Authors: Yan, F.-J., Wang, Y.-J., Yan, S.-R., Lu, J., Zheng, Y.-L. Tags: Research Articles Source Type: research

Double trouble? Gag in conjunction with double insert in HIV protease contributes to reduced DRV susceptibility
HIV protease is essential for processing the Gag polyprotein to produce infectious virions and is a major target in antiretroviral therapy. We have identified an unusual HIV-1 subtype C variant that contains insertions of leucine and asparagine (L38NL) in the hinge region of protease at position 38. This was isolated from a protease inhibitor naìˆve infant. Isothermal titration calorimetry showed that 10% less of L38NL protease was in the active conformation as compared with a reference strain. L38NL protease displayed a ±50% reduction in KM and kcat. The catalytic efficiency (kcat/KM) of L38NL protease was not sig...
Source: Biochemical Journal - January 31, 2019 Category: Biochemistry Authors: Williams, A., Basson, A., Achilonu, I., Dirr, H. W., Morris, L., Sayed, Y. Tags: Research Articles Source Type: research

Progress in targeting RAS with small molecule drugs
RAS proteins have traditionally been deemed undruggable, as they do not possess an active site to which small molecules could bind but small molecules that target one form of oncogenic RAS, KRAS G12C, are already in preclinical and clinical trials, and several other compounds that bind to different RAS proteins at distinct sites are in earlier stage evaluation. KRAS is the major clinical target, as it is by far the most significant form of RAS in terms of cancer incidence. Unfortunately, KRAS exists in two isoforms, each with unique biochemical properties. This complicates efforts to target KRAS specifically. KRAS is also ...
Source: Biochemical Journal - January 31, 2019 Category: Biochemistry Authors: McCormick, F. Tags: Review Articles Source Type: research

Phosphofructokinase controls the acetaldehyde-induced phase shift in isolated yeast glycolytic oscillators
The response of oscillatory systems to external perturbations is crucial for emergent properties such as synchronisation and phase locking and can be quantified in a phase response curve (PRC). In individual, oscillating yeast cells, we characterised experimentally the phase response of glycolytic oscillations for external acetaldehyde pulses and followed the transduction of the perturbation through the system. Subsequently, we analysed the control of the relevant system components in a detailed mechanistic model. The observed responses are interpreted in terms of the functional coupling and regulation in the reaction netw...
Source: Biochemical Journal - January 31, 2019 Category: Biochemistry Authors: van Niekerk, D. D., Gustavsson, A.-K., Mojica-Benavides, M., Adiels, C. B., Goksör, M., Snoep, J. L. Tags: Research Articles Source Type: research

The KH-type splicing regulatory protein (KSRP) regulates type III interferon expression post-transcriptionally
Type III interferons (IFNs) are the latest members of the IFN family. They play an important role in immune defense mechanisms, especially in antiviral responses at mucosal sites. Moreover, they control inflammatory reactions by modulating neutrophil and dendritic cell functions. Therefore, it is important to identify cellular mechanisms involved in the control of type III IFN expression. All IFN family members contain AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) of their mRNAs that determine mRNA half-life and consequently the expressional level of these cytokines. mRNA stability is controlled by differ...
Source: Biochemical Journal - January 31, 2019 Category: Biochemistry Authors: Schmidtke, L., Schrick, K., Saurin, S., Käfer, R., Gather, F., Weinmann-Menke, J., Kleinert, H., Pautz, A. Tags: Research Articles Source Type: research

The peroxisomal zebrafish SCP2-thiolase (type-1) is a weak transient dimer as revealed by crystal structures and native mass spectrometry
The SCP2 (sterol carrier protein 2)-thiolase (type-1) functions in the vertebrate peroxisomal, bile acid synthesis pathway, converting 24-keto-THC-CoA and CoA into choloyl-CoA and propionyl-CoA. This conversion concerns the β-oxidation chain shortening of the steroid fatty acyl-moiety of 24-keto-THC-CoA. This class of dimeric thiolases has previously been poorly characterized. High-resolution crystal structures of the zebrafish SCP2-thiolase (type-1) now reveal an open catalytic site, shaped by residues of both subunits. The structure of its non-dimerized monomeric form has also been captured in the obtained crystals....
Source: Biochemical Journal - January 25, 2019 Category: Biochemistry Authors: Kiema, T.-R., Thapa, C. J., Laitaoja, M., Schmitz, W., Maksimainen, M. M., Fukao, T., Rouvinen, J., Jänis, J., Wierenga, R. K. Tags: Research Articles Source Type: research

Sorting nexin 27 rescues neuroligin 2 from lysosomal degradation to control inhibitory synapse number
Retromer is an evolutionarily conserved endosomal trafficking complex that mediates the retrieval of cargo proteins from a degradative pathway for sorting back to the cell surface. To promote cargo recycling, the core retromer trimer of VPS (vacuolar protein sorting)26, VPS29 and VPS35 recognises cargo either directly, or through an adaptor protein, the most well characterised of which is the PDZ [postsynaptic density 95 (PSD95), disk large, zona occludens] domain-containing sorting nexin SNX27. Neuroligins (NLGs) are postsynaptic trans-synaptic scaffold proteins that function in the clustering of postsynaptic proteins to ...
Source: Biochemical Journal - January 25, 2019 Category: Biochemistry Authors: Binda, C. S., Nakamura, Y., Henley, J. M., Wilkinson, K. A. Tags: Research Articles Source Type: research

Adaptation of the Staphylococcus aureus leukocidin LukGH for the rabbit host by protein engineering
Host defense against Staphylococcus aureus greatly depends on bacterial clearance by phagocytic cells. LukGH (or LukAB) is the most potent staphylococcal leukocidin towards human phagocytes in vitro, but its role in pathogenesis is obscured by the lack of suitable small animal models because LukGH has limited or no cytotoxicity towards rodent and rabbit compared with human polymorphonuclear cells (PMNs) likely due to an impaired interaction with its cellular receptor, CD11b. We aimed at adapting LukGH for the rabbit host by improving binding to the rabbit homolog of CD11b, specifically its I-domain (CD11b-I). Targeted amin...
Source: Biochemical Journal - January 25, 2019 Category: Biochemistry Authors: Trstenjak, N., Stulik, L., Rouha, H., Zmajkovic, J., Zerbs, M., Nagy, E., Badarau, A. Tags: Research Articles Source Type: research

Validation of anti-glucocerebrosidase antibodies for western blot analysis on protein lysates of murine and human cells
Gaucher disease (GD) is a rare lysosomal storage disorder caused by mutations in the GBA1 gene, encoding the lysosome-resident glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in GBA1 and the development of synucleinopathies, including Parkinson disease, has directed attention to glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the glucocerebrosidase enzyme. The use of various commercial and custom-made glucocerebrosidase antibo...
Source: Biochemical Journal - January 25, 2019 Category: Biochemistry Authors: Qi, W., Davidson, B. A., Nguyen, M., Lindstrom, T., Grey, R. J., Burnett, R., Aflaki, E., Sidransky, E., Westbroek, W. Tags: Research Articles Source Type: research

Repurposing of idebenone as a potential anti-cancer agent
Glioblastoma (GB) represents the most common and aggressive form of malignant primary brain tumour associated with high rates of morbidity and mortality. In the present study, we considered the potential use of idebenone (IDE), a Coenzyme Q10 analogue, as a novel chemotherapeutic agent for GB. On two GB cell lines, U373MG and U87MG, IDE decreased the viable cell number and enhanced the cytotoxic effects of two known anti-proliferative agents: temozolomide and oxaliplatin. IDE also affected the clonogenic and migratory capacity of both GB cell lines, at 25 and 50 µM, a concentration equivalent to that transiently...
Source: Biochemical Journal - January 25, 2019 Category: Biochemistry Authors: Damiani, E., Yuecel, R., Wallace, H. M. Tags: Research Articles Source Type: research

Elevated hypertrophy, growth plate maturation, glycosaminoglycan deposition, and exostosis formation in the Hspg2 exon 3 null mouse intervertebral disc
Heparan sulfate (HS) regulates diverse cell signalling events in intervertebral disc development and homeostasis. The aim of the present study was to investigate the effect of ablation of perlecan HS/CS on murine intervertebral disc development. Genetic models carrying mutations in genes encoding HS biosynthetic enzymes have identified multiple roles for HS in tissue homeostasis. In the present study, we utilised an Hspg2 exon 3 null HS/CS-deficient mouse to assess the role of perlecan HS in disc cell regulation. HS makes many important contributions to growth factor sequestration, stabilisation/delivery, and activation of...
Source: Biochemical Journal - January 18, 2019 Category: Biochemistry Authors: Shu, C. C., Smith, S. M., Little, C. B., Melrose, J. Tags: Research Articles Source Type: research

Structural insights into the molecular mechanism underlying Sirt5-catalyzed desuccinylation of histone peptides
Histone modification is a ubiquitous regulatory mechanism involved in a variety of biological processes, including gene expression, DNA damage repair, cell differentiation, and ontogenesis. Succinylation sites on histones have been identified and may have functional consequences. Here, we demonstrate that human sirtuin 5 (Sirt5) catalyzes the sequence-selective desuccinylation of numerous histone succinyl sites. Structural studies of Sirt5 in complex with four succinyl peptides indicate an essential role for the conserved main chain hydrogen bonds formed by the succinyl lysine (0), +1, and +3 sites for substrate-enzyme rec...
Source: Biochemical Journal - January 18, 2019 Category: Biochemistry Authors: Hang, T., Chen, W., Wu, M., Zhan, L., Wang, C., Jia, N., Zhang, X., Zang, J. Tags: Research Articles Source Type: research

Ca2+-dependent calmodulin binding to cardiac ryanodine receptor (RyR2) calmodulin-binding domains
The Ca2+ sensor calmodulin (CaM) regulates cardiac ryanodine receptor (RyR2)-mediated Ca2+ release from the sarcoplasmic reticulum. CaM inhibits RyR2 in a Ca2+-dependent manner and aberrant CaM-dependent inhibition results in life-threatening cardiac arrhythmias. However, the molecular details of the CaM–RyR2 interaction remain unclear. Four CaM-binding domains (CaMBD1a, -1b, -2, and -3) in RyR2 have been proposed. Here, we investigated the Ca2+-dependent interactions between CaM and these CaMBDs by monitoring changes in the fluorescence anisotropy of carboxytetramethylrhodamine (TAMRA)-labeled CaMBD peptides during ...
Source: Biochemical Journal - January 18, 2019 Category: Biochemistry Authors: Brohus, M., Sondergaard, M. T., Wayne Chen, S. R., van Petegem, F., Overgaard, M. T. Tags: Research Articles Source Type: research

The kinetic characteristics of human and trypanosomatid phosphofructokinases for the reverse reaction
Eukaryotic ATP-dependent phosphofructokinases (PFKs) are often considered unidirectional enzymes catalysing the transfer of a phospho moiety from ATP to fructose 6-phosphate to produce ADP and fructose 1,6-bisphosphate. The reverse reaction is not generally considered to occur under normal conditions and has never been demonstrated for any eukaryotic ATP-dependent PFKs, though it does occur in inorganic pyrophosphate-dependent PFKs and has been experimentally shown for bacterial ATP-dependent PFKs. The evidence is provided via two orthogonal assays that all three human PFK isoforms can catalyse the reverse reaction in vitr...
Source: Biochemical Journal - January 18, 2019 Category: Biochemistry Authors: Fernandes, P. M., Kinkead, J., McNae, I. W., Bringaud, F., Michels, P. A. M., Walkinshaw, M. D. Tags: Research Articles Source Type: research

Structural basis for two efficient modes of agropinic acid opine import into the bacterial pathogen Agrobacterium tumefaciens
Agrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. These opines serve as nutrients and are imported into bacteria via periplasmic-binding proteins (PBPs) in association with ABC transporters. Structural and affinity data on agropine and agropinic acid opines bound to PBPs are currently lacking. Here, we investigated the molecular basis of AgtB and AgaA, proposed as the specific PBP for agropine and agropinic acid import, respectively. Using genetic approaches...
Source: Biochemical Journal - January 15, 2019 Category: Biochemistry Authors: Marty, L., Vigouroux, A., Aumont-Nicaise, M., Pelissier, F., Meyer, T., Lavire, C., Dessaux, Y., Morera, S. Tags: Research Articles Source Type: research

Engineering Arabidopsis long-chain acyl-CoA synthetase 9 variants with enhanced enzyme activity
Long-chain acyl-CoA synthetase (LACS, EC 6.2.1.3) catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which, in turn, serves as the major acyl donor for various lipid metabolic pathways. Increasing the size of acyl-CoA pool by enhancing LACS activity appears to be a useful approach to improve the production and modify the composition of fatty acid-derived compounds, such as triacylglycerol. In the present study, we aimed to improve the enzyme activity of Arabidopsis thaliana LACS9 (AtLACS9) by introducing random mutations into its cDNA using error-prone PCR. Two AtLACS9 variants containing multiple ...
Source: Biochemical Journal - January 15, 2019 Category: Biochemistry Authors: Xu, Y., Caldo, K. M. P., Holic, R., Mietkiewska, E., Ozga, J., Rizvi, S. M., Chen, G., Weselake, R. J. Tags: Research Articles Source Type: research

Ribavirin-induced down-regulation of CCAAT/enhancer-binding protein {alpha} leads to suppression of lipogenesis
Recently, we demonstrated that the anti-viral drug ribavirin (RBV) had the ability to suppress lipogenesis through down-regulation of retinoid X receptor α (RXRα) under the control of the intracellular GTP-level and AMP-activated protein kinase-related kinases, especially microtubule affinity regulating kinase 4 (MARK4). RXRα-overexpression attenuated but did not abolish lipogenesis suppression by RBV, implying that additional factor(s) were involved in this suppressive effect. In the present study, we found that the protein level, but not the mRNA level, of CCAAT/enhancer-binding protein α (C/EBP&a...
Source: Biochemical Journal - January 15, 2019 Category: Biochemistry Authors: Satoh, S., Onomura, D., Ueda, Y., Dansako, H., Honda, M., Kaneko, S., Kato, N. Tags: Research Articles Source Type: research

Mechanism of metal ion-induced activation of a two-component sensor kinase
Two-component systems (TCSs) are essential for bacteria to sense, respond, and adapt to changing environments, such as elevation of Cu(I)/Ag(I) ions in the periplasm. In Escherichia coli, the CusS–CusR TCS up-regulates the cusCFBA genes under increased periplasmic Cu(I)/Ag(I) concentrations to help maintain metal ion homeostasis. The CusS histidine kinase is a homodimeric integral membrane protein that binds to periplasmic Cu(I)/Ag(I) and transduces a signal to its cytoplasmic kinase domain. However, the mechanism of how metal binding in the periplasm activates autophosphorylation in the cytoplasm is unknown. Here, w...
Source: Biochemical Journal - January 15, 2019 Category: Biochemistry Authors: Affandi, T., McEvoy, M. M. Tags: Research Articles Source Type: research

New structural insights into anomeric carbohydrate recognition by frutalin: an {alpha}-D-galactose-binding lectin from breadfruit seeds
Frutalin (FTL) is a multiple-binding lectin belonging to the jacalin-related lectin (JRL) family and derived from Artocarpus incisa (breadfruit) seeds. This lectin specifically recognizes and binds α-d-galactose. FTL has been successfully used in immunobiological research for the recognition of cancer-associated oligosaccharides. However, the molecular bases by which FTL promotes these specific activities remain poorly understood. Here, we report the whole 3D structure of FTL for the first time, as determined by X-ray crystallography. The obtained crystals diffracted to 1.81 Å (Apo-frutalin) and 1.65 Å (f...
Source: Biochemical Journal - January 11, 2019 Category: Biochemistry Authors: Vieira Neto, A. E., de Sousa, F. D., Pereira, H. D., Moreno, F. B. M. B., Lourenzoni, M. R., Grangeiro, T. B., Monteiro Moreira, A. C. d. O., Moreira, R. d. A. Tags: Research Articles Source Type: research

Glycerol-3-phosphate acyltransferases 3 and 4 direct glycerolipid synthesis and affect functionality in activated macrophages
Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo2-lipid A, w...
Source: Biochemical Journal - January 11, 2019 Category: Biochemistry Authors: Quiroga, I. Y., Pellon-Maison, M., Suchanek, A. L., Coleman, R. A., Gonzalez-Baro, M. R. Tags: Research Articles Source Type: research

Using backbone-cyclized Cys-rich polypeptides as molecular scaffolds to target protein-protein interactions
The use of disulfide-rich backbone-cyclized polypeptides, as molecular scaffolds to design a new generation of bioimaging tools and drugs that are potent and specific, and thus might have fewer side effects than traditional small-molecule drugs, is gaining increasing interest among the scientific and in the pharmaceutical industries. Highly constrained macrocyclic polypeptides are exceptionally more stable to chemical, thermal and biological degradation and show better biological activity when compared with their linear counterparts. Many of these relatively new scaffolds have been also found to be highly tolerant to seque...
Source: Biochemical Journal - January 11, 2019 Category: Biochemistry Authors: Chaudhuri, D., Aboye, T., Camarero, J. A. Tags: Review Articles Source Type: research

Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein
The LRR (leucine-rich repeat)–Roc (Ras of complex proteins)–COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer–monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5'-diphosphate)-bound states and monomeric in the GTP (guanosine-5'-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucl...
Source: Biochemical Journal - January 7, 2019 Category: Biochemistry Authors: Deyaert, E., Leemans, M., Singh, R. K., Gallardo, R., Steyaert, J., Kortholt, A., Lauer, J., Versees, W. Tags: Research Articles Source Type: research

Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling
Up-regulation of epidermal growth factor receptor (EGFR) is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here, we report the isolation and functional characterization of novel camelid single-domain antibodies (sdAbs or VHHs) directed against human EGFR. The source of these VHHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost), which is notable in that genetic immunization of large, outbred animals is generally poorly effective. The VHHs targeted multiple sites on the receptor's surface with high...
Source: Biochemical Journal - January 7, 2019 Category: Biochemistry Authors: Rossotti, M. A., Henry, K. A., van Faassen, H., Tanha, J., Callaghan, D., Hussack, G., Arbabi-Ghahroudi, M., MacKenzie, C. R. Tags: Research Articles Source Type: research

Strangers in strange lands: mitochondrial proteins found at extra-mitochondrial locations
The mitochondrial proteome is estimated to contain ~1100 proteins, the vast majority of which are nuclear-encoded, with only 13 proteins encoded by the mitochondrial genome. The import of these nuclear-encoded proteins into mitochondria was widely believed to be unidirectional, but recent discoveries have revealed that many these ‘mitochondrial’ proteins are exported, and have extra-mitochondrial activities divergent from their mitochondrial function. Surprisingly, three of the exported proteins discovered thus far are mitochondrially encoded and have significantly different extra-mitochondrial roles than those...
Source: Biochemical Journal - January 7, 2019 Category: Biochemistry Authors: Scanlon, D. P., Salter, M. W. Tags: Review Articles Source Type: research

Understanding phosphoinositides: rare, dynamic, and essential membrane phospholipids
Polyphosphoinositides (PPIs) are essential phospholipids located in the cytoplasmic leaflet of eukaryotic cell membranes. Despite contributing only a small fraction to the bulk of cellular phospholipids, they make remarkable contributions to practically all aspects of a cell's life and death. They do so by recruiting cytoplasmic proteins/effectors or by interacting with cytoplasmic domains of membrane proteins at the membrane–cytoplasm interface to organize and mold organelle identity. The present study summarizes aspects of our current understanding concerning the metabolism, manipulation, measurement, and intimate ...
Source: Biochemical Journal - January 7, 2019 Category: Biochemistry Authors: Dickson, E. J., Hille, B. Tags: Review Articles Source Type: research

The multifunctional role of phospho-calmodulin in pathophysiological processes
Calmodulin (CaM) is a versatile Ca2+-sensor/transducer protein that modulates hundreds of enzymes, channels, transport systems, transcription factors, adaptors and other structural proteins, controlling in this manner multiple cellular functions. In addition to its capacity to regulate target proteins in a Ca2+-dependent and Ca2+-independent manner, the posttranslational phosphorylation of CaM by diverse Ser/Thr- and Tyr-protein kinases has been recognized as an important additional manner to regulate this protein by fine-tuning its functionality. In this review, we shall cover developments done in recent years in which ph...
Source: Biochemical Journal - December 21, 2018 Category: Biochemistry Authors: Villalobo, A. Tags: Review Articles Source Type: research

HDAC1 overexpression enhances {beta}-cell proliferation by down-regulating Cdkn1b/p27
The homeobox transcription factor Nkx6.1 is sufficient to increase functional β-cell mass, where functional β-cell mass refers to the combination of β-cell proliferation, glucose-stimulated insulin secretion (GSIS) and β-cell survival. Here, we demonstrate that the histone deacetylase 1 (HDAC1), which is an early target of Nkx6.1, is sufficient to increase functional β-cell mass. We show that HDAC activity is necessary for Nkx6.1-mediated proliferation, and that HDAC1 is sufficient to increase β-cell proliferation in primary rat islets and the INS-1 832/13 β-cell line. The increase in HDA...
Source: Biochemical Journal - December 19, 2018 Category: Biochemistry Authors: Draney, C., Austin, M. C., Leifer, A. H., Smith, C. J., Kener, K. B., Aitken, T. J., Hess, K. H., Haines, A. C., Lett, L. A., Hernandez-Carretero, A., Fueger, P. T., Arlotto, M., Tessem, J. S. Tags: Research Articles Source Type: research

CosR is an oxidative stress sensing a MarR-type transcriptional repressor in Corynebacterium glutamicum
The MarR family is unique to both bacteria and archaea. The members of this family, one of the most prevalent families of transcriptional regulators in bacteria, enable bacteria to adapt to changing environmental conditions, such as the presence of antibiotics, toxic chemicals, or reactive oxygen species (ROS), mainly by thiol-disulfide switches. Although the genome of Corynebacterium glutamicum encodes a large number of the putative MarR-type transcriptional regulators, their physiological and biochemical functions have so far been limited to only two proteins, regulator of oxidative stress response RosR and quinone oxido...
Source: Biochemical Journal - December 19, 2018 Category: Biochemistry Authors: Si, M., Chen, C., Su, T., Che, C., Yao, S., Liang, G., Li, G., Yang, G. Tags: Research Articles Source Type: research

Structural enzymology reveals the molecular basis of substrate regiospecificity and processivity of an exemplar bacterial glycoside hydrolase family 74 endo-xyloglucanase
Paenibacillus odorifer produces a single multimodular enzyme containing a glycoside hydrolase (GH) family 74 module (AIQ73809). Recombinant production and characterization of the GH74 module (PoGH74cat) revealed a highly specific, processive endo-xyloglucanase that can hydrolyze the polysaccharide backbone at both branched and unbranched positions. X-ray crystal structures obtained for the free enzyme and oligosaccharide complexes evidenced an extensive hydrophobic binding platform — the first in GH74 extending from subsites –4 to +6 — and unique mobile active-site loops. Site-directed mutagenesis reveale...
Source: Biochemical Journal - December 19, 2018 Category: Biochemistry Authors: Arnal, G., Stogios, P. J., Asohan, J., Skarina, T., Savchenko, A., Brumer, H. Tags: Research Articles Source Type: research

Structure of the sensory domain of McpX from Sinorhizobium meliloti, the first known bacterial chemotactic sensor for quaternary ammonium compounds
The α-proteobacterium Sinorhizobium meliloti can live freely in the soil or engage in a symbiosis with its legume host. S. meliloti facilitates nitrogen fixation in root nodules, thus providing pivotal, utilizable nitrogen to the host. The organism has eight chemoreceptors, namely McpT to McpZ and IcpA that facilitate chemotaxis. McpX is the first known bacterial sensor of quaternary ammonium compounds (QACs) such as choline and betaines. Because QACs are exuded at chemotaxis-relevant concentrations by germinating alfalfa seeds, McpX has been proposed to contribute to host-specific chemotaxis. We have determined the ...
Source: Biochemical Journal - December 14, 2018 Category: Biochemistry Authors: Shrestha, M., Compton, K. K., Mancl, J. M., Webb, B. A., Brown, A. M., Scharf, B. E., Schubot, F. D. Tags: Research Articles Source Type: research

Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction
The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T),...
Source: Biochemical Journal - December 14, 2018 Category: Biochemistry Authors: Da'as, S. I., Fakhro, K., Thanassoulas, A., Krishnamoorthy, N., Saleh, A., Calver, B. L., Safieh-Garabedian, B., Toft, E., Nounesis, G., Lai, F. A., Nomikos, M. Tags: Research Articles Source Type: research

Co-repressor, co-activator and general transcription factor: the many faces of the Sin3 histone deacetylase (HDAC) complex
At face value, the Sin3 histone deacetylase (HDAC) complex appears to be a prototypical co-repressor complex, that is, a multi-protein complex recruited to chromatin by DNA bound repressor proteins to facilitate local histone deacetylation and transcriptional repression. While this is almost certainly part of its role, Sin3 stubbornly refuses to be pigeon-holed in quite this way. Genome-wide mapping studies have found that Sin3 localises predominantly to the promoters of actively transcribed genes. While Sin3 knockout studies in various species result in a combination of both up- and down-regulated genes. Furthermore, gene...
Source: Biochemical Journal - December 14, 2018 Category: Biochemistry Authors: Adams, G. E., Chandru, A., Cowley, S. M. Tags: Review Articles Source Type: research

Piercing the lipid raft: the case of Vibrio cholerae cytolysin
In a recent issue of Biochemical Journal, Kathuria et al. [Biochem. J. (2018) 475, 3039–3055] report that membrane binding of the pore-forming toxin Vibrio cholerae cytolysin (VCC) is facilitated by the presence of cholesterol, and the presence of this sterol within the lipid bilayer is key for the formation of a functional pore. Yet, in the presence of accessory non-lipid components, VCC retains its membrane-binding capability likely through membrane lipid raft structures. In light of their results, the authors provide new insights into the roles of cholesterol and of membrane microstructures in the binding, the oli...
Source: Biochemical Journal - December 14, 2018 Category: Biochemistry Authors: Cyr, N. Tags: Commentaries Source Type: research

On mechanisms of colicin import: the outer membrane quandary
Discussion of channel-forming colicins focuses mainly on colicin E1 for which BtuB is receptor and the OM TolC protein the proposed translocator. The ability of TolC, part of a multidrug efflux pump, for which there is no precedent for an import function, to provide a trans-periplasmic import pathway for colicin E1, is questioned on the basis of an unfavorable hairpin conformation of colicin N-terminal peptides inserted into TolC. (Source: Biochemical Journal)
Source: Biochemical Journal - December 12, 2018 Category: Biochemistry Authors: Cramer, W. A., Sharma, O., Zakharov, S. D. Tags: Review Articles Source Type: research

Rational engineering of a malate dehydrogenase for microbial production of 2,4-dihydroxybutyric acid via homoserine pathway
A synthetic pathway for the production of 2,4-dihydroxybutyric acid from homoserine (HMS), composed of two consecutive enzymatic reaction steps has been recently reported. An important step in this pathway consists in the reduction in 2-keto-4-hydroxybutyrate (OHB) into (l)-dihydroxybutyrate (DHB), by an enzyme with OHB reductase activity. In the present study, we used a rational approach to engineer an OHB reductase by using the cytosolic (l)-malate dehydrogenase from Escherichia coli (Ec-Mdh) as the template enzyme. Structural analysis of (l)-malate dehydrogenase and (l)-lactate dehydrogenase enzymes acting on sterically...
Source: Biochemical Journal - December 12, 2018 Category: Biochemistry Authors: Frazao, C. J. R., Topham, C. M., Malbert, Y., Francois, J. M., Walther, T. Tags: Research Articles Source Type: research

Specificity and mechanism of carbohydrate demethylation by cytochrome P450 monooxygenases
Degradation of carbohydrates by bacteria represents a key step in energy metabolism that can be inhibited by methylated sugars. Removal of methyl groups, which is critical for further processing, poses a biocatalytic challenge because enzymes need to overcome a high energy barrier. Our structural and computational analysis revealed how a member of the cytochrome P450 family evolved to oxidize a carbohydrate ligand. Using structural biology, we ascertained the molecular determinants of substrate specificity and revealed a highly specialized active site complementary to the substrate chemistry. Invariance of the residues inv...
Source: Biochemical Journal - December 12, 2018 Category: Biochemistry Authors: Robb, C. S., Reisky, L., Bornscheuer, U. T., Hehemann, J.-H. Tags: Research Articles Source Type: research

Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity
Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400–657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cell...
Source: Biochemical Journal - December 11, 2018 Category: Biochemistry Authors: Gomez, A., Bindesboll, C., Satheesh, S. V., Grimaldi, G., Hutin, D., MacPherson, L., Ahmed, S., Tamblyn, L., Cho, T., Nebb, H. I., Moen, A., Anonsen, J. H., Grant, D. M., Matthews, J. Tags: Research Articles Source Type: research

Identification of active site residues implies a two-step catalytic mechanism for acyl-ACP thioesterase
In plants and bacteria that use a Type II fatty acid synthase, isozymes of acyl-acyl carrier protein (ACP) thioesterase (TE) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In the present study, the sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed ...
Source: Biochemical Journal - December 10, 2018 Category: Biochemistry Authors: Jing, F., Yandeau-Nelson, M. D., Nikolau, B. J. Tags: Research Articles Source Type: research

DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors
Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomo...
Source: Biochemical Journal - December 10, 2018 Category: Biochemistry Authors: Kozisek, M., Navratil, V., Rojikova, K., Pokorna, J., Berenguer Albinana, C., Pachl, P., Zemanova, J., Machara, A., Sacha, P., Hudlicky, J., Cisarova, I., Rezacova, P., Konvalinka, J. Tags: Research Articles Source Type: research

GPCR structure and function relationship: identification of a biased apelin receptor mutant
Biased ligands of G protein-coupled receptors (GPCRs) may have improved therapeutic benefits and safety profiles. However, the molecular mechanism of GPCR biased signaling remains largely unknown. Using apelin receptor (APJ) as a model, we systematically investigated the potential effects of amino acid residues around the orthosteric binding site on biased signaling. We discovered that a single residue mutation I109A (I1093.32) in the transmembrane domain 3 (TM3) located in the deep ligand-binding pocket was sufficient to convert a balanced APJ into a G protein signaling biased receptor. APJ I109A mutant receptor retained ...
Source: Biochemical Journal - December 6, 2018 Category: Biochemistry Authors: Ban, T., Li, X., Ma, X., Yang, H., Song, Y., Sun, Y., Shen, M., Li, N., Zhang, M.-Y., Ma, Y., Zhong, W., Zhang, M., Hu, L. A. Tags: Research Articles Source Type: research

Polyamines stimulate the CHSY1 synthesis through the unfolding of the RNA G-quadruplex at the 5'-untraslated region
Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the ...
Source: Biochemical Journal - December 6, 2018 Category: Biochemistry Authors: Yamaguchi, K., Asakura, K., Imamura, M., Kawai, G., Sakamoto, T., Furihata, T., Linhardt, R. J., Igarashi, K., Toida, T., Higashi, K. Tags: Research Articles Source Type: research

Rhodobacter sphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates
Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chai...
Source: Biochemical Journal - December 6, 2018 Category: Biochemistry Authors: Tarrago, L., Grosse, S., Siponen, M. I., Lemaire, D., Alonso, B., Miotello, G., Armengaud, J., Arnoux, P., Pignol, D., Sabaty, M. Tags: Research Articles Source Type: research

Solution structure of TbCentrin4 from Trypanosoma brucei and its interactions with Ca2+ and other centrins
Centrin is a conserved calcium-binding protein that plays an important role in diverse cellular biological processes such as ciliogenesis, gene expression, DNA repair and signal transduction. In Trypanosoma brucei, TbCentrin4 is mainly localized in basal bodies and bi-lobe structure, and is involved in the processes coordinating karyokinesis and cytokinesis. In the present study, we solved the solution structure of TbCentrin4 using NMR (nuclear magnetic resonance) spectroscopy. TbCentrin4 contains four EF-hand motifs consisting of eight α-helices. Isothermal titration calorimetry experiment showed that TbCentrin4 has...
Source: Biochemical Journal - December 6, 2018 Category: Biochemistry Authors: Shan, F., Ye, K., Zhang, J., Liao, S., Zhang, X., Xu, C., Tu, X. Tags: Research Articles Source Type: research

LIMK2-1, a new isoform of human LIMK2, regulates actin cytoskeleton remodeling via a different signaling pathway than that of its two homologs, LIMK2a and LIMK2b
LIMK1 and LIMK2 (LIMKs, LIM kinases) are kinases that play a crucial role in cytoskeleton dynamics by independently regulating both actin filament and microtubule remodeling. LIMK1 and, more recently, LIMK2 have been shown to be involved in cancer development and metastasis, resistance of cancer cells to microtubule-targeted treatments, neurological diseases, and viral infection. LIMKs have thus recently emerged as new therapeutic targets. Databanks describe three isoforms of human LIMK2: LIMK2a, LIMK2b, and LIMK2-1. Evidence suggests that they may not have completely overlapping functions. We biochemically characterized t...
Source: Biochemical Journal - December 6, 2018 Category: Biochemistry Authors: Vallee, B., Cuberos, H., Doudeau, M., Godin, F., Gosset, D., Vourc'h, P., Andres, C. R., Benedetti, H. Tags: Research Articles Source Type: research