Identification of adducin-binding residues on the cytoplasmic domain of erythrocyte membrane protein, band 3
Two major complexes form structural bridges that connect the erythrocyte membrane to its underlying spectrin-based cytoskeleton. Although the band 3–ankyrin bridge may account for most of the membrane-to-cytoskeleton interactions, the linkage between the cytoplasmic domain of band 3 (cdb3) and adducin has also been shown to be critical to membrane integrity. In the present paper, we demonstrate that adducin, a major component of the spectrin–actin junctional complex, binds primarily to residues 246–264 of cdb3, and mutation of two exposed glutamic acid residues within this sequence completely abrogates bo...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Franco, T., Chu, H., Low, P. S. Tags: Research Articles Source Type: research

pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport
Multidrug resistance 1 (MDR1) is a member of the major facilitator superfamily that contributes to MDR of Candida albicans. This antiporter belongs to the drug/H+ antiporter 1 family, pairing the downhill gradient of protons to drug extrusion. Hence, drug efflux from cytosol to extracellular space and the parallel import of H+ towards cytosol are inextricably linked processes. For monitoring the drug/H+ antiporter activity of Mdr1p, we developed a new system, exploiting a GFP variant pHluorin, which changes its fluorescence properties with pH. This enabled us to measure the cytosolic pH correlated to drug efflux. Since pro...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Redhu, A. K., Khandelwal, N. K., Banerjee, A., Moreno, A., Falson, P., Prasad, R. Tags: Research Articles Source Type: research

Structure of purotoxin-2 from wolf spider: modular design and membrane-assisted mode of action in arachnid toxins
Traditionally, arachnid venoms are known to contain two particularly important groups of peptide toxins. One is disulfide-rich neurotoxins with a predominance of β-structure that specifically target protein receptors in neurons or muscle cells. The other is linear cationic cytotoxins that form amphiphilic α-helices and exhibit rather non-specific membrane-damaging activity. In the present paper, we describe the first 3D structure of a modular arachnid toxin, purotoxin-2 (PT2) from the wolf spider Alopecosa marikovskyi (Lycosidae), studied by NMR spectroscopy. PT2 is composed of an N-terminal inhibitor cystine kn...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Oparin, P. B., Nadezhdin, K. D., Berkut, A. A., Arseniev, A. S., Grishin, E. V., Vassilevski, A. A. Tags: Research Articles Source Type: research

Two independent evolutionary routes to Na+/H+ cotransport function in membrane pyrophosphatases
Membrane-bound pyrophosphatases (mPPases) hydrolyze pyrophosphate (PPi) to transport H+, Na+ or both and help organisms to cope with stress conditions, such as high salinity or limiting nutrients. Recent elucidation of mPPase structure and identification of subfamilies that have fully or partially switched from Na+ to H+ pumping have established mPPases as versatile models for studying the principles governing the mechanism, specificity and evolution of cation transporters. In the present study, we constructed an accurate phylogenetic map of the interface of Na+-transporting PPases (Na+-PPases) and Na+- and H+-transporting...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Nordbo, E., Luoto, H. H., Baykov, A. A., Lahti, R., Malinen, A. M. Tags: Research Articles Source Type: research

Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1
Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro. Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 s...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Muleya, V., Marondedze, C., Wheeler, J. I., Thomas, L., Mok, Y.-F., Griffin, M. D. W., Manallack, D. T., Kwezi, L., Lilley, K. S., Gehring, C., Irving, H. R. Tags: Research Articles Source Type: research

Hot1 factor recruits co-activator Sub1 and elongation complex Spt4/5 to osmostress genes
Hyperosmotic stress response involves the adaptative mechanisms needed for cell survival. Under high osmolarity conditions, many stress response genes are activated by several unrelated transcription factors that are controlled by the Hog1 kinase. Osmostress transcription factor Hot1 regulates the expression of several genes involved in glycerol biosynthesis, and the presence of this transcription factor in their promoters is essential for RNApol II recruitment. The physical association between Hog1 and Hot1 activates this transcription factor and directs the RNA polymerase II localization at these promoters. We, herein, d...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Gomar-Alba, M., del Olmo, M. Tags: Research Articles Source Type: research

Structural basis of arginine asymmetrical dimethylation by PRMT6
PRMT6 is a type I protein arginine methyltransferase, generating the asymmetric dimethylarginine mark on proteins such as histone H3R2. Asymmetric dimethylation of histone H3R2 by PRMT6 acts as a repressive mark that antagonizes trimethylation of H3 lysine 4 by the MLL histone H3K4 methyltransferase. PRMT6 is overexpressed in several cancer types, including prostate, bladder and lung cancers; therefore, it is of great interest to develop potent and selective inhibitors for PRMT6. Here, we report the synthesis of a potent bisubstrate inhibitor GMS [6'-methyleneamine sinefungin, an analog of sinefungin (SNF)], and the crysta...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Wu, H., Zheng, W., Eram, M. S., Vhuiyan, M., Dong, A., Zeng, H., He, H., Brown, P., Frankel, A., Vedadi, M., Luo, M., Min, J. Tags: Research Articles Source Type: research

Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport
Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP7), are conserved eukaryotic signaling molecules that possess pyrophosphate and monophosphate moieties. Generated predominantly by inositol hexakisphosphate kinases (IP6Ks), inositol pyrophosphates can modulate protein function by posttranslational serine pyrophosphorylation. Here, we report inositol pyrophosphates as novel regulators of cytoplasmic dynein-driven vesicle transport. Mammalian cells lacking IP6K1 display defects in dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance. Expression of c...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Chanduri, M., Rai, A., Malla, A. B., Wu, M., Fiedler, D., Mallik, R., Bhandari, R. Tags: Research Articles Source Type: research

Ablation of CD44 induces glycolysis-to-oxidative phosphorylation transition via modulation of the c-Src-Akt-LKB1-AMPK{alpha} pathway
Cluster of differentiation 44 (CD44) is a transmembrane glycoprotein that has been identified as a cancer stem cell marker in various cancer cells. Although many studies have focused on CD44 as a cancer stem cell marker, its effect on cancer cell metabolism remains unclear. To investigate the role of CD44 on cancer cell metabolism, we established CD44 knock-down cells via retroviral delivery of shRNA against CD44 in human breast cancer cells. Silencing of CD44 decreased the glycolytic phenotype of cancer cells, affecting glucose uptake, ATP production, and lactate production. We also found that ablation of the CD44-induced...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Nam, K., Oh, S., Shin, I. Tags: Research Articles Source Type: research

Identification of PNGase-dependent ERAD substrates in Saccharomyces cerevisiae
We report herein the identification of endogenous substrates for the cytoplasmic PNGase in Saccharomyces cerevisiae. Using an isotope-coded glycosylation site-specific tagging (IGOT) method-based LC/MS analysis, 11 glycoproteins were specifically detected in the cytosol of PNGase-deletion cells (png1). Among these molecules, at least five glycoproteins were clearly identified as ERAD substrates in vivo. Moreover, four out of the five proteins were found to be either deglycosylated by PNGase in vivo or the overall degradation was delayed in a png1 mutant. Our results clearly indicate that the IGOT method promises to be a po...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Hosomi, A., Fujita, M., Tomioka, A., Kaji, H., Suzuki, T. Tags: Research Articles Source Type: research

TPL2 meets p38MAPK: emergence of a novel positive feedback loop in inflammation
The activation of p38MAPK by Toll-like receptor signalling is essential for the inflammatory response of innate immunity due to its role in post-transcriptional regulation of TNFα and cytokine biosynthesis. p38MAPK activation proceeds by the upstream MAP2Ks, MAPK kinase (MKK)3/6 as well as MKK4, which in turn are substrates for MAP3Ks, such as TGFβ-activated protein kinase-1 (TAK1). In contrast, TPL2 has been described as an exclusive MAP3K of MKK1/2-triggering activation of the classical ERKs, ERK1/2. In the recent issue of the Biochemical Journal, Pattison et al. report their screening for TPL2 substrates in L...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Menon, M. B., Gaestel, M. Tags: Commentaries Source Type: research

Molecular signaling cascades involved in nonmelanoma skin carcinogenesis
Nonmelanoma skin cancer (NMSC) is the most common cancer worldwide and the incidence continues to rise, in part due to increasing numbers in high-risk groups such as organ transplant recipients and those taking photosensitizing medications. The most significant risk factor for NMSC is ultraviolet radiation (UVR) from sunlight, specifically UVB, which is the leading cause of DNA damage, photoaging, and malignant transformation in the skin. Activation of apoptosis following UVR exposure allows the elimination of irreversibly damaged cells that may harbor oncogenic mutations. However, UVR also activates signaling cascades tha...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Feehan, R. P., Shantz, L. M. Tags: Review Articles Source Type: research

The role of the mtDNA set point in differentiation, development and tumorigenesis
Mitochondrial DNA replication is critical for maintaining mtDNA copy number to generate sufficient cellular energy that is required for development and for functional cells. In early development, mtDNA copy number is strictly regulated at different stages, and, as a result, the establishment of the mtDNA set point is required for sequential cell lineage commitment. The failure to establish the mtDNA set point results in incomplete differentiation or embryonic arrest. The regulation of mtDNA copy number during differentiation is closely associated with cellular gene expression, especially with the pluripotency network, and ...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Sun, X., St. John, J. C. Tags: Review Articles Source Type: research

Targeting polyamine metabolism for cancer therapy and prevention
The chemically simple, biologically complex eukaryotic polyamines, spermidine and spermine, are positively charged alkylamines involved in many crucial cellular processes. Along with their diamine precursor putrescine, their normally high intracellular concentrations require fine attenuation by multiple regulatory mechanisms to keep these essential molecules within strict physiologic ranges. Since the metabolism of and requirement for polyamines are frequently dysregulated in neoplastic disease, the metabolic pathway and functions of polyamines provide rational drug targets; however, these targets have been difficult to ex...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Murray-Stewart, T. R., Woster, P. M., Casero, R. A. Tags: Review Articles Source Type: research

The role of TREX in gene expression and disease
TRanscription and EXport (TREX) is a conserved multisubunit complex essential for embryogenesis, organogenesis and cellular differentiation throughout life. By linking transcription, mRNA processing and export together, it exerts a physiologically vital role in the gene expression pathway. In addition, this complex prevents DNA damage and regulates the cell cycle by ensuring optimal gene expression. As the extent of TREX activity in viral infections, amyotrophic lateral sclerosis and cancer emerges, the need for a greater understanding of TREX function becomes evident. A complete elucidation of the composition, function an...
Source: Biochemical Journal - September 27, 2016 Category: Biochemistry Authors: Heath, C. G., Viphakone, N., Wilson, S. A. Tags: Review Articles Source Type: research

Isoform-specific function of calpains in cell adhesion disruption: studies in postlactational mammary gland and breast cancer
Cleavage of adhesion proteins is the first step for physiological clearance of undesired cells during postlactational regression of the mammary gland, but also for cell migration in pathological states such as breast cancer. The intracellular Ca2+-dependent proteases, calpains (CAPNs), are known to cleave adhesion proteins. The isoform-specific function of CAPN1 and CAPN2 was explored and compared in two models of cell adhesion disruption: mice mammary gland during weaning-induced involution and breast cancer cell lines according to tumor subtype classification. In both models, E-cadherin, β-catenin, p-120, and talin-...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Rodriguez-Fernandez, L., Ferrer-Vicens, I., Garcia, C., Oltra, S. S., Zaragoza, R., Vina, J. R., Garcia-Trevijano, E. R. Tags: Research Articles Source Type: research

Use of CRISPR/Cas9-engineered INS-1 pancreatic {beta} cells to define the pharmacology of dual GIPR/GLP-1R agonists
Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindro...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Naylor, J., Suckow, A. T., Seth, A., Baker, D. J., Sermadiras, I., Ravn, P., Howes, R., Li, J., Snaith, M. R., Coghlan, M. P., Hornigold, D. C. Tags: Research Articles Source Type: research

Structural insights into the interaction of p97 N-terminus domain and VBM in rhomboid protease, RHBDL4
RHBDL4 is an active rhomboid that specifically recognizes and cleaves atypical, positively charged transmembrane endoplasmic reticulum-associated degradation (ERAD) substrates. Interaction of valosin-containing protein (p97/VCP) and RHBDL4 is crucial to retrotranslocate polyubiquitinated substrates for ERAD pathway. Here, we report the first complex structure of VCP-binding motif (VBM) with p97 N-terminal domain (p97N) at 1.88 Å resolution. Consistent with p97 adaptor proteins including p47-ubiquitin regulatory X (UBX), gp78-VCP-interacting motif (VIM), OTU1-UBX-like element, and FAF1-UBX, RHBDL4 VBM also binds ...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Lim, J. J., Lee, Y., Ly, T. T., Kang, J. Y., Lee, J.-G., An, J. Y., Youn, H.-S., Park, K. R., Kim, T. G., Yang, J. K., Jun, Y., Eom, S. H. Tags: Research Articles Source Type: research

TLR and TNF-R1 activation of the MKK3/MKK6-p38{alpha} axis in macrophages is mediated by TPL-2 kinase
Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-β-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8D270A/D270A mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Pattison, M. J., Mitchell, O., Flynn, H. R., Chen, C.-S., Yang, H.-T., Ben-Addi, H., Boeing, S., Snijders, A. P., Ley, S. C. Tags: Research Articles Source Type: research

In the absence of phosphate shuttling, exercise reveals the in vivo importance of creatine-independent mitochondrial ADP transport
The transport of cytosolic adenosine diphosphate (ADP) into the mitochondria is a major control point in metabolic homeostasis, as ADP concentrations directly affect glycolytic flux and oxidative phosphorylation rates within mitochondria. A large contributor to the efficiency of this process is thought to involve phosphocreatine (PCr)/Creatine (Cr) shuttling through mitochondrial creatine kinase (Mi-CK), whereas the biological importance of alterations in Cr-independent ADP transport during exercise remains unknown. Therefore, we utilized an Mi-CK knockout (KO) model to determine whether in vivo Cr-independent mechanisms a...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Miotto, P. M., Holloway, G. P. Tags: Research Articles Source Type: research

Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence
Mammalian glutamate dehydrogenase (GDH), a nuclear-encoded enzyme central to cellular metabolism, is among the most abundant mitochondrial proteins (constituting up to 10% of matrix proteins). To attain such high levels, GDH depends on very efficient mitochondrial targeting that, for human isoenzymes hGDH1 and hGDH2, is mediated by an unusually long cleavable presequence (N53). Here, we studied the mitochondrial transport of these proteins using isolated yeast mitochondria and human cell lines. We found that both hGDHs were very rapidly imported and processed in isolated mitochondria, with their presequences (N53) alone be...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Kalef-Ezra, E., Kotzamani, D., Zaganas, I., Katrakili, N., Plaitakis, A., Tokatlidis, K. Tags: Research Articles Source Type: research

Structural and biophysical analysis of nuclease protein antibiotics
Protein antibiotics (bacteriocins) are a large and diverse family of multidomain toxins that kill specific Gram-negative bacteria during intraspecies competition for resources. Our understanding of the mechanism of import of such potent toxins has increased significantly in recent years, especially with the reporting of several structures of bacteriocin domains. Less well understood is the structural biochemistry of intact bacteriocins and how these compare across bacterial species. Here, we focus on endonuclease (DNase) bacteriocins that target the genomes of Escherichia coli and Pseudomonas aeruginosa, known as E-type co...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Klein, A., Wojdyla, J. A., Joshi, A., Josts, I., McCaughey, L. C., Housden, N. G., Kaminska, R., Byron, O., Walker, D., Kleanthous, C. Tags: Research Articles Source Type: research

Cdk2 catalytic activity is essential for meiotic cell division in vivo
Cyclin-dependent kinases (Cdks) control the eukaryotic cell cycle by phosphorylating serine and threonine residues in key regulatory proteins, but some Cdk family members may exert kinase-independent functions that cannot easily be assessed using gene knockout approaches. While Cdk2-deficient mice display near-normal mitotic cell proliferation due to the compensatory activities of Cdk1 and Cdk4, they are unable to undergo meiotic generation of gametes and are consequently sterile. To investigate whether Cdk2 regulates meiosis via protein phosphorylation or by alternative kinase-independent mechanisms, we generated two diff...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Chauhan, S., Diril, M. K., Lee, J. H. S., Bisteau, X., Manoharan, V., Adhikari, D., Ratnacaram, C. K., Janela, B., Noffke, J., Ginhoux, F., Coppola, V., Liu, K., Tessarollo, L., Kaldis, P. Tags: Research Articles Source Type: research

Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin
Ezrin is a member of the ERM (ezrin–radixin–moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ez...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Phang, J. M., Harrop, S. J., Duff, A. P., Sokolova, A. V., Crossett, B., Walsh, J. C., Beckham, S. A., Nguyen, C. D., Davies, R. B., Glöckner, C., Bromley, E. H. C., Wilk, K. E., Curmi, P. M. G. Tags: Research Articles Source Type: research

'Up with the LRRK: a phosphorylated Rab10 assay for evaluation of LRRK2 activity and inhibitor engagement
Protein kinases catalyse the addition of phosphate groups to Ser/Thr and Tyr residues in cognate substrates and are mutated or hyperactive in a variety of diseases, making them important targets for rationally designed drugs. A good example is the Parkinson's disease-associated kinase, leucine-rich repeat kinase 2 (LRRK2), which is mutated (and probably hyperactive) in a small, but significant, subset of patients. An exciting new approach for personalised therapy is the development of central nervous system (CNS)-active small-molecule kinase inhibitors, which could be employed to ‘normalise’ LRRK2 signalling in...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Eyers, P. A. Tags: Commentaries Source Type: research

Molecular regulation of insulin granule biogenesis and exocytosis
Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia, insulin resistance and hyperinsulinemia in early disease stages but a relative insulin insufficiency in later stages. Insulin, a peptide hormone, is produced in and secreted from pancreatic β-cells following elevated blood glucose levels. Upon its release, insulin induces the removal of excessive exogenous glucose from the bloodstream primarily by stimulating glucose uptake into insulin-dependent tissues as well as promoting hepatic glycogenesis. Given the increasing prevalence of T2DM worldwide, elucidating the underlying mechanis...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Röder, P. V., Wong, X., Hong, W., Han, W. Tags: Review Articles Source Type: research

HCN2 ion channels: basic science opens up possibilities for therapeutic intervention in neuropathic pain
Nociception — the ability to detect painful stimuli — is an invaluable sense that warns against present or imminent damage. In patients with chronic pain, however, this warning signal persists in the absence of any genuine threat and affects all aspects of everyday life. Neuropathic pain, a form of chronic pain caused by damage to sensory nerves themselves, is dishearteningly refractory to drugs that may work in other types of pain and is a major unmet medical need begging for novel analgesics. Hyperpolarisation-activated cyclic nucleotide (HCN)-modulated ion channels are best known for their fundamental pacema...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Tsantoulas, C., Mooney, E. R., McNaughton, P. A. Tags: Review Articles Source Type: research

Nectin spot: a novel type of nectin-mediated cell adhesion apparatus
Nectins are Ca2+-independent immunoglobulin (Ig) superfamily cell adhesion molecules constituting a family with four members, all of which have three Ig-like loops at their extracellular regions. Nectins play roles in the formation of a variety of cell–cell adhesion apparatuses. There are at least three types of nectin-mediated cell adhesions: afadin- and cadherin-dependent, afadin-dependent and cadherin-independent, and afadin- and cadherin-independent. In addition, nectins trans-interact with nectin-like molecules (Necls) with three Ig-like loops and other Ig-like molecules with one to three Ig-like loops. Furtherm...
Source: Biochemical Journal - September 12, 2016 Category: Biochemistry Authors: Mizutani, K., Takai, Y. Tags: Review Articles Source Type: research

A paradigm shift in oxygen sensing with a twist in the tale!
AMP-activated protein kinase (AMPK) is pivotal to metabolic homoeostasis in eukaryotes, serving as a critical energy sensor. Increased AMPK activity during oxygen deprivation (hypoxia) protects against potentially catastrophic deficits in ATP supply. Although the nervous system circuitry for elaboration of the complex cardiorespiratory response to hypoxia has been understood in some detail for many decades, there is continued and considerable interest in the molecular machinery underpinning the mechanism(s) of oxygen sensing. In this issue of the Biochemical Journal, Evans et al. [(2016) Biochem. J.] review their recent wo...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: O'Halloran, K. D. Tags: Commentaries Source Type: research

Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors
Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phospho...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Ito, G., Katsemonova, K., Tonelli, F., Lis, P., Baptista, M. A. S., Shpiro, N., Duddy, G., Wilson, S., Ho, P. W.-L., Ho, S.-L., Reith, A. D., Alessi, D. R. Tags: Research articles Source Type: research

Proteasome regulates turnover of toxic human amylin in pancreatic cells
Toxic human amylin (hA) oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Although recent studies demonstrated a causal connection between hA uptake and toxicity in pancreatic cells, the mechanism of amylin's clearance following its internalization and its relationship to toxicity is yet to be determined, and hence was investigated here. Using pancreatic rat insulinoma β-cells and human islets as model systems, we show that hA, following its internalization, first accumulates in the cytosol followed by its translocation into nucleus, and to a lesser extent lysosomes, ...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Singh, S., Trikha, S., Sarkar, A., Jeremic, A. M. Tags: Research articles Source Type: research

Only the complex N559-glycan in the synaptic vesicle glycoprotein 2C mediates high affinity binding to botulinum neurotoxin serotype A1
In conclusion, we show the importance of the complex N559-glycan of SV2C-LD4, adding a third anchor point beside a ganglioside and the SV2C-LD4 peptide, for BoNT/A neuronal cell surface binding and uptake. (Source: Biochemical Journal)
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Mahrhold, S., Bergstrom, T., Stern, D., Dorner, B. G., Astot, C., Rummel, A. Tags: Research articles Source Type: research

The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine
We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2'-deoxycytidine-5'-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP ...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Requena, C. E., Perez-Moreno, G., Horvath, A., Vertessy, B. G., Ruiz-Perez, L. M., Gonzalez-Pacanowska, D., Vidal, A. E. Tags: Research articles Source Type: research

Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana
Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana. To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Cabassa-Hourton, C., Schertl, P., Bordenave-Jacquemin, M., Saadallah, K., Guivarc'h, A., Lebreton, S., Planchais, S., Klodmann, J., Eubel, H., Crilat, E., Lefebvre-De Vos, D., Ghelis, T., Richard, L., Abdelly, C., Carol, P., Braun, H.-P., Savoure, A. Tags: Research articles Source Type: research

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation
Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5–ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Fujimoto, S., Tsuji, T., Fujiwara, T., Takeda, T.-a., Merriman, C., Fukunaka, A., Nishito, Y., Fu, D., Hoch, E., Sekler, I., Fukue, K., Miyamae, Y., Masuda, S., Nagao, M., Kambe, T. Tags: Research articles Source Type: research

Critical role of XBP1 in cancer signalling is regulated by PIN1
XBP1 (X-box-binding protein 1) is activated in cancer and has a pivotal role in tumorigenesis and progression of human cancer. In particular, the XBP1 transcriptional regulatory network is well known to drive cancer development, but little is known about whether the stability of XBP1 is regulated and, if so, what controls the stability of XBP1. In the present study we show that PIN1 prolyl isomerase interacts with the active form of XBP1 (XBP1s) in a phosphorylation-dependent manner and promotes XBP1s-induced cell proliferation and transformation through the regulation of XBP1 stability. By contrast, depletion of Pin1 ...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Chae, U., Park, S.-J., Kim, B., Wei, S., Min, J.-S., Lee, J.-H., Park, S. H., Lee, A.-H., Lu, K. P., Lee, D.-S., Min, S.-H. Tags: Research articles Source Type: research

ACAP3 regulates neurite outgrowth through its GAP activity specific to Arf6 in mouse hippocampal neurons
ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Miura, Y., Hongu, T., Yamauchi, Y., Funakoshi, Y., Katagiri, N., Ohbayashi, N., Kanaho, Y. Tags: Research articles Source Type: research

Targeting protein function: the expanding toolkit for conditional disruption
A major objective in biological research is to understand spatial and temporal requirements for any given gene, especially in dynamic processes acting over short periods, such as catalytically driven reactions, subcellular transport, cell division, cell rearrangement and cell migration. The interrogation of such processes requires the use of rapid and flexible methods of interfering with gene function. However, many of the most widely used interventional approaches, such as RNAi or CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated 9), operate at the level of the gene or its transcri...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Campbell, A. E., Bennett, D. Tags: Review articles Source Type: research

The emerging role of AMPK in the regulation of breathing and oxygen supply
Regulation of breathing is critical to our capacity to accommodate deficits in oxygen availability and demand during, for example, sleep and ascent to altitude. It is generally accepted that a fall in arterial oxygen increases afferent discharge from the carotid bodies to the brainstem and thus delivers increased ventilatory drive, which restores oxygen supply and protects against hypoventilation and apnoea. However, the precise molecular mechanisms involved remain unclear. We recently identified as critical to this process the AMP-activated protein kinase (AMPK), which is key to the cell-autonomous regulation of metabolic...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Evans, A. M., Mahmoud, A. D., Moral-Sanz, J., Hartmann, S. Tags: Review articles Source Type: research

When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches
Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a ...
Source: Biochemical Journal - August 29, 2016 Category: Biochemistry Authors: Munoz, V., Cerminara, M. Tags: Review articles Source Type: research

Zinc transporter ZIP10 forms a heteromer with ZIP6 which regulates embryonic development and cell migration
There is growing evidence that zinc and its transporters are involved in cell migration during development and in cancer. In the present study, we show that zinc transporter ZIP10 (SLC39A10) stimulates cell motility and proliferation, both in mammalian cells and in the zebrafish embryo. This is associated with inactivation of GSK (glycogen synthase kinase)-3α and -3β and down-regulation of E-cadherin (CDH1). Morpholino-mediated knockdown of zip10 causes delayed epiboly and deformities of the head, eye, heart and tail. Furthermore, zip10 deficiency results in overexpression of cdh1, zip6 and stat3, the latter gen...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Taylor, K. M., Muraina, I. A., Brethour, D., Schmitt-Ulms, G., Nimmanon, T., Ziliotto, S., Kille, P., Hogstrand, C. Tags: Research articles Source Type: research

Function and expression study uncovered hepatocyte plasma membrane ecto-ATP synthase as a novel player in liver regeneration
ATP synthase, canonically mitochondrially located, is reported to be ectopically expressed on the plasma membrane outer face of several cell types. We analysed, for the first time, the expression and catalytic activities of the ecto- and mitochondrial ATP synthase during liver regeneration. Liver regeneration was induced in rats by two-thirds partial hepatectomy. The protein level and the ATP synthase and/or hydrolase activities of the hepatocyte ecto- and mitochondrial ATP synthase were analysed on freshly isolated hepatocytes and mitochondria from control, sham-operated and partial hepatectomized rats. During the priming...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Taurino, F., Giannoccaro, C., Sardanelli, A. M., Cavallo, A., De Luca, E., Santacroce, S., Papa, S., Zanotti, F., Gnoni, A. Tags: Research articles Source Type: research

The Ca2+/Mn2+-transporting SPCA2 pump is regulated by oxygen and cell density in colon cancer cells
The mammalian SPCA1 and SPCA2 ATPases localize in membranes of the secretory pathway and transport ions of Ca2+ and Mn2+. The role of tissue-specific SPCA2 isoform, highly expressed in lungs, mammary gland and gastrointestinal tract, is poorly understood. To elucidate the function of SPCA2, we studied human colon cancer HCT116 cells, grown under ambient and decreased O2 levels. We found that in contrast with other Ca2+-ATPase isoforms the expression of SPCA2 was up-regulated under hypoxia (3% O2), in both adherent (2D) and spheroid (3D) cultures. In spheroids, experiencing lowest O2 levels (30–50 μM, measured...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Jenkins, J., Papkovsky, D. B., Dmitriev, R. I. Tags: Research articles Source Type: research

Lachesana tarabaevi, an expert in membrane-active toxins
In the present study, we show that venom of the ant spider Lachesana tarabaevi is unique in terms of molecular composition and toxicity. Whereas venom of most spiders studied is rich in disulfide-containing neurotoxic peptides, L. tarabaevi relies on the production of linear (no disulfide bridges) cytolytic polypeptides. We performed full-scale peptidomic examination of L. tarabaevi venom supported by cDNA library analysis. As a result, we identified several dozen components, and a majority (~80% of total venom protein) exhibited membrane-active properties. In total, 33 membrane-interacting polypeptides (length of 18&ndash...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Kuzmenkov, A. I., Sachkova, M. Y., Kovalchuk, S. I., Grishin, E. V., Vassilevski, A. A. Tags: Research articles Source Type: research

Hypertrophic cardiomyopathy mutations in the calponin-homology domain of ACTN2 affect actin binding and cardiomyocyte Z-disc incorporation
α-Actinin-2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients, A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain (ABD) of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full l...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Haywood, N. J., Wolny, M., Rogers, B., Trinh, C. H., Shuping, Y., Edwards, T. A., Peckham, M. Tags: Research articles Source Type: research

Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli
Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli. HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we d...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Reimann, S., Poschmann, G., Kanonenberg, K., Stuhler, K., Smits, S. H. J., Schmitt, L. Tags: Research articles Source Type: research

Proximity-dependent biotin labelling in yeast using the engineered ascorbate peroxidase APEX2
The engineered ascorbate peroxidase (APEX2) has been effectively employed in mammalian cells to identify protein–protein interactions. APEX2 fused to a protein of interest covalently tags nearby proteins with biotin-phenol (BP) when H2O2 is added to the cell culture medium. Subsequent affinity purification of biotinylated proteins allows for identification by MS. BP labelling occurs in 1 min, providing temporal control of labelling. The APEX2 tool enables proteomic mapping of subcellular compartments as well as identification of dynamic protein complexes, and has emerged as a new methodology for proteomic analys...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Hwang, J., Espenshade, P. J. Tags: Research articles Source Type: research

Ubiquitin C-terminal hydrolase L1 (UCH-L1): structure, distribution and roles in brain function and dysfunction
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is an extremely abundant protein in the brain where, remarkably, it is estimated to make up 1–5% of total neuronal protein. Although it comprises only 223 amino acids it has one of the most complicated 3D knotted structures yet discovered. Beyond its expression in neurons UCH-L1 has only very limited expression in other healthy tissues but it is highly expressed in several forms of cancer. Although UCH-L1 is classed as a deubiquitinating enzyme (DUB) the direct functions of UCH-L1 remain enigmatic and a wide ar...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Bishop, P., Rocca, D., Henley, J. M. Tags: Review articles Source Type: research

Mechanisms of Hsp90 regulation
Heat shock protein 90 (Hsp90) is a molecular chaperone that is involved in the activation of disparate client proteins. This implicates Hsp90 in diverse biological processes that require a variety of co-ordinated regulatory mechanisms to control its activity. Perhaps the most important regulator is heat shock factor 1 (HSF1), which is primarily responsible for upregulating Hsp90 by binding heat shock elements (HSEs) within Hsp90 promoters. HSF1 is itself subject to a variety of regulatory processes and can directly respond to stress. HSF1 also interacts with a variety of transcriptional factors that help integrate biologic...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Prodromou, C. Tags: Review articles Source Type: research

Physiological and pathophysiological functions of Swiprosin-1/EFhd2 in the nervous system
Synaptic dysfunction and dysregulation of Ca2+ are linked to neurodegenerative processes and behavioural disorders. Our understanding of the causes and factors involved in behavioural disorders and neurodegeneration, especially Alzheimer's disease (AD), a tau-related disease, is on the one hand limited and on the other hand controversial. Here, we review recent data about the links between the Ca2+-binding EF-hand-containing cytoskeletal protein Swiprosin-1/EFhd2 and neurodegeneration. Specifically, we summarize the functional biochemical data obtained in vitro with the use of recombinant EFhd2 protein, and integrated...
Source: Biochemical Journal - August 11, 2016 Category: Biochemistry Authors: Mielenz, D., Gunn-Moore, F. Tags: Review articles Source Type: research