Hotspots of age-related protein degradation: the importance of neighboring residues for the formation of non-disulfide crosslinks derived from cysteine
Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemized, truncated, and covalently cross-linked. One reaction responsible for age-related protein cross-linking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence for this was found in many lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent cross-links than others. Foremost among them was Cys5 in βA4 crystallin....
Source: Biochemical Journal - July 11, 2017 Category: Biochemistry Authors: Friedrich, M. G., Wang, Z., Oakley, A. J., Schey, K. L., Truscott, R. J. W. Tags: Research Articles Source Type: research

Transcription regulation mechanism of the syntaxin 1A gene via protein kinase A
Syntaxin 1A (Stx1a) is primarily involved in the docking of synaptic vesicles at active zones in neurons. Its gene is a TATA-less gene, with several transcription initiation sites, which is activated by the binding of Sp1 and acetylated histone H3 (H3) in the core promoter region (CPR) through the derepression of class I histone deacetylase (HDAC). In the present study, to clarify the factor characterizing Stx1a gene expression via the protein kinase A (PKA) pathway inducing the Stx1a mRNA, we investigated whether the epigenetic process is involved in the Stx1a gene transcription induced by PKA signaling. We found that the...
Source: Biochemical Journal - July 11, 2017 Category: Biochemistry Authors: Nakayama, T., Akagawa, K. Tags: Research Articles Source Type: research

HIV-1 Tat potently stabilises Mdm2 and enhances viral replication
This study highlights the importance of post-translational modifications of host cellular factors in HIV-1 replication and pathogenesis. (Source: Biochemical Journal)
Source: Biochemical Journal - July 11, 2017 Category: Biochemistry Authors: Raja, R., Ronsard, L., Lata, S., Trivedi, S., Banerjea, A. C. Tags: Research Articles Source Type: research

Molecular basis of function and the unusual antioxidant activity of a cyanobacterial cysteine desulfurase
Cysteine desulfurases, which supply sulfur for iron–sulfur cluster biogenesis, are broadly distributed in all phyla including cyanobacteria, the progenitors of plant chloroplasts. The SUF (sulfur utilization factor) system is responsible for Fe–S cluster biosynthesis under stress. The suf operon from cyanobacterium Anabaena PCC 7120 showed the presence of a cysteine desulfurase, sufS (alr2495), but not the accessory sulfur-accepting protein (SufE). However, an open reading frame (alr3513) encoding a SufE-like protein (termed AsaE, Anabaena sulfur acceptor E) was found at a location distinct from the suf operon....
Source: Biochemical Journal - July 6, 2017 Category: Biochemistry Authors: Banerjee, M., Chakravarty, D., Ballal, A. Tags: Research Articles Source Type: research

Biochemical and functional characterization of the human tissue kallikrein 9
We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+. KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified c...
Source: Biochemical Journal - July 6, 2017 Category: Biochemistry Authors: Filippou, P. S., Farkona, S., Brinc, D., Yu, Y., Prassas, I., Diamandis, E. P. Tags: Research Articles Source Type: research

Characterization of the oligomeric states of the CK2 {alpha}2{beta}2 holoenzyme in solution
The regulatory mechanism of protein kinase CK2 has still to be fully clarified. The prevailing hypothesis is that CK2 is controlled by a self-polymerisation mechanism leading to inactive supramolecular assemblies that, when needed, can be disassembled into the α2β2 monomer, the active form of the holoenzyme. In vitro, monomeric α2β2 seems present only at high ionic strengths, typically 0.35–0.50 M NaCl, while at lower salt concentrations oligomers are formed. In the present study, size-exclusion chromatography (SEC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS) and mu...
Source: Biochemical Journal - July 6, 2017 Category: Biochemistry Authors: Lolli, G., Naressi, D., Sarno, S., Battistutta, R. Tags: Research Articles Source Type: research

Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity
Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has ...
Source: Biochemical Journal - July 6, 2017 Category: Biochemistry Authors: Lorenz, R., Moon, E.-W., Kim, J. J., Schmidt, S. H., Sankaran, B., Pavlidis, I. V., Kim, C., Herberg, F. W. Tags: Research Articles Source Type: research

A self-consistent structural perturbation approach for determining the magnitude and extent of allosteric coupling in proteins
Elucidating the extent of energetic coupling between residues in single-domain proteins, which is a fundamental determinant of allostery, information transfer and folding cooperativity, has remained a grand challenge. While several sequence- and structure-based approaches have been proposed, a self-consistent description that is simultaneously compatible with unfolding thermodynamics is lacking. We recently developed a simple structural perturbation protocol that captures the changes in thermodynamic stabilities induced by point mutations within the protein interior. Here, we show that a fundamental residue-specific compon...
Source: Biochemical Journal - July 6, 2017 Category: Biochemistry Authors: Rajasekaran, N., Naganathan, A. N. Tags: Research Articles Source Type: research

Functional characterization of the copper transcription factor AfMac1 from Aspergillus fumigatus
In this report, we investigated the copper transcription factor AfMac1 (Aspergillus fumigatus Mac1 homolog) and identified its regulatory mechanism in A. fumigatus. AfMac1 has domains homologous to the DNA-binding and copper-binding domains of Mac1 from Saccharomyces cerevisiae, and AfMac1 efficiently complemented Mac1 in S. cerevisiae. Expression of Afmac1 resulted in CTR1 up-regulation, and mutation of the DNA-binding domain of Afmac1 failed to activate CTR1 expression in S. cerevisiae. The Afmac1 deletion strain of A. fumigatus failed to grow in copper-limited media, and its growth was restored by introducing ctrC. We f...
Source: Biochemical Journal - July 3, 2017 Category: Biochemistry Authors: Park, Y.-S., Kim, T.-H., Yun, C.-W. Tags: Research Articles Source Type: research

Phorbol-12-myristate-13-acetate-mediated stabilization of leukemia inhibitory factor (lif) mRNA: involvement of Nucleolin and PCBP1
Leukemia inhibitory factor (LIF) is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of LIF in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region of human lif mRNA revealed several conserved sequences with conventional cis-acting elements. A 216 nucleotide containing proximal cis-element with two AUUUA pentamers and four ...
Source: Biochemical Journal - July 3, 2017 Category: Biochemistry Authors: Chakraborty, A., Mukherjee, S., Saha, S., De, S., Sengupta (Bandyopadhyay), S. Tags: Research Articles Source Type: research

Role of glycosylation in nucleating protein folding and stability
Glycosylation constitutes one of the most common, ubiquitous and complex forms of post-translational modification. It commences with the synthesis of the protein and plays a significant role in deciding its folded state, oligomerization and thus its function. Recent studies have demonstrated that N-linked glycans help proteins to fold as the stability and folding kinetics are altered with the removal of the glycans from them. Several studies have shown that it alters not only the thermodynamic stability but also the structural features of the folded proteins modulating their interactions and functions. Their inhibition and...
Source: Biochemical Journal - July 3, 2017 Category: Biochemistry Authors: Jayaprakash, N. G., Surolia, A. Tags: Review Articles Source Type: research

Heme A synthesis and CcO activity are essential for Trypanosoma cruzi infectivity and replication
Trypanosoma cruzi, the causative agent of Chagas disease, presents a complex life cycle and adapts its metabolism to nutrients’ availability. Although T. cruzi is an aerobic organism, it does not produce heme. This cofactor is acquired from the host and is distributed and inserted into different heme-proteins such as respiratory complexes in the parasite's mitochondrion. It has been proposed that T. cruzi's energy metabolism relies on a branched respiratory chain with a cytochrome c oxidase-type aa3 (CcO) as the main terminal oxidase. Heme A, the cofactor for all eukaryotic CcO, is synthesized via two sequential enzy...
Source: Biochemical Journal - June 27, 2017 Category: Biochemistry Authors: Merli, M. L., Cirulli, B. A., Menendez-Bravo, S. M., Cricco, J. A. Tags: Research Articles Source Type: research

G-protein-coupled receptor kinase-2 is a critical regulator of TNF{alpha} signaling in colon epithelial cells
G-protein-coupled receptor kinase-2 (GRK2) belongs to the GRK family of serine/threonine protein kinases critical in the regulation of G-protein-coupled receptors. Apart from this canonical role, GRK2 is also involved in several signaling pathways via distinct intracellular interactomes. In the present study, we examined the role of GRK2 in TNFα signaling in colon epithelial cell–biological processes including wound healing, proliferation, apoptosis, and gene expression. Knockdown of GRK2 in the SW480 human colonic cells significantly enhanced TNFα-induced epithelial cell wound healing without any effect ...
Source: Biochemical Journal - June 27, 2017 Category: Biochemistry Authors: Steury, M. D., Lucas, P. C., McCabe, L. R., Parameswaran, N. Tags: Research Articles Source Type: research

Evolution of biosynthetic diversity
Since the emergence of the last common ancestor from which all extant life evolved, the metabolite repertoire of cells has increased and diversified. Not only has the metabolite cosmos expanded, but the ways in which the same metabolites are made have diversified. Enzymes catalyzing the same reaction have evolved independently from different protein folds; the same protein fold can produce enzymes recognizing different substrates, and enzymes performing different chemistries. Genes encoding useful enzymes can be transferred between organisms and even between the major domains of life. Organisms that live in metabolite-rich...
Source: Biochemical Journal - June 27, 2017 Category: Biochemistry Authors: Michael, A. J. Tags: Review Articles Source Type: research

Combining properties of different classes of PI3K{alpha} inhibitors to understand the molecular features that confer selectivity
In conclusion, we have used a structure-based design approach to combine features from two different compound classes to create new PI3Kα-selective inhibitors. This provides new insights into the contribution of different chemical units and interactions with different parts of the active site to the selectivity and potency of PI3Kα inhibitors. (Source: Biochemical Journal)
Source: Biochemical Journal - June 26, 2017 Category: Biochemistry Authors: Gong, G. Q., Kendall, J. D., Dickson, J. M. J., Rewcastle, G. W., Buchanan, C. M., Denny, W. A., Shepherd, P. R., Flanagan, J. U. Tags: Research Articles Source Type: research

Identification of a conformational heparin-recognition motif on the peptide hormone secretin: key role for cell surface binding
Secretin is a peptide hormone that exerts pleiotropic physiological functions by specifically binding to its cognate membrane-bound receptor. The membrane catalysis model of peptide–receptor interactions states that soluble peptidic ligands initially interact with the plasma membrane. This interaction increases the local concentration and structures the peptide, enhancing the rate of receptor binding. However, this model does not consider the dense network of glycosaminoglycans (GAGs) at the surface of eukaryotic cells. These sulfated polysaccharide chains are known to sequester numerous proteic signaling molecules. ...
Source: Biochemical Journal - June 26, 2017 Category: Biochemistry Authors: Quittot, N., Nguyen, P. T., Neree, A. T., Lussier, M. P., Bourgault, S. Tags: Research Articles Source Type: research

Interleukin-1 and TRAF6-dependent activation of TAK1 in the absence of TAB2 and TAB3
Interleukin-1 (IL-1) signaling induces the formation of Lys63-linked ubiquitin (K63-Ub) chains, which are thought to activate the ‘master’ protein kinase TGFβ-activated kinase 1 (TAK1) by interacting with its TAK1-binding 2 (TAB2) and TAB3 subunits. Here, we report that IL-1β can also activate the TAB1–TAK1 heterodimer present in TAB2/TAB3 double knockout (DKO) IL-1 receptor-expressing cells. The IL-1β-dependent activation of the TAB1–TAK1 heterodimer in TAB2/3 DKO cells is required for the expression and E3 ligase activity of tumor necrosis factor receptor-associated factor 6 (TRAF6)...
Source: Biochemical Journal - June 26, 2017 Category: Biochemistry Authors: Zhang, J., Macartney, T., Peggie, M., Cohen, P. Tags: Research Articles Source Type: research

Comparative functional analysis of ribonuclease 1 homologs: molecular insights into evolving vertebrate physiology
Pancreatic-type ribonucleases (ptRNases) comprise a class of highly conserved secretory endoribonucleases in vertebrates. The prototype of this enzyme family is ribonuclease 1 (RNase 1). Understanding the physiological roles of RNase 1 is becoming increasingly important, as engineered forms of the enzyme progress through clinical trials as chemotherapeutic agents for cancer. Here, we present an in-depth biochemical characterization of RNase 1 homologs from a broad range of mammals (human, bat, squirrel, horse, cat, mouse, and cow) and nonmammalian species (chicken, lizard, and frog). We discover that the human homolog of R...
Source: Biochemical Journal - June 21, 2017 Category: Biochemistry Authors: Lomax, J. E., Eller, C. H., Raines, R. T. Tags: Research Articles Source Type: research

Non-linearity of the collagen triple helix in solution and implications for collagen function
Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly)n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG)10unblocked, was com...
Source: Biochemical Journal - June 16, 2017 Category: Biochemistry Authors: Walker, K. T., Nan, R., Wright, D. W., Gor, J., Bishop, A. C., Makhatadze, G. I., Brodsky, B., Perkins, S. J. Tags: Research Articles Source Type: research

Systematic identification of functional residues of Artemisia annua amorpha-4,11-diene synthase
Terpene synthases (TPSs) are responsible for the extremely diversified and complex structure of terpenoids. Amorpha-4,11-diene synthase (ADS) has a high (90%) fidelity in generating the sesquiterpene precursor for the biosynthesis of artemisinin, an antimalarial drug, however, little is known about how active site residues of ADS are involved in carbocation rearrangement and cyclization reactions. Here, we identify seven residues that are key to most of the catalytic steps in ADS. By structural modeling and amino acid sequence alignments of ADS with two functionally relevant sesquiterpene synthases from Artemisia annua, we...
Source: Biochemical Journal - June 16, 2017 Category: Biochemistry Authors: Fang, X., Li, J.-X., Huang, J.-Q., Xiao, Y.-L., Zhang, P., Chen, X.-Y. Tags: Research Articles Source Type: research

Cellular and disease functions of the Prader-Willi Syndrome gene MAGEL2
Melanoma antigen L2 (MAGEL2 or MAGE-L2) is a member of the MAGE family of ubiquitin ligase regulators. It is maternally imprinted and often paternally deleted or mutated in the related neurodevelopmental syndromes, Prader–Willi Syndrome (PWS) and Schaaf–Yang Syndrome (SHFYNG). MAGEL2 is highly expressed in the hypothalamus and plays an important role in a fundamental cellular process that recycles membrane proteins from endosomes through the retromer sorting pathway. MAGEL2 is part of a multi-subunit protein complex consisting of MAGEL2, the TRIM27 E3 ubiquitin ligase, and the USP7 deubiquitinating enzyme. The ...
Source: Biochemical Journal - June 16, 2017 Category: Biochemistry Authors: Tacer, K. F., Potts, P. R. Tags: Review Articles Source Type: research

Ligand-induced activation of human TRPM2 requires the terminal ribose of ADPR and involves Arg1433 and Tyr1349
TRPM2 (transient receptor potential channel, subfamily melastatin, member 2) is a Ca2+-permeable non-selective cation channel activated by the binding of adenosine 5'-diphosphoribose (ADPR) to its cytoplasmic NUDT9H domain (NUDT9 homology domain). Activation of TRPM2 by ADPR downstream of oxidative stress has been implicated in the pathogenesis of many human diseases, rendering TRPM2 an attractive novel target for pharmacological intervention. However, the structural basis underlying this activation is largely unknown. Since ADP (adenosine 5'-diphosphate) alone did not activate or antagonize the channel, we used a chemical...
Source: Biochemical Journal - June 16, 2017 Category: Biochemistry Authors: Fliegert, R., Watt, J. M., Schöbel, A., Rozewitz, M. D., Moreau, C., Kirchberger, T., Thomas, M. P., Sick, W., Araujo, A. C., Harneit, A., Potter, B. V. L., Guse, A. H. Tags: Research Articles Source Type: research

The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome
Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethyl...
Source: Biochemical Journal - June 13, 2017 Category: Biochemistry Authors: Rozanska, A., Richter-Dennerlein, R., Rorbach, J., Gao, F., Lewis, R. J., Chrzanowska-Lightowlers, Z. M., Lightowlers, R. N. Tags: Research Articles Source Type: research

P2X7 receptor cross-talk regulates ATP-induced pannexin 1 internalization
In the nervous system, extracellular ATP levels transiently increase in physiological and pathophysiological circumstances, effecting key signalling pathways in plasticity and inflammation through purinergic receptors. Pannexin 1 (Panx1) forms ion- and metabolite-permeable channels that mediate ATP release and are particularly enriched in the nervous system. Our recent study demonstrated that elevation of extracellular ATP triggers Panx1 internalization in a concentration- and time-dependent manner. Notably, this effect was sensitive to inhibition of ionotropic P2X7 purinergic receptors (P2X7Rs). Here, we report our novel ...
Source: Biochemical Journal - June 13, 2017 Category: Biochemistry Authors: Boyce, A. K. J., Swayne, L. A. Tags: Research Articles Source Type: research

Light harvesting in phototrophic bacteria: structure and function
This review serves as an introduction to the variety of light-harvesting (LH) structures present in phototrophic prokaryotes. It provides an overview of the LH complexes of purple bacteria, green sulfur bacteria (GSB), acidobacteria, filamentous anoxygenic phototrophs (FAP), and cyanobacteria. Bacteria have adapted their LH systems for efficient operation under a multitude of different habitats and light qualities, performing both oxygenic (oxygen-evolving) and anoxygenic (non-oxygen-evolving) photosynthesis. For each LH system, emphasis is placed on the overall architecture of the pigment–protein complex, as well as...
Source: Biochemical Journal - June 13, 2017 Category: Biochemistry Authors: Saer, R. G., Blankenship, R. E. Tags: Review Articles Source Type: research

1-N-histidine phosphorylation of ChlD by the AAA+ ChlI2 stimulates magnesium chelatase activity in chlorophyll synthesis
Magnesium chelatase (Mg-chelatase) inserts magnesium into protoporphyrin during the biosynthesis of chlorophyll and bacteriochlorophyll. Enzyme activity is reconstituted by forming two separate preactivated complexes consisting of a GUN4/ChlH/protoporphyrin IX substrate complex and a ChlI/ChlD enzyme ‘motor’ complex. Formation of the ChlI/ChlD complex in both Chlamydomonas reinhardtii and Oryza sativa is accompanied by phosphorylation of ChlD by ChlI, but the orthologous protein complex from Rhodobacter capsulatus, BchI/BchD, gives no detectable phosphorylation of BchD. Phosphorylation produces a 1-N-phospho-hi...
Source: Biochemical Journal - June 9, 2017 Category: Biochemistry Authors: Sawicki, A., Zhou, S., Kwiatkowski, K., Luo, M., Willows, R. D. Tags: Research Articles Source Type: research

The Slo(w) path to identifying the mitochondrial channels responsible for ischemic protection
Mitochondria play an important role in tissue ischemia and reperfusion (IR) injury, with energetic failure and the opening of the mitochondrial permeability transition pore being the major causes of IR-induced cell death. Thus, mitochondria are an appropriate focus for strategies to protect against IR injury. Two widely studied paradigms of IR protection, particularly in the field of cardiac IR, are ischemic preconditioning (IPC) and volatile anesthetic preconditioning (APC). While the molecular mechanisms recruited by these protective paradigms are not fully elucidated, a commonality is the involvement of mitochondrial K+...
Source: Biochemical Journal - June 9, 2017 Category: Biochemistry Authors: Smith, C. O., Nehrke, K., Brookes, P. S. Tags: Review Articles Source Type: research

Encephalomyocarditis virus 3C protease attenuates type I interferon production through disrupting the TANK-TBK1-IKK{varepsilon}-IRF3 complex
TRAF family member-associated NF-B activator (TANK) is a scaffold protein that assembles into the interferon (IFN) regulator factor 3 (IRF3)-phosphorylating TANK-binding kinase 1 (TBK1)–(IB) kinase (IKK) complex, where it is involved in regulating phosphorylation of the IRF3 and IFN production. However, the functions of TANK in encephalomyocarditis virus (EMCV) infection-induced type I IFN production are not fully understood. Here, we demonstrated that, instead of stimulating type I IFN production, the EMCV-HB10 strain infection potently inhibited Sendai virus- and polyI:C-induced IRF3 phosphorylation and type I IFN ...
Source: Biochemical Journal - June 9, 2017 Category: Biochemistry Authors: Huang, L., Xiong, T., Yu, H., Zhang, Q., Zhang, K., Li, C., Hu, L., Zhang, Y., Zhang, L., Liu, Q., Wang, S., He, X., Bu, Z., Cai, X., Cui, S., Li, J., Weng, C. Tags: Research Articles Source Type: research

{alpha}-Synuclein may cross-bridge v-SNARE and acidic phospholipids to facilitate SNARE-dependent vesicle docking
Misfolded α-synuclein (A-syn) is widely recognized as the primal cause of neurodegenerative diseases including Parkinson's disease and dementia with Lewy bodies. The normal cellular function of A-syn has, however, been elusive. There is evidence that A-syn plays multiple roles in the exocytotic pathway in the neuron, but the underlying molecular mechanisms are unclear. A-syn has been known to interact with negatively charged phospholipids and with vesicle SNARE protein VAMP2. Using single-vesicle docking/fusion assays, we find that A-syn promotes SNARE-dependent vesicles docking significantly at 2.5 µM. Wh...
Source: Biochemical Journal - June 6, 2017 Category: Biochemistry Authors: Lou, X., Kim, J., Hawk, B. J., Shin, Y.-K. Tags: Research Articles Source Type: research

The mechanism of activation of IRAK1 and IRAK4 by interleukin-1 and Toll-like receptor agonists
We have developed the first assays that measure the protein kinase activities of interleukin-1 receptor-associated kinase 1 (IRAK1) and IRAK4 reliably in human cell extracts, by employing Pellino1 as a substrate in conjunction with specific pharmacological inhibitors of IRAK1 and IRAK4. We exploited these assays to show that IRAK4 was constitutively active and that its intrinsic activity towards Pellino1 was not increased significantly by stimulation with interleukin-1 (IL-1) in IL-1R-expressing HEK293 cells, Pam3CSK4-stimulated human THP1 monocytes or primary human macrophages. Our results, in conjunction with those of ot...
Source: Biochemical Journal - June 6, 2017 Category: Biochemistry Authors: Vollmer, S., Strickson, S., Zhang, T., Gray, N., Lee, K. L., Rao, V. R., Cohen, P. Tags: Research Articles Source Type: research

Cytosine methylation by DNMT2 facilitates stability and survival of HIV-1 RNA in the host cell during infection
The enigmatic methyltransferase, DNMT2 (DNA methyltransferase 2), structurally resembles a DNA methyltransferase, but has been shown to be a tRNA methyltransferase targeting cytosine within a specific CpG in different tRNA molecules. We had previously shown that, during environmental stress conditions, DNMT2 is re-localized from the nucleus to the cytoplasmic stress granules (SGs) and is associated with RNA-processing proteins. In the present study, we show that DNMT2 binds and methylates various mRNA species in a sequence-independent manner and gets re-localized to SGs in a phosphorylation-dependent manner. Importantly, o...
Source: Biochemical Journal - June 6, 2017 Category: Biochemistry Authors: Dev, R. R., Ganji, R., Singh, S. P., Mahalingam, S., Banerjee, S., Khosla, S. Tags: Research Articles Source Type: research

Probing native metal ion association sites through quenching of fluorophores in the nucleotide-binding domains of the ABC transporter MsbA
ATP-binding cassette (ABC) transporters are ubiquitously present in prokaryotic and eukaryotic cells. Binding of ATP to the nucleotide-binding domains (NBDs) elicits major conformational changes of the transporters resulting in the transport of the substrate across the membrane. The availability of a crystal structure of the NBDs enabled us to elucidate the local structure and small-scale dynamics in the NBDs. Here, we labeled the ABC transporter MsbA, a homodimeric flippase from Escherichia coli, with a fluorescent probe, Alexa532, within the NBDs. ATP application elicited collisional quenching, whereas no quenching was o...
Source: Biochemical Journal - May 30, 2017 Category: Biochemistry Authors: Tatsumi, D., Nanatani, K., Koike, Y., Kamagata, K., Takahashi, S., Konno, A., Furuta, T., Sakurai, M., Uozumi, N. Tags: Research Articles Source Type: research

Global functions of extracellular, transmembrane and cytoplasmic domains of organic solute transporter {beta}-subunit
Transport of bile acids across the basolateral membrane of the intestinal enterocyte is carried out by the organic solute transporter (Ost) composed of a seven-transmembrane domain (TMD) subunit (Ostα) and an ancillary single TMD subunit (Ostβ). Although previous investigations have demonstrated the importance of the TMD of Ostβ for its activity, further studies were conducted to assess the contributions of other regions of the Ostβ subunit. Transport activity was retained when Ostβ was truncated to contain only the TMD with 15 additional residues on each side and co-expressed with Ostα, whe...
Source: Biochemical Journal - May 25, 2017 Category: Biochemistry Authors: Christian, W. V., Hinkle, P. M. Tags: Research Articles Source Type: research

Proteoglycans, ion channels and cell-matrix adhesion
Cell surface proteoglycans comprise a transmembrane or membrane-associated core protein to which one or more glycosaminoglycan chains are covalently attached. They are ubiquitous receptors on nearly all animal cell surfaces. In mammals, the cell surface proteoglycans include the six glypicans, CD44, NG2 (CSPG4), neuropilin-1 and four syndecans. A single syndecan is present in invertebrates such as nematodes and insects. Uniquely, syndecans are receptors for many classes of proteins that can bind to the heparan sulphate chains present on syndecan core proteins. These range from cytokines, chemokines, growth factors and morp...
Source: Biochemical Journal - May 25, 2017 Category: Biochemistry Authors: Mitsou, I., Multhaupt, H. A. B., Couchman, J. R. Tags: Review Articles Source Type: research

Amino acid homeostasis and signalling in mammalian cells and organisms
Cells have a constant turnover of proteins that recycle most amino acids over time. Net loss is mainly due to amino acid oxidation. Homeostasis is achieved through exchange of essential amino acids with non-essential amino acids and the transfer of amino groups from oxidised amino acids to amino acid biosynthesis. This homeostatic condition is maintained through an active mTORC1 complex. Under amino acid depletion, mTORC1 is inactivated. This increases the breakdown of cellular proteins through autophagy and reduces protein biosynthesis. The general control non-derepressable 2/ATF4 pathway may be activated in addition, res...
Source: Biochemical Journal - May 25, 2017 Category: Biochemistry Authors: Bröer, S., Bröer, A. Tags: Review Articles Source Type: research

Spatial intensity distribution analysis quantifies the extent and regulation of homodimerization of the secretin receptor
Previous studies have indicated that the G-protein-coupled secretin receptor is present as a homodimer, organized through symmetrical contacts in transmembrane domain IV, and that receptor dimerization is critical for high-potency signalling by secretin. However, whether all of the receptor exists in the dimeric form or if this is regulated is unclear. We used measures of quantal brightness of the secretin receptor tagged with monomeric enhanced green fluorescent protein (mEGFP) and spatial intensity distribution analysis to assess this. Calibration using cells expressing plasma membrane-anchored forms of mEGFP initially a...
Source: Biochemical Journal - May 24, 2017 Category: Biochemistry Authors: Ward, R. J., Pediani, J. D., Harikumar, K. G., Miller, L. J., Milligan, G. Tags: Research Articles Source Type: research

Dual histone reader ZMYND8 inhibits cancer cell invasion by positively regulating epithelial genes
Enhanced migratory potential and invasiveness of cancer cells contribute crucially to cancer progression. These phenotypes are achieved by precise alteration of invasion-associated genes through local epigenetic modifications which are recognized by a class of proteins termed a chromatin reader. ZMYND8 [zinc finger MYND (myeloid, Nervy and DEAF-1)-type containing 8], a key component of the transcription regulatory network, has recently been shown to be a novel reader of H3.1K36Me2/H4K16Ac marks. Through differential gene expression analysis upon silencing this chromatin reader, we identified a subset of genes involved in c...
Source: Biochemical Journal - May 19, 2017 Category: Biochemistry Authors: Basu, M., Sengupta, I., Khan, M. W., Srivastava, D. K., Chakrabarti, P., Roy, S., Das, C. Tags: Research Articles Source Type: research

CCM2 and PAK4 act downstream of atrial natriuretic peptide signaling to promote cell spreading
Atrial natriuretic peptide (ANP) is a cardiac hormone released by the atrium in response to stretching forces. Via its receptor, guanylyl cyclase-A (GC-A), ANP maintains cardiovascular homeostasis by exerting diuretic, natriuretic, and hypotensive effects mediated, in part, by endothelial cells. Both in vivo and in vitro, ANP enhances endothelial barrier function by reducing RhoA activity and reorganizing the actin cytoskeleton. We established mouse endothelial cells that stably express GC-A and used them to analyze the molecular mechanisms responsible for actin reorganization. Stimulation by ANP resulted in phosphorylatio...
Source: Biochemical Journal - May 19, 2017 Category: Biochemistry Authors: Miura, K., Nojiri, T., Akitake, Y., Ando, K., Fukuhara, S., Zenitani, M., Kimura, T., Hino, J., Miyazato, M., Hosoda, H., Kangawa, K. Tags: Research Articles Source Type: research

Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry
Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved m...
Source: Biochemical Journal - May 16, 2017 Category: Biochemistry Authors: Masson, G. R., Maslen, S. L., Williams, R. L. Tags: Research Articles Source Type: research

Molecular basis for the interaction between stress-inducible phosphoprotein 1 (STIP1) and S100A1
Stress-inducible phosphoprotein 1 (STIP1) is a cellular co-chaperone, which regulates heat-shock protein 70 (Hsp70) and Hsp90 activity during client protein folding. Members of the S100 family of dimeric calcium-binding proteins have been found to inhibit Hsp association with STIP1 through binding of STIP1 tetratricopeptide repeat (TPR) domains, possibly regulating the chaperone cycle. Here, we investigated the molecular basis of S100A1 binding to STIP1. We show that three S100A1 dimers associate with one molecule of STIP1 in a calcium-dependent manner. Isothermal titration calorimetry revealed that individual STIP1 TPR do...
Source: Biochemical Journal - May 16, 2017 Category: Biochemistry Authors: Maciejewski, A., Prado, V. F., Prado, M. A. M., Choy, W.-Y. Tags: Research Articles Source Type: research

1,N6-{alpha}-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase
1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein – an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A -> C and A -> T transversions and, less frequently, A -> G transitions...
Source: Biochemical Journal - May 16, 2017 Category: Biochemistry Authors: Dylewska, M., Kusmierek, J. T., Pilzys, T., Poznanski, J., Maciejewska, A. M. Tags: Research Articles Source Type: research

Introduction to the human gut microbiota
The human gastrointestinal (GI) tract harbours a complex and dynamic population of microorganisms, the gut microbiota, which exert a marked influence on the host during homeostasis and disease. Multiple factors contribute to the establishment of the human gut microbiota during infancy. Diet is considered as one of the main drivers in shaping the gut microbiota across the life time. Intestinal bacteria play a crucial role in maintaining immune and metabolic homeostasis and protecting against pathogens. Altered gut bacterial composition (dysbiosis) has been associated with the pathogenesis of many inflammatory diseases and i...
Source: Biochemical Journal - May 16, 2017 Category: Biochemistry Authors: Thursby, E., Juge, N. Tags: Review Articles Source Type: research

Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p
The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport...
Source: Biochemical Journal - May 16, 2017 Category: Biochemistry Authors: Zulkifli, M., Bachhawat, A. K. Tags: Research Articles Source Type: research

Hiding in plain sight: immune evasion by the staphylococcal protein SdrE
The human immune system is responsible for identification and destruction of invader cells, such as the bacterial pathogen Staphylococcus aureus. In response, S. aureus brings to the fight a large number of virulence factors, including several that allow it to evade the host immune response. The staphylococcal surface protein SdrE was recently reported to bind to complement Factor H, an important regulator of complement activation. Factor H attaches to the surface of host cells to inhibit complement activation and amplification, preventing the destruction of the host cell. SdrE binding to Factor H allows S. aureus to mimic...
Source: Biochemical Journal - May 10, 2017 Category: Biochemistry Authors: Herr, A. B., Thorman, A. W. Tags: Commentaries Source Type: research

Arabidopsis fructokinase-like protein associations are regulated by ATP
The Arabidopsis thaliana fructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated. Homology modeling using pfkB X-ray structures, sequenc...
Source: Biochemical Journal - May 10, 2017 Category: Biochemistry Authors: Riggs, J. W., Callis, J. Tags: Research Articles Source Type: research

Transcriptional and translational adaptation to aerobic nitrate anabolism in the denitrifier Paracoccus denitrificans
Transcriptional adaptation to nitrate-dependent anabolism by Paracoccus denitrificans PD1222 was studied. A total of 74 genes were induced in cells grown with nitrate as N-source compared with ammonium, including nasTSABGHC and ntrBC genes. The nasT and nasS genes were cotranscribed, although nasT was more strongly induced by nitrate than nasS. The nasABGHC genes constituted a transcriptional unit, which is preceded by a non-coding region containing hairpin structures involved in transcription termination. The nasTS and nasABGHC transcripts were detected at similar levels with nitrate or glutamate as N-source, but nasABGHC...
Source: Biochemical Journal - May 10, 2017 Category: Biochemistry Authors: Luque-Almagro, V. M., Manso, I., Sullivan, M. J., Rowley, G., Ferguson, S. J., Moreno-Vivian, C., Richardson, D. J., Gates, A. J., Roldan, M. D. Tags: Research Articles Source Type: research

RUNX transcription factors at the interface of stem cells and cancer
The RUNX1 transcription factor is a critical regulator of normal haematopoiesis and its functional disruption by point mutations, deletions or translocations is a major causative factor leading to leukaemia. In the majority of cases, genetic changes in RUNX1 are linked to loss of function classifying it broadly as a tumour suppressor. Despite this, several recent studies have reported the need for a certain level of active RUNX1 for the maintenance and propagation of acute myeloid leukaemia and acute lymphoblastic leukaemia cells, suggesting an oncosupportive role of RUNX1. Furthermore, in solid cancers, RUNX1 is overexpre...
Source: Biochemical Journal - May 10, 2017 Category: Biochemistry Authors: Deltcheva, E., Nimmo, R. Tags: Review Articles Source Type: research

Effect of different {gamma}-subunit isoforms on the regulation of AMPK
AMP-activated protein kinase (AMPK) plays a key role in integrating metabolic pathways in response to energy demand. AMPK activation results in a wide range of downstream responses, many of which are associated with improved metabolic outcome, making AMPK an attractive target for the treatment of metabolic diseases. AMPK is a heterotrimeric complex consisting of a catalytic subunit (α) and two regulatory subunits (β and ). The -subunit harbours the nucleotide-binding sites and plays an important role in AMPK regulation in response to cellular energy levels. In mammals, there are three isoforms of the -subunit an...
Source: Biochemical Journal - May 9, 2017 Category: Biochemistry Authors: Willows, R., Navaratnam, N., Lima, A., Read, J., Carling, D. Tags: Research Articles Source Type: research

Roles of Leu28 side chain intercalation in the interaction between Cren7 and DNA
Crenarchaeal chromatin protein Cren7 binds double-stranded DNA in the minor groove, introducing a sharp single-step DNA kink. The side chain of Leu28, a residue conserved among all Cren7 homologs, intercalates into the kinked DNA step. In the present study, we replaced Leu28 with a residue containing a hydrophobic side chain of different sizes (i.e. L28A, L28V, L28I, L28M and L28F). Both the stability of the Cren7–DNA complex and the ability of Cren7 to constrain DNA supercoils correlated well with the size of the intercalated side chain. Structural analysis shows that L28A induces a kink (~43°), nearly as sharp ...
Source: Biochemical Journal - May 9, 2017 Category: Biochemistry Authors: Zhang, Z., Zhao, M., Wang, L., Chen, Y., Dong, Y., Gong, Y., Huang, L. Tags: Research Articles Source Type: research

Effect of position-specific single-point mutations and biophysical characterization of amyloidogenic peptide fragments identified from lattice corneal dystrophy patients
Corneal stromal dystrophies are a group of genetic disorders that may be caused by mutations in the transforming growth factor β-induced (TGFBI) gene which results in the aggregation and deposition of mutant proteins in various layers of the cornea. The type of amino acid substitution dictates the age of onset, anatomical location of the deposits, morphological features of deposits (amyloid, amorphous powder or a mixture of both forms) and the severity of disease presentation. It has been suggested that abnormal turnover and aberrant proteolytic processing of the mutant proteins result in the accumulation of insoluble...
Source: Biochemical Journal - May 9, 2017 Category: Biochemistry Authors: Anandalakshmi, V., Murugan, E., Leng, E. G. T., Ting, L. W., Chaurasia, S. S., Yamazaki, T., Nagashima, T., George, B. L., Peh, G. S. L., Pervushin, K., Lakshminarayanan, R., Mehta, J. S. Tags: Research Articles Source Type: research