Ezrin-anchored PKA phosphorylates serine 369 and 373 on connexin 43 to enhance gap junction assembly, communication, and cell fusion
A limited number of human cells can fuse to form multinucleated syncytia. In the differentiation of human placenta, mononuclear cytotrophoblasts fuse to form an endocrinologically active, non-proliferative, multinucleated syncytium. This syncytium covers the placenta and manages the exchange of nutrients and gases between maternal and fetal circulation. We recently reported protein kinase A (PKA) to be part of a macromolecular signaling complex with ezrin and gap junction protein connexin 43 (Cx43) that provides cAMP-mediated control of gap junction communication. Here, we examined the associated phosphorylation events. In...
Source: Biochemical Journal - January 31, 2018 Category: Biochemistry Authors: Dukic, A. R., Gerbaud, P., Guibourdenche, J., Thiede, B., Tasken, K., Pidoux, G. Tags: Research Articles Source Type: research

Molecular control of stomatal development
Plants have evolved developmental plasticity which allows the up- or down-regulation of photosynthetic and water loss capacities as new leaves emerge. This developmental plasticity enables plants to maximise fitness and to survive under differing environments. Stomata play a pivotal role in this adaptive process. These microscopic pores in the epidermis of leaves control gas exchange between the plant and its surrounding environment. Stomatal development involves regulated cell fate decisions that ensure optimal stomatal density and spacing, enabling efficient gas exchange. The cellular patterning process is regulated by a...
Source: Biochemical Journal - January 31, 2018 Category: Biochemistry Authors: Zoulias, N., Harrison, E. L., Casson, S. A., Gray, J. E. Tags: Review Articles Source Type: research

Identification of a second binding site on the TRIM25 B30.2 domain
The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5'-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerizat...
Source: Biochemical Journal - January 23, 2018 Category: Biochemistry Authors: D'Cruz, A. A., Kershaw, N. J., Hayman, T. J., Linossi, E. M., Chiang, J. J., Wang, M. K., Dagley, L. F., Kolesnik, T. B., Zhang, J.-G., Masters, S. L., Griffin, M. D. W., Gack, M. U., Murphy, J. M., Nicola, N. A., Babon, J. J., Nicholson, S. E. Tags: Research Articles Source Type: research

Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens
The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a...
Source: Biochemical Journal - January 23, 2018 Category: Biochemistry Authors: Owen, R. A., Fyfe, P. K., Lodge, A., Biboy, J., Vollmer, W., Hunter, W. N., Sargent, F. Tags: Research Articles Source Type: research

Decoding the molecular switches of natriuretic peptides which differentiate its vascular and renal functions
Heart failure (HF) is associated with high morbidity and mortality. Dysfunction of blood pressure and/or volume homeostatic processes result in lower perfusion and/or congestion. Treatment strategies exerting differential effects on pressure and volume mechanisms are critical in handling patients with HF. Atrial natriuretic peptides (ANPs) are a key hormone in maintaining circulation. It binds to NP receptor-A (NPR-A) on vasculature, kidneys and nervous system to lowers blood pressure and volume. It exerts a concentration-dependent pharmacological activity, and only increased renal excretion of water and sodium at low dose...
Source: Biochemical Journal - January 23, 2018 Category: Biochemistry Authors: Sridharan, S., Kini, R. M. Tags: Research Articles Source Type: research

Topoisomerases as anticancer targets
Many cancer type-specific anticancer agents have been developed and significant advances have been made toward precision medicine in cancer treatment. However, traditional or nonspecific anticancer drugs are still important for the treatment of many cancer patients whose cancers either do not respond to or have developed resistance to cancer-specific anticancer agents. DNA topoisomerases, especially type IIA topoisomerases, are proved therapeutic targets of anticancer and antibacterial drugs. Clinically successful topoisomerase-targeting anticancer drugs act through topoisomerase poisoning, which leads to replication fork ...
Source: Biochemical Journal - January 23, 2018 Category: Biochemistry Authors: Delgado, J. L., Hsieh, C.-M., Chan, N.-L., Hiasa, H. Tags: Review Articles Source Type: research

JMJD6 induces HOTAIR, an oncogenic lincRNA, by physically interacting with its proximal promoter
Using microarray analysis, we found that HOX transcript antisense intergenic RNA (HOTAIR) is up-regulated by Jumonji domain containing-6 (JMJD6), a bifunctional lysyl hydroxylase and arginine demethylase. In breast cancer, both JMJD6 and HOTAIR RNAs increase tumor growth and associate with poor prognosis but no molecular relationship between them is known. We show that overexpression of JMJD6 increased HOTAIR expression and JMJD6 siRNAs suppressed it in ER+ MCF-7, triple negative MDA-MB-231 and non-breast cancer HEK 293 cells. Therefore, JMJD6 regulates HOTAIR independent of ER status. Using various deletion constructs spa...
Source: Biochemical Journal - January 15, 2018 Category: Biochemistry Authors: Biswas, A., Shettar, A., Mukherjee, G., Kondaiah, P., Desai, K. V. Tags: Research Articles Source Type: research

The stress sigma factor of RNA polymerase RpoS/{sigma}S is a solvent-exposed open molecule in solution
In bacteria, one primary and multiple alternative sigma () factors associate with the RNA polymerase core enzyme (E) to form holoenzymes (E) with different promoter recognition specificities. The alternative factor RpoS/S is produced in stationary phase and under stress conditions and reprograms global gene expression to promote bacterial survival. To date, the three-dimensional structure of a full-length free factor remains elusive. The current model suggests that extensive interdomain contacts in a free factor result in a compact conformation that masks the DNA-binding determinants of , explaining why a free factor does ...
Source: Biochemical Journal - January 15, 2018 Category: Biochemistry Authors: Cavaliere, P., Brier, S., Filipenko, P., Sizun, C., Raynal, B., Bonnete, F., Levi-Acobas, F., Bellalou, J., England, P., Chamot-Rooke, J., Mayer, C., Norel, F. Tags: Research Articles Source Type: research

Assaying kinase activity of the TPL-2/NF-{kappa}B1 p105/ABIN-2 complex using an optimal peptide substrate
The MKK1/2 kinase tumour progression locus 2 (TPL-2) is critical for the production of tumour necrosis factor alpha (TNFα) in innate immune responses and a potential anti-inflammatory drug target. Several earlier pharmaceutical company screens with the isolated TPL-2 kinase domain have identified small-molecule inhibitors that specifically block TPL-2 signalling in cells, but none of these have progressed to clinical development. We have previously shown that TPL-2 catalytic activity regulates TNF production by macrophages while associated with NF-B1 p105 and ABIN-2, independently of MKK1/2 phosphorylation via an unk...
Source: Biochemical Journal - January 11, 2018 Category: Biochemistry Authors: Kümper, S., Gantke, T., Chen, C.-S., Soneji, Y., Pattison, M. J., Chakravarty, P., Kjaer, S., Thomas, D., Haslam, C., Leavens, B. J., House, D., Powell, D. J., Ley, S. C. Tags: Research Articles Source Type: research

Investigation of specific interactions between T7 promoter and T7 RNA polymerase by force spectroscopy using atomic force microscope
The specific recognition and binding of promoter and RNA polymerase is the first step of transcription initiation in bacteria and largely determines transcription activity. Therefore, direct analysis of the interaction between promoter and RNA polymerase in vitro may be a new strategy for promoter characterization, to avoid interference due to the cell's biophysical condition and other regulatory elements. In the present study, the specific interaction between T7 promoter and T7 RNA polymerase was studied as a model system using force spectroscopy based on atomic force microscope (AFM). The specific interaction between T7 ...
Source: Biochemical Journal - January 11, 2018 Category: Biochemistry Authors: Zhang, X., Yao, Z., Duan, Y., Zhang, X., Shi, J., Xu, Z. Tags: Research Articles Source Type: research

Degradation pathway of plant complex-type N-glycans: identification and characterization of a key {alpha}1,3-fucosidase from glycoside hydrolase family 29
Plant complex-type N-glycans are characterized by the presence of α1,3-linked fucose towards the proximal N-acetylglucosamine residue and β1,2-linked xylose towards the β-mannose residue. These glycans are ultimately degraded by the activity of several glycoside hydrolases. However, the degradation pathway of plant complex-type N-glycans has not been entirely elucidated because the gene encoding α1,3-fucosidase, a glycoside hydrolase acting on plant complex-type N-glycans, has not yet been identified, and its substrate specificity remains to be determined. In the present study, we found that AtFUC1 (a...
Source: Biochemical Journal - January 11, 2018 Category: Biochemistry Authors: Kato, S., Hayashi, M., Kitagawa, M., Kajiura, H., Maeda, M., Kimura, Y., Igarashi, K., Kasahara, M., Ishimizu, T. Tags: Research Articles Source Type: research

Functional characterization of the Ca2+-ATPase SMA1 from Schistosoma mansoni
Schistosoma mansoni is a parasite that causes bilharzia, a neglected tropical disease affecting hundreds of millions of people each year worldwide. In 2012, S. mansoni had been identified as the only invertebrate possessing two SERCA-type Ca2+-ATPases, SMA1 and SMA2. However, our analysis of recent genomic data shows that the presence of two SERCA pumps is rather frequent in parasitic flatworms. To understand the reasons of this redundancy in S. mansoni, we compared SMA1 and SMA2 at different levels. In terms of sequence and organization, the genes SMA1 and SMA2 are similar, suggesting that they might be the result of a du...
Source: Biochemical Journal - January 11, 2018 Category: Biochemistry Authors: Marechal, X., De Mendonca, R., Miras, R., Revilloud, J., Catty, P. Tags: Research Articles Source Type: research

Multiple substitutions lead to increased loop flexibility and expanded specificity in Acinetobacter baumannii carbapenemase OXA-239
OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced ~3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) ...
Source: Biochemical Journal - January 11, 2018 Category: Biochemistry Authors: Harper, T. M., June, C. M., Taracila, M. A., Bonomo, R. A., Powers, R. A., Leonard, D. A. Tags: Research Articles Source Type: research

An unusual diphosphatase from the PhnP family cleaves reactive FAD photoproducts
Flavins are notoriously photolabile, but while the photoproducts derived from the iso-alloxazine ring are well known the other photoproducts are not. In the case of FAD, typically the main cellular flavin, the other photoproducts are predicted to include four- and five-carbon sugars linked to ADP. These FAD photoproducts were shown to be potent glycating agents, more so than ADP-ribose. Such toxic compounds would require disposal via an ADP-sugar diphosphatase or other route. Comparative analysis of bacterial genomes uncovered a candidate disposal gene that is chromosomally clustered with genes for FAD synthesis or transpo...
Source: Biochemical Journal - January 11, 2018 Category: Biochemistry Authors: Beaudoin, G. A. W., Li, Q., Bruner, S. D., Hanson, A. D. Tags: Research Articles Source Type: research

A dimeric catalytic core relates the short and long forms of ATP-phosphoribosyltransferase
Adenosine triphosphate (ATP) phosphoribosyltransferase (ATP-PRT) catalyses the first committed step of histidine biosynthesis in plants and microorganisms. Two forms of ATP-PRT have been reported, which differ in their molecular architecture and mechanism of allosteric regulation. The short-form ATP-PRT is a hetero-octamer, with four HisG chains that comprise only the catalytic domains and four separate chains of HisZ required for allosteric regulation by histidine. The long-form ATP-PRT is homo-hexameric, with each chain comprising two catalytic domains and a covalently linked regulatory domain that binds histidine as an ...
Source: Biochemical Journal - January 5, 2018 Category: Biochemistry Authors: Mittelstädt, G., Jiao, W., Livingstone, E. K., Moggre, G.-J., Nazmi, A. R., Parker, E. J. Tags: Research Articles Source Type: research

Characterization of the catalytic signature of Scabin toxin, a DNA-targeting ADP-ribosyltransferase
Scabin was previously identified as a novel DNA-targeting mono-ADP-ribosyltransferase (mART) toxin from the plant pathogen 87.22 strain of Streptomyces scabies. Scabin is a member of the Pierisin-like subgroup of mART toxins, since it targets DNA. An in-depth characterization of both the glycohydrolase and transferase enzymatic activities of Scabin was conducted. Several protein variants were developed based on an initial Scabin·DNA molecular model. Consequently, three residues were deemed important for DNA-binding and transferase activity. Trp128 and Trp155 are important for binding the DNA substrate and participat...
Source: Biochemical Journal - January 5, 2018 Category: Biochemistry Authors: Lyons, B., Lugo, M. R., Carlin, S., Lidster, T., Merrill, A. R. Tags: Research Articles Source Type: research

Revisiting paradigms of Ca2+ signaling protein kinase regulation in plants
Calcium (Ca2+) serves as a universal second messenger in eukaryotic signal transduction. Understanding the Ca2+ activation kinetics of Ca2+ sensors is critical to understanding the cellular signaling mechanisms involved. In this review, we discuss the regulatory properties of two sensor classes: the Ca2+-dependent protein kinases (CPKs/CDPKs) and the calcineurin B-like (CBL) proteins that control the activity of CBL-interacting protein kinases (CIPKs) and identify emerging topics and some foundational points that are not well established experimentally. Most plant CPKs are activated by physiologically relevant Ca2+ concent...
Source: Biochemical Journal - January 5, 2018 Category: Biochemistry Authors: Bender, K. W., Zielinski, R. E., Huber, S. C. Tags: Review Articles Source Type: research

Structural and biochemical characterization of the catalytic domains of GdpP reveals a unified hydrolysis mechanism for the DHH/DHHA1 phosphodiesterase
The Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterases (PDEs) that catalyze degradation of cyclic di-adenosine monophosphate (c-di-AMP) could be subdivided into two subfamilies based on the final product [5'-phosphadenylyl-adenosine (5'-pApA) or AMP]. In a previous study, we revealed that Rv2837c, a stand-alone DHH/DHHA1 PDE, employs a 5'-pApA internal flipping mechanism to produce AMPs. However, why the membrane-bound DHH/DHHA1 PDE can only degrade c-di-AMP to 5'-pApA remains obscure. Here, we report the crystal structure of the DHH/DHHA1 domain of GdpP (GdpP-C), and structures in comp...
Source: Biochemical Journal - January 5, 2018 Category: Biochemistry Authors: Wang, F., He, Q., Su, K., Wei, T., Xu, S., Gu, L. Tags: Research Articles Source Type: research

Back to the future: new target-validated Rab antibodies for evaluating LRRK2 signalling in cell biology and Parkinson's disease
The addition of phosphate groups to substrates allows protein kinases to regulate a myriad of biological processes, and contextual analysis of protein-bound phosphate is important for understanding how kinases contribute to physiology and disease. Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase linked to familial and sporadic cases of Parkinson's disease (PD). Recent work established that multiple Rab GTPases are physiological substrates of LRRK2, with Rab10 in particular emerging as a human substrate whose site-specific phosphorylation mirrors hyperactive LRRK2 lesions associated with PD. However, current assays ...
Source: Biochemical Journal - January 5, 2018 Category: Biochemistry Authors: Eyers, P. A. Tags: Commentaries Source Type: research

Reduced expression of cardiac ryanodine receptor protects against stress-induced ventricular tachyarrhythmia, but increases the susceptibility to cardiac alternans
Reduced protein expression of the cardiac ryanodine receptor type 2 (RyR2) is thought to affect the susceptibility to stress-induced ventricular tachyarrhythmia (VT) and cardiac alternans, but direct evidence for the role of RyR2 protein expression in VT and cardiac alternans is lacking. Here, we used a mouse model (crrm1) that expresses a reduced level of the RyR2 protein to determine the impact of reduced RyR2 protein expression on the susceptibility to VT, cardiac alternans, cardiac hypertrophy, and sudden death. Electrocardiographic analysis revealed that after the injection of relatively high doses of caffeine and epi...
Source: Biochemical Journal - January 5, 2018 Category: Biochemistry Authors: Zhong, X., Vallmitjana, A., Sun, B., Xiao, Z., Guo, W., Wei, J., Ni, M., Chen, Y., O'Brien, E. R., Gillis, A. M., Hoshijima, M., Takeshima, H., Hove-Madsen, L., Benitez, R., Belke, D., Wayne Chen, S. R. Tags: Research Articles Source Type: research

Molecular basis for the integration of environmental signals by FurB from Anabaena sp. PCC 7120
FUR (Ferric uptake regulator) proteins are among the most important families of transcriptional regulators in prokaryotes, often behaving as global regulators. In the cyanobacterium Anabaena PCC 7120, FurB (Zur, Zinc uptake regulator) controls zinc and redox homeostasis through the repression of target genes in a zinc-dependent manner. In vitro, non-specific binding of FurB to DNA elicits protection against oxidative damage and avoids cleavage by deoxyribonuclease I. The present study provides, for the first time, evidence of the influence of redox environment in the interaction of FurB with regulatory zinc and its consequ...
Source: Biochemical Journal - January 5, 2018 Category: Biochemistry Authors: Sein-Echaluce, V. C., Pallares, M. C., Lostao, A., Yruela, I., Velazquez-Campoy, A., Luisa Peleato, M., Fillat, M. F. Tags: Research Articles Source Type: research

Plant DHDPR forms a dimer with unique secondary structure features that preclude higher-order assembly
Dihydrodipicolinate reductase (DHDPR) catalyses the second reaction in the diaminopimelate pathway of lysine biosynthesis in bacteria and plants. In contrast with the tetrameric bacterial DHDPR enzymes, we show that DHDPR from Vitis vinifera (grape) and Selaginella moellendorffii are dimeric in solution. In the present study, we have also determined the crystal structures of DHDPR enzymes from the plants Arabidopsis thaliana and S. moellendorffii, which are the first dimeric DHDPR structures. The analysis of these models demonstrates that the dimer forms through the intra-strand interface, and that unique secondary feature...
Source: Biochemical Journal - January 5, 2018 Category: Biochemistry Authors: Watkin, S. A. J., Keown, J. R., Richards, E., Goldstone, D. C., Devenish, S. R. A., Grant Pearce, F. Tags: Research Articles Source Type: research

Mechanism of activation of SGK3 by growth factors via the Class 1 and Class 3 PI3Ks
Derailment of the PI3K-AGC protein kinase signalling network contributes to many human diseases including cancer. Recent work has revealed that the poorly studied AGC kinase family member, SGK3, promotes resistance to cancer therapies that target the Class 1 PI3K pathway, by substituting for loss of Akt kinase activity. SGK3 is recruited and activated at endosomes, by virtue of its phox homology domain binding to PtdIns(3)P. Here, we demonstrate that endogenous SGK3 is rapidly activated by growth factors such as IGF1, through pathways involving both Class 1 and Class 3 PI3Ks. We provide evidence that IGF1 enhances endosoma...
Source: Biochemical Journal - January 2, 2018 Category: Biochemistry Authors: Malik, N., Macartney, T., Hornberger, A., Anderson, K. E., Tovell, H., Prescott, A. R., Alessi, D. R. Tags: Research Articles Source Type: research

Chloroplast division protein ARC3 acts on FtsZ2 by preventing filament bundling and enhancing GTPase activity
Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (Filamentous temperature sensitive Z) proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts, except that MinC was lost and replaced by the eukaryotic ARC3 (accumulation and replication of chloroplasts). Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPa...
Source: Biochemical Journal - January 2, 2018 Category: Biochemistry Authors: Shaik, R. S., Sung, M. W., Vitha, S., Holzenburg, A. Tags: Research Articles Source Type: research

Leishmania LABCG2 transporter is involved in ATP-dependent transport of thiols
The Leishmania LABCG2 transporter has a key role in the redox metabolism of these protozoan parasites. Recently, the involvement of LABCG2 in virulence, autophagy and oxidative stress has been described. Null mutant parasites for LABCG2 present an increase in the intracellular levels of glutathione (GSH) and trypanothione [T(SH)2]. On the other hand, parasites overexpressing LABCG2 transporter export non-protein thiols to the extracellular medium. To explore if LABCG2 may mediate an active transport of non-protein thiols, the effect of these molecules on ATPase activity of LABCG2 as well as the ability of LABCG2 to transpo...
Source: Biochemical Journal - January 2, 2018 Category: Biochemistry Authors: Perea, A., Manzano, J. I., Kimura, Y., Ueda, K., Castanys, S., Gamarro, F. Tags: Research Articles Source Type: research

Steroidogenic abnormalities in translocator protein knockout mice and significance in the aging male
The translocator protein (TSPO) has been proposed to act as a key component in a complex important for mitochondrial cholesterol importation, which is the rate-limiting step in steroid hormone synthesis. However, TSPO function in steroidogenesis has recently been challenged by the development of TSPO knockout (TSPO-KO) mice, as they exhibit normal baseline gonadal testosterone and adrenal corticosteroid production. Here, we demonstrate that despite normal androgen levels in young male TSPO-KO mice, TSPO deficiency alters steroidogenic flux and results in reduced total steroidogenic output. Specific reductions in the levels...
Source: Biochemical Journal - January 2, 2018 Category: Biochemistry Authors: Barron, A. M., Ji, B., Kito, S., Suhara, T., Higuchi, M. Tags: Research Articles Source Type: research

SUMO chain formation relies on the amino-terminal region of SUMO-conjugating enzyme and has dedicated substrates in plants
The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for in vitro activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO m...
Source: Biochemical Journal - January 2, 2018 Category: Biochemistry Authors: Tomanov, K., Nehlin, L., Ziba, I., Bachmair, A. Tags: Research Articles Source Type: research

Biochemical characterization of INTS3 and C9ORF80, two subunits of hNABP1/2 heterotrimeric complex in nucleic acid binding
Human nucleic acid-binding protein 1 and 2 (hNABP1 and hNABP2, also known as hSSB2 and hSSB1 respectively) form two separate and independent complexes with two identical proteins, integrator complex subunit 3 (INTS3) and C9ORF80. We and other groups have demonstrated that hNABP1 and 2 are single-stranded (ss) DNA- and RNA-binding proteins, and function in DNA repair; however, the function of INTS3 and C9OFR80 remains elusive. In the present study, we purified recombinant proteins INTS3 and C9ORF80 to near homogeneity. Both proteins exist as a monomer in solution; however, C9ORF80 exhibits anomalous behavior on SDS–PA...
Source: Biochemical Journal - January 2, 2018 Category: Biochemistry Authors: Vidhyasagar, V., He, Y., Guo, M., Talwar, T., Singh, R. S., Yadav, M., Katselis, G., Vizeacoumar, F. J., Lukong, K. E., Wu, Y. Tags: Research Articles Source Type: research

Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils
There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable, brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here, we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of us...
Source: Biochemical Journal - January 2, 2018 Category: Biochemistry Authors: Fan, Y., Howden, A. J. M., Sarhan, A. R., Lis, P., Ito, G., Martinez, T. N., Brockmann, K., Gasser, T., Alessi, D. R., Sammler, E. M. Tags: Research Articles Source Type: research

Development of phospho-specific Rab protein antibodies to monitor in vivo activity of the LRRK2 Parkinson's disease kinase
Mutations that activate the LRRK2 (leucine-rich repeat protein kinase 2) protein kinase predispose to Parkinson's disease, suggesting that LRRK2 inhibitors might have therapeutic benefit. Recent work has revealed that LRRK2 phosphorylates a subgroup of 14 Rab proteins, including Rab10, at a specific residue located at the centre of its effector-binding switch-II motif. In the present study, we analyse the selectivity and sensitivity of polyclonal and monoclonal phospho-specific antibodies raised against nine different LRRK2-phosphorylated Rab proteins (Rab3A/3B/3C/3D, Rab5A/5B/5C, Rab8A/8B, Rab10, Rab12, Rab29[T71], Rab29[...
Source: Biochemical Journal - January 2, 2018 Category: Biochemistry Authors: Lis, P., Burel, S., Steger, M., Mann, M., Brown, F., Diez, F., Tonelli, F., Holton, J. L., Ho, P. W., Ho, S.-L., Chou, M.-Y., Polinski, N. K., Martinez, T. N., Davies, P., Alessi, D. R. Tags: Research Articles Source Type: research

Correction: Substrate-specifying determinants of the nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP2
(Source: Biochemical Journal)
Source: Biochemical Journal - December 14, 2017 Category: Biochemistry Authors: Cimpean, A., Stefan, C., Gijsbers, R., Stalmans, W., Bollen, M. Tags: Corrections Source Type: research

Correction: Integration of flux measurements to resolve changes in anabolic and catabolic metabolism in cardiac myocytes
(Source: Biochemical Journal)
Source: Biochemical Journal - December 14, 2017 Category: Biochemistry Authors: Gibb, A. A., Lorkiewicz, P. K., Zheng, Y.-T., Zhang, X., Bhatnagar, A., Jones, S. P., Hill, B. G. Tags: Corrections Source Type: research

Correction: The drug ornidazole inhibits photosynthesis in a different mechanism described for protozoa and anaerobic bacteria
(Source: Biochemical Journal)
Source: Biochemical Journal - December 14, 2017 Category: Biochemistry Authors: Marcus, Y., Tal, N., Ronen, M., Carmieli, R., Gurevitz, M. Tags: Corrections Source Type: research

Spectroscopic and calorimetric characterization of spermine oxidase and its association forms
Spermine oxidase (SMOX) is a flavin-containing enzyme that oxidizes spermine to produce spermidine, 3-aminopropanaldehyde, and hydrogen peroxide. SMOX has been shown to play key roles in inflammation and carcinogenesis; indeed, it is differentially expressed in several human cancer types. Our previous investigation has revealed that SMOX purified after heterologous expression in Escherichia coli actually consists of monomers, covalent homodimers, and other higher-order forms. All association forms oxidize spermine and, after treatment with dithiothreitol, revert to SMOX monomer. Here, we report a detailed investigation on ...
Source: Biochemical Journal - December 14, 2017 Category: Biochemistry Authors: Leonetti, A., Cervoni, L., Polticelli, F., Kanamori, Y., Yurtsever, Z. N., Agostinelli, E., Mariottini, P., Stano, P., Cervelli, M. Tags: Research Articles Source Type: research

Therapeutic targeting of non-coding RNAs in cancer
The majority of the human genome encodes RNAs that do not code for proteins. These non-coding RNAs (ncRNAs) affect normal expression of the genes, including oncogenes and tumour suppressive genes, which make them a new class of targets for drug development in cancer. Although microRNAs (miRNAs) are the most studied regulatory ncRNAs to date, and miRNA-targeted therapeutics have already reached clinical development, including the mimics of the tumour suppressive miRNAs miR-34 and miR-16, which reached phase I clinical trials for the treatment of liver cancer and mesothelioma, the importance of long non-coding RNAs (lncRNAs)...
Source: Biochemical Journal - December 14, 2017 Category: Biochemistry Authors: Slaby, O., Laga, R., Sedlacek, O. Tags: Review Articles Source Type: research

Microvesicles derived from human Wharton's Jelly mesenchymal stem cells ameliorate ischemia-reperfusion-induced renal fibrosis by releasing from G2/M cell cycle arrest
In conclusion, MVs derived from hWJMSCs ameliorate IR-induced renal fibrosis by inducing G2/M cell cycle arrest via Erk1/2 signaling. (Source: Biochemical Journal)
Source: Biochemical Journal - December 14, 2017 Category: Biochemistry Authors: Chen, W., Yan, Y., Song, C., Ding, Y., Du, T. Tags: Research Articles Source Type: research

First biochemical and crystallographic characterization of a fast-performing ferritin from a marine invertebrate
Ferritin, a multimeric cage-like enzyme, is integral to iron metabolism across all phyla through the sequestration and storage of iron through efficient ferroxidase activity. While ferritin sequences from ~900 species have been identified, crystal structures from only 50 species have been reported, the majority from bacterial origin. We recently isolated a secreted ferritin from the marine invertebrate Chaetopterus sp. (parchment tube worm), which resides in muddy coastal seafloors. Here, we present the first ferritin from a marine invertebrate to be crystallized and its biochemical characterization. The initial ferroxidas...
Source: Biochemical Journal - December 11, 2017 Category: Biochemistry Authors: De Meulenaere, E., Bailey, J. B., Tezcan, F. A., Deheyn, D. D. Tags: Research Articles Source Type: research

Engineering oxidative stability in human hemoglobin based on the Hb providence ({beta}K82D) mutation and genetic cross-linking
Previous work suggested that hemoglobin (Hb) tetramer formation slows autoxidation and hemin loss and that the naturally occurring mutant, Hb Providence (HbProv; βK82D), is much more resistant to degradation by H2O2. We have examined systematically the effects of genetic cross-linking of Hb tetramers with and without the HbProv mutation on autoxidation, hemin loss, and reactions with H2O2, using native HbA and various wild-type recombinant Hbs as controls. Genetically cross-linked Hb Presbyterian (βN108K) was also examined as an example of a low oxygen affinity tetramer. Our conclusions are: (a) at low concentrat...
Source: Biochemical Journal - December 11, 2017 Category: Biochemistry Authors: Strader, M. B., Bangle, R., Parker Siburt, C. J., Varnado, C. L., Soman, J., Benitez Cardenas, A. S., Samuel, P. P., Singleton, E. W., Crumbliss, A. L., Olson, J. S., Alayash, A. I. Tags: Research Articles Source Type: research

Nutlin-3 plus tanshinone IIA exhibits synergetic anti-leukemia effect with imatinib by reactivating p53 and inhibiting the AKT/mTOR pathway in Ph+ ALL
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is triggered by BCR/ABL kinase. Recent efforts focused on the development of more potent tyrosine kinase inhibitors (TKIs) that also inhibit mutant tyrosine kinases such as nilotinib and dasatinib. Although major advances in the treatment of this aggressive disease with potent inhibitors of the BCR/ABL kinases, patients in remission frequently relapse due to drug resistance possibly mediated, at least in part, by compensatory activation of growth-signaling pathways and protective feedback signaling of leukemia cells in response to TKI treatment. Contin...
Source: Biochemical Journal - December 6, 2017 Category: Biochemistry Authors: Guo, Y., Li, Y., Xiang, B., Huang, X.-O., Ma, H.-B., Wang, F.-F., Gong, Y.-P. Tags: Research Articles Source Type: research

Omics of bifidobacteria: research and insights into their health-promoting activities
Members of the genus Bifidobacterium include gut commensals that are particularly abundant among the microbial communities residing in the gut of healthy breast-fed infants, where their presence has been linked to many beneficial host effects. Next-generation DNA sequencing and comparative and functional genome methodologies have been shown to be particularly useful in exploring the diversity of this genus. These combined approaches have allowed the identification of genetic features related to bifidobacterial establishment in the gut, involving host–microbe as well as microbe–microbe interactions. Among these,...
Source: Biochemical Journal - December 6, 2017 Category: Biochemistry Authors: Bottacini, F., van Sinderen, D., Ventura, M. Tags: Review Articles Source Type: research

Dual phosphorylation in response regulator protein PrrA is crucial for intracellular survival of mycobacteria consequent upon transcriptional activation
The remarkable ability of Mycobacterium tuberculosis (Mtb) to survive inside human macrophages is attributed to the presence of a complex sensory and regulatory network. PrrA is a DNA-binding regulatory protein, belonging to an essential two-component system (TCS), PrrA/B, which is required for early phase intracellular replication of Mtb. Despite its importance, the mechanism of PrrA/B-mediated signaling is not well understood. In the present study, we demonstrate that the binding of PrrA on the promoter DNA and its consequent activation is cumulatively controlled via dual phosphorylation of the protein. We have further c...
Source: Biochemical Journal - December 6, 2017 Category: Biochemistry Authors: Mishra, A. K., Yabaji, S. M., Dubey, R. K., Dhamija, E., Srivastava, K. K. Tags: Research Articles Source Type: research

Regulation of insulin-like growth factor receptors by Ubiquilin1
Insulin-like growth factor-1 receptor (IGF1R) is a receptor tyrosine kinase that mediates growth, proliferation and survival. Dysregulation of IGF pathway contributes to the initiation, progression and metastasis of cancer and is also involved in diseases of glucose metabolism, such as diabetes. We have identified Ubiquilin1 (UBQLN1) as a novel interaction partner of IGF1R, IGF2R and insulin receptor (INSR). UBQLN family of proteins have been studied primarily in the context of protein quality control and in the field of neurodegenerative disorders. Our laboratory discovered a link between UBQLN1 function and tumorigenesis...
Source: Biochemical Journal - December 6, 2017 Category: Biochemistry Authors: Kurlawala, Z., Dunaway, R., Shah, P. P., Gosney, J. A., Siskind, L. J., Ceresa, B. P., Beverly, L. J. Tags: Research Articles Source Type: research

Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-{alpha}
The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP–NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein...
Source: Biochemical Journal - December 6, 2017 Category: Biochemistry Authors: Bernardes, N. E., Takeda, A. A. S., Dreyer, T. R., Cupertino, F. B., Virgilio, S., Pante, N., Bertolini, M. C., Fontes, M. R. M. Tags: Research Articles Source Type: research

GPI-anchored proteins are confined in subdiffraction clusters at the apical surface of polarized epithelial cells
Spatio-temporal compartmentalization of membrane proteins is critical for the regulation of diverse vital functions in eukaryotic cells. It was previously shown that, at the apical surface of polarized MDCK cells, glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are organized in small cholesterol-independent clusters of single GPI-AP species (homoclusters), which are required for the formation of larger cholesterol-dependent clusters formed by multiple GPI-AP species (heteroclusters). This clustered organization is crucial for the biological activities of GPI-APs; hence, understanding the spatio-temporal prop...
Source: Biochemical Journal - December 1, 2017 Category: Biochemistry Authors: Paladino, S., Lebreton, S., Lelek, M., Riccio, P., De Nicola, S., Zimmer, C., Zurzolo, C. Tags: Research Articles Source Type: research

GPR4 knockout improves renal ischemia-reperfusion injury and inhibits apoptosis via suppressing the expression of CHOP
The aim of the present study was to investigate the effects and molecular mechanisms of GPR4 (G-protein-coupled receptor 4) in cell apoptosis and renal ischemia–reperfusion (IR) injury in vivo and in vitro. GPR4–/– mice and wild-type (WT) mice underwent renal IR or sham procedures. For hypoxia/reoxygenation (HR), human umbilical vein endothelial cells (HUVECs) were subjected to 4 h of hypoxia, followed by 6 h of reoxygenation. Renal histological changes were observed by periodic acid-Schiff staining and myeloperoxidase activity. Apoptosis was detected by TUNEL staining. GPR4, C/EBP-homologous pr...
Source: Biochemical Journal - December 1, 2017 Category: Biochemistry Authors: Dong, B., Zhang, X., Fan, Y., Cao, S., Zhang, X. Tags: Research Articles Source Type: research

Interplay between {sigma} region 3.2 and secondary channel factors during promoter escape by bacterial RNA polymerase
In bacterial RNA polymerase (RNAP), conserved region 3.2 of the subunit was proposed to contribute to promoter escape by interacting with the 5'-end of nascent RNA, thus facilitating dissociation. RNAP activity during transcription initiation can also be modulated by protein factors that bind within the secondary channel and reach the enzyme active site. To monitor the kinetics of promoter escape in real time, we used a molecular beacon assay with fluorescently labeled 70 subunit of Escherichia coli RNAP. We show that substitutions and deletions in region 3.2 decrease the rate of promoter escape and lead to accumulation of...
Source: Biochemical Journal - December 1, 2017 Category: Biochemistry Authors: Petushkov, I., Esyunina, D., Mekler, V., Severinov, K., Pupov, D., Kulbachinskiy, A. Tags: Research Articles Source Type: research

Direct visualization of interaction between calmodulin and connexin45
Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of -family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer....
Source: Biochemical Journal - November 27, 2017 Category: Biochemistry Authors: Zou, J., Salarian, M., Chen, Y., Zhuo, Y., Brown, N. E., Hepler, J. R., Yang, J. J. Tags: Research Articles Source Type: research

Mitochondrial DNA density homeostasis accounts for a threshold effect in a cybrid model of a human mitochondrial disease
Mitochondrial dysfunction is involved in a wide array of devastating diseases, but the heterogeneity and complexity of the symptoms of these diseases challenges theoretical understanding of their causation. With the explosion of omics data, we have the unprecedented opportunity to gain deep understanding of the biochemical mechanisms of mitochondrial dysfunction. This goal raises the outstanding need to make these complex datasets interpretable. Quantitative modelling allows us to translate such datasets into intuition and suggest rational biomedical treatments. Taking an interdisciplinary approach, we use a recently publi...
Source: Biochemical Journal - November 24, 2017 Category: Biochemistry Authors: Aryaman, J., Johnston, I. G., Jones, N. S. Tags: Research Articles Source Type: research

Synthesis and degradation of cAMP in Giardia lamblia: possible role and characterization of a nucleotidyl cyclase with a single cyclase homology domain
Despite its importance in the regulation of growth and differentiation processes of a variety of organisms, the mechanism of synthesis and degradation of cAMP (cyclic AMP) has not yet been described in Giardia lamblia. In this work, we measured significant quantities of cAMP in trophozoites of G. lamblia incubated in vitro and later detected how it increases during the first hours of encystation, and how it then returns to basal levels at 24 h. Through an analysis of the genome of G. lamblia, we found sequences of three putative enzymes — one phosphodiesterase (gPDE) and two nucleotidyl cyclases (gNC1 and gNC2) ...
Source: Biochemical Journal - November 21, 2017 Category: Biochemistry Authors: Saraullo, V., Di Siervi, N., Jerez, B., Davio, C., Zurita, A. Tags: Research Articles Source Type: research

TSPO mutations in rats and a human polymorphism impair the rate of steroid synthesis
In this report, we show that TSPO deletion mutations in rat and its corresponding rs6971 polymorphism in humans alter adrenocorticotropic hormone-induced plasma corticosteroid concentrations. Rat tissues examined show increased cholesteryl ester accumulation, and neurosteroid formation was undetectable in homozygous rats. These results also support a role for TSPO ligands in diseases with steroid-dependent stress and anxiety elements. (Source: Biochemical Journal)
Source: Biochemical Journal - November 21, 2017 Category: Biochemistry Authors: Owen, D. R., Fan, J., Campioli, E., Venugopal, S., Midzak, A., Daly, E., Harlay, A., Issop, L., Libri, V., Kalogiannopoulou, D., Oliver, E., Gallego-Colon, E., Colasanti, A., Huson, L., Rabiner, E. A., Suppiah, P., Essagian, C., Matthews, P. M., Papadopou Tags: Research Articles Source Type: research