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 naïve 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 n...
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
