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1.  Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33 
The Biochemical journal  2011;436(2):313-319.
GTPases act as molecular switches to control many cellular processes, including signalling, protein translation and targeting. Switch activity can be regulated by external effector proteins or intrinsic properties, such as dimerization. The recognition and translocation of pre-proteins into chloroplasts [via the TOC/TIC (translocator at the outer envelope membrane of chloroplasts/inner envelope membrane of chloroplasts)] is controlled by two homologous receptor GTPases, Toc33 and Toc159, whose reversible dimerization is proposed to regulate translocation of incoming proteins in a GTP-dependent manner. Toc33 is a homodimerizing GTPase. Functional analysis suggests that homodimerization is a key step in the translocation process, the molecular functions of which, as well as the elements regulating this event, are largely unknown. In the present study, we show that homodimerization reduces the rate of nucleotide exchange, which is consistent with the observed orientation of the monomers in the crystal structure. Pre-protein binding induces a dissociation of the Toc33 homodimer and results in the exchange of GDP for GTP. Thus homodimerization does not serve to activate the GTPase activity as discussed many times previously, but to control the nucleotide-loading state. We discuss this novel regulatory mode and its impact on the current models of protein import into the chloroplast.
doi:10.1042/BJ20110246
PMCID: PMC4086448  PMID: 21434866
dimeric GTPase; GDP-dissociation-inhibitor function (GDI function); G-protein; protein translocation; substrate-based regulation
2.  New methods for capturing the mystery lipid, PtdIns5P 
The Biochemical journal  2010;428(3):e1-e2.
The enormous versatility of phosphatidylinositol as a mediator of intracellular signalling is due to its variable phosphorylation on every combination of the 3′, 4′ and 5′ positions, as well as an even more complex range of phosphorylated products when inositol phosphate is released by phospholipase C activity. The phosphoinositides are produced by distinct enzymes in distinct intracellular membranes, and recruit and regulate downstream signalling proteins containing binding domains [PH (pleckstrin homology), PX (Phox homology), FYVE etc.] that are relatively specific for these lipids. Specific recruitment of downstream proteins presumably involves a coincidence detection mechanism, in which a combination of lipid–protein and protein–protein interactions define specificity. Of the seven intrucellular phosphoinositide, quantification of PtdIns5P levels in intact cells has remained difficult. In this issue of the Biochemical Journal, Sarkes and Rameh describe a novel HPLC-based approach which makes possible an analysis of the subcellular distribution of PtdIns5P and other phosphoinositides.
doi:10.1042/BJ20100688
PMCID: PMC4082335  PMID: 20504279
3.  Protein Kinase C Pharmacology: Refining the Toolbox 
The Biochemical journal  2013;452(2):195-209.
SYNOPSIS
Protein kinase C (PKC) has been in the limelight since the discovery three decades ago that it acts as a major receptor for the tumor-promoting phorbol esters. Phorbol esters, with their potent ability to activate two of the three classes of PKC isozymes, have remained the best pharmacological tool for directly modulating PKC activity. However, with the discovery of other phorbol ester-responsive proteins, the advent of various small-molecule and peptide modulators, and the need to distinguish isozyme-specific activity, the pharmacology of PKC has become increasingly complex. Not surprisingly, many of the compounds originally touted as direct modulators of PKC have subsequently been shown to hit many other cellular targets and, in some cases, not even directly modulate PKC. The complexities and reversals in PKC pharmacology have led to widespread confusion about the current status of the pharmacological tools available to control PKC activity. Here, we aim to clarify the cacophony in the literature regarding the current state of bona fide and discredited cellular PKC modulators, including activators, small-molecule inhibitors, and peptides, and also address the use of genetically-encoded reporters and of PKC mutants to measure the effects of these drugs on the spatiotemporal dynamics of signaling by specific isozymes.
doi:10.1042/BJ20130220
PMCID: PMC4079666  PMID: 23662807
4.  Lipin1 regulates PPARγ transcriptional activity 
The Biochemical journal  2013;453(1):49-60.
PPARγ (peroxisome proliferator-activated receptor-γ) is a master transcription factor involved in adipogenesis through regulating adipocyte-specific gene expression. Recently, lipin1 was found to act as a key factor for adipocyte maturation and maintenance by modulating the C/EBPα (CCAAT/enhancer-binding protein α) and PPARγ network; however, the precise mechanism by which lipin1 affects the transcriptional activity of PPARγ is largely unknown. The results of the present study show that lipin1 activates PPARγ by releasing co-repressors, NcoR1 (nuclear receptor co-repressor 1) and SMRT (silencing mediator of retinoid and thyroid hormone receptor), from PPARγ in the absence of the ligand rosiglitazone. We also identified a novel lipin1 TAD (transcriptional activation domain), between residues 217 and 399, which is critical for the activation of PPARγ, but not PPARα. Furthermore, this TAD is unique to lipin1 since this region does not show any homology with the other lipin isoforms, lipin2 and lipin3. The activity of the lipin1 TAD is enhanced by p300 and SRC-1 (steroid receptor co-activator 1), but not by PCAF (p300/CBP-associated factor) and PGC-1α (PPAR co-activator 1α). The physical interaction between lipin1 and PPARγ occurs at the lipin1 C-terminal region from residues 825 to 926, and the VXXLL motif at residue 885 is critical for binding with and the activation of PPARγ. The action of lipin1 as a co-activator of PPARγ enhanced adipocyte differentiation; the TAD and VXXLL motif played critical roles, but the catalytic activity of lipin1 was not directly involved. Collectively, these data suggest that lipin1 functions as a key regulator of PPARγ activity through its ability to release co-repressors and recruit co-activators via a mechanism other than PPARα activation.
doi:10.1042/BJ20121598
PMCID: PMC3690191  PMID: 23627357
co-activator; co-repressor; lipin1; peroxisome proliferator-activated receptor (PPAR)
5.  Describing Sequence-Ensemble Relationships for Intrinsically Disordered Proteins 
The Biochemical journal  2013;449(2):307-318.
Synopsis
Intrinsically disordered proteins participate in important protein-protein and protein-nucleic acid interactions and control cellular phenotypes through their prominence as dynamic organizers of transcriptional, post-transcriptional, and signaling networks. These proteins challenge the tenets of the structure-function paradigm and their functional mechanisms remain a mystery given that they fail to fold autonomously into specific structures. Solving this mystery requires a first principles understanding of the quantitative relationships between information encoded in the sequences of disordered proteins and the ensemble of conformations they sample. Advances in quantifying sequence-ensemble relationships have been facilitated through a four-way synergy between bioinformatics, biophysical experiments, computer simulations, and polymer physics theories. Here, we review these advances and the resultant insights that allow us to develop a concise quantitative framework for describing sequence-ensemble relationships of intrinsically disordered proteins.
doi:10.1042/BJ20121346
PMCID: PMC4074364  PMID: 23240611
6.  RESPONSES OF HYPERTROPHIED MYOCYTES TO REACTIVE SPECIES: IMPLICATIONS FOR GLYCOLYSIS AND ELECTROPHILE METABOLISM 
The Biochemical journal  2011;435(2):519-528.
Synopsis
During cardiac remodeling, the heart generates higher levels of reactive species; yet an intermediate “compensatory” stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischemia or in the failing heart. For this, we measured energetics in control and phenylephrine (PE)-treated rat neonatal cardiac myocytes (NRCMs) under basal conditions and when stressed with 4-hydroxynonenal (HNE). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a twofold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homeostasis under conditions of increased oxidative stress.
doi:10.1042/BJ20101390
PMCID: PMC4072126  PMID: 21275902
cardiac hypertrophy; mitochondria; glycolysis; aldehyde dehydrogenase; extracellular flux; 4-hydroxynonenal
7.  Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides 
The Biochemical journal  2009;421(1):79-86.
Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 μM respectively. Structure–activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases.
doi:10.1042/BJ20090309
PMCID: PMC4058678  PMID: 19379130
hypochlorous acid; myeloperoxidase; neutrophil; nitroxide; protein oxidation; superoxide
8.  Structural Model of a Putrescine-Cadaverine Permease from Trypanosoma cruzi Predicts Residues Vital for Transport and Ligand Binding 
The Biochemical journal  2013;452(3):423-432.
The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the amino acid, polyamine, organocation (APC) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key amino acid residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed based upon the atomic coordinates of the E. coli AdiC arginine-agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first 10 helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that residues Trp241, and a Glu247-Arg403 salt bridge participate in a gating system and that residues Asn245, Tyr148 and Tyr400 contribute to the putrescine binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine binding pocket.
doi:10.1042/BJ20130350
PMCID: PMC3952013  PMID: 23535070
Polyamines; putrescine; transport; parasites; Trypanosoma cruzi; homology modeling
9.  Differential conformational dynamics in the closely homologous FK506-binding domains of FKBP51 and FKBP52 
Biochemical Journal  2014;461(Pt 1):115-123.
As co-chaperones of Hsp90 (heat-shock protein 90), FKBP51 (FK506-binding protein of 51 kDa) and FKBP52 (FK506-binding protein of 52 kDa) act as antagonists in regulating the hormone affinity and nuclear transport of steroid receptor complexes. Exchange of Leu119 in FKBP51 for Pro119 in FKBP52 has been shown to largely reverse the steroid receptor activities of FKBP51 and FKBP52. To examine whether differences in conformational dynamics/plasticity might correlate with changes in the reported receptor activities, 15N-NMR relaxation measurements were carried out on the N-terminal FKBP domains of FKBP51 and FKBP52 as well as their residue-swapped variants. Both proteins exhibit a similar pattern of motion in the picosecond–nanosecond timeframe as well as a small degree of 15N line-broadening, indicative of motion in the microsecond–millisecond timeframe, in the β3a strand of the central sheet. Only the FKBP51 domain exhibits much larger line-broadening in the adjacent β3 bulge (40′s loop of FKBP12) and throughout the long β4–β5 loop (80′s loop of FKBP12). The L119P mutation at the tip of the β4–β5 loop completely suppressed the line-broadening in this loop while partially suppressing the line-broadening in the neighbouring β2 and β3a strands. The complementary P119L and P119L/P124S variants of FKBP52 yielded similar patterns of line-broadening for the β4–β5 loop as that for FKBP51, although only 20% and 60% as intense respectively. However, despite the close structural similarity in the packing interactions between the β4–β5 loop and the β3a strand for FKBP51 and FKBP52, the line-broadening in the β3a strand is unaffected by the P119L or P119L/P124S mutations in FKBP52.
Unlike FKBP52, the FK1 domain of FKBP51 exhibits microsecond–millisecond conformational dynamics in the β3 bulge and the β4–β5 loop, known sites of protein signalling interactions. Swapping residue 119 yields altered conformational dynamics in a pattern reminiscent of reported modulations in steroid receptor activity.
doi:10.1042/BJ20140232
PMCID: PMC4060953  PMID: 24749623
conformational dynamics; differential line-broadening; FK506-binding protein of 51 kDa (FKBP51); FK506-binding protein of 52 kDa (FKBP52); mutational analysis; nuclear magnetic resonance (NMR); FKBP, FK506-binding protein; FRB, FKBP12–rapamycin-binding; Hsp, heat-shock protein; LBD, ligand-binding domain; NF-κB, nuclear factor κB; TPR, tetratricopeptide
10.  Cleavage of Notch1 by granzyme B disables its transcriptional activity 
The Biochemical journal  2011;437(2):313-322.
Granzyme-mediated cell death is the main pathway for cytotoxic lymphocytes to kill virus-infected and tumour cells. A major player in this process is GrB (granzyme B), which triggers apoptosis in both caspase-dependent and caspase-independent pathways. A caspase-independent substrate of GrB is the highly conserved transmembrane receptor Notch1. The GrB cleavage sites in Notch1 and functional consequences of Notch1 cleavage by GrB were unknown. In the present study, we confirmed that Notch1 is a direct and caspase-independent substrate of GrB. We demonstrate that GrB cleaved the intracellular Notch1 domain at least twice at two distinct aspartic acids, Asp1860 and Asp1961. GrB cleavage of Notch1 can occur in all subcellular compartments, during maturation of the receptor, at the membrane, and in the nucleus. GrB also displayed perforin-independent functions by cleaving the extracellular domain of Notch1. Overall, cleavage of Notch1 by GrB resulted in a loss of transcriptional activity, independent of Notch1 activation. We conclude that GrB disables Notch1 function, probably resulting in anti-cellular proliferation and cell death signals.
doi:10.1042/BJ20110226
PMCID: PMC4050498  PMID: 21548883
cleavage; granzyme B; Notch signalling; proteolysis; γ-secretase; serine protease
11.  Intestinal absorption of water-soluble vitamins in health and disease 
The Biochemical journal  2011;437(3):357-372.
Our knowledge of the mechanisms and regulation of intestinal absorption of water-soluble vitamins under normal physiological conditions, and of the factors/conditions that affect and interfere with theses processes has been significantly expanded in recent years as a result of the availability of a host of valuable molecular/cellular tools. Although structurally and functionally unrelated, the water-soluble vitamins share the feature of being essential for normal cellular functions, growth and development, and that their deficiency leads to a variety of clinical abnormalities that range from anaemia to growth retardation and neurological disorders. Humans cannot synthesize water-soluble vitamins (with the exception of some endogenous synthesis of niacin) and must obtain these micronutrients from exogenous sources. Thus body homoeostasis of these micronutrients depends on their normal absorption in the intestine. Interference with absorption, which occurs in a variety of conditions (e.g. congenital defects in the digestive or absorptive system, intestinal disease/resection, drug interaction and chronic alcohol use), leads to the development of deficiency (and sub-optimal status) and results in clinical abnormalities. It is well established now that intestinal absorption of the water-soluble vitamins ascorbate, biotin, folate, niacin, pantothenic acid, pyridoxine, riboflavin and thiamin is via specific carrier-mediated processes. These processes are regulated by a variety of factors and conditions, and the regulation involves transcriptional and/or post-transcriptional mechanisms. Also well recognized now is the fact that the large intestine possesses specific and efficient uptake systems to absorb a number of water-soluble vitamins that are synthesized by the normal microflora. This source may contribute to total body vitamin nutrition, and especially towards the cellular nutrition and health of the local colonocytes. The present review aims to outline our current understanding of the mechanisms involved in intestinal absorption of water-soluble vitamins, their regulation, the cell biology of the carriers involved and the factors that negatively affect these absorptive events.
doi:10.1042/BJ20110326
PMCID: PMC4049159  PMID: 21749321
ascorbate; biotin; folate; intestinal transport; riboflavin; thiamin
12.  Assembly of a membrane receptor complex: roles of the uroplakin II prosequence in regulating uroplakin bacterial receptor oligomerization 
The Biochemical journal  2008;414(2):195-203.
The apical surface of the mammalian urothelium is almost completely covered by two-dimensional protein crystals (known as urothelial plaques) of hexagonally packed 16 nm particles consisting of two UP (uroplakin) heterodimers, i.e. UPs Ia/II and Ib/III pairs. UPs are functionally important as they contribute to the urothelial permeability barrier function, and UPIa may serve as the receptor for the uropathogenic Escherichia coli that causes over 90% of urinary tract infections. We study here how the UP proteins are assembled and targeted to the urothelial apical surface, paying special attention to the roles of the prosequence of UPII in UP oligomerization. We show that (i) the formation of the UPIa/UPII heterodimer, necessary for ER (endoplasmic reticulum) exit, requires disulfide formation in the prosequence domain of proUPII (the immature form of UPII still containing its prosequence); (ii) differentiation-dependent N-glycosylation of the prosequence leads to UP stabilization; (iii) a failure to form tetramers in cultured urothelial cells, in part due to altered glycosylation of the prosequence, may block two-dimensional crystal formation; and (iv) the prosequence of UPII remains attached to the mature protein complex on the urothelial apical surface even after it has been cleaved by the trans-Golgi-network-associated furin. Our results indicate that proper secondary modifications of the prosequence of UPII play important roles in regulating the oligomerization and function of the UP protein complex.
doi:10.1042/BJ20080550
PMCID: PMC4048928  PMID: 18481938
disulfide formation; glycosylation; integral membrane protein; prosequence; protein assembly; uroplakin
13.  Oxygen regulates the band 3–ankyrin bridge in the human erythrocyte membrane 
The Biochemical journal  2013;449(1):143-150.
The oxygenation state of erythrocytes is known to impact several cellular processes. As the only known O2-binding protein in red blood cells, haemoglobin has been implicated in the oxygenation-mediated control of cell pathways and properties. Band 3, an integral membrane protein linked to the spectrin/actin cytoskeleton, preferentially binds deoxygenated haemoglobin at its N-terminus, and has been postulated to participate in the mechanism by which oxygenation controls cellular processes. Because the ankyrin-binding site on band 3 is located near the deoxyHb (deoxygenated haemoglobin)-binding site, we hypothesized that deoxyHb might impact the association between band 3 and the underlying erythrocyte cytoskeleton, a link that is primarily established through band 3–ankyrin bridging. In the present paper we show that deoxygenation of human erythrocytes results in displacement of ankyrin from band 3, leading to release of the spectrin/actin cytoskeleton from the membrane. This weakening of membrane–cytoskeletal interactions during brief periods of deoxygenation could prove beneficial to blood flow, but during episodes of prolonged deoxygenation, such as during sickle cell occlusive crises, could promote unwanted membrane vesiculation.
doi:10.1042/BJ20120869
PMCID: PMC4049537  PMID: 23013433
band 3–ankyrin bridge; deoxygenated haemoglobin (deoxyHb); erythrocyte membrane; membrane mechanical properties; red cell cytoskeleton
14.  A highly efficient peptide substrate for EGFR activates the kinase by inducing aggregation 
The Biochemical journal  2013;453(3):337-344.
Synopsis
Formation of an asymmetric dimer by the epidermal growth factor receptor (EGFR) kinase domains results in allosteric activation. Since this dimer does not readily form in solution, the EGFR kinase domain phosphorylates most peptide substrates with a relatively low catalytic efficiency. Peptide C is a synthetic peptide substrate of EGFR developed by others that is phosphorylated with a significantly higher catalytic efficiency, and we sought to understand the basis for this. Peptide C was found to increase EGFR kinase activity by promoting formation of the EGFR kinase domain asymmetric dimer. Activation of the kinase domain by Peptide C also enhances phosphorylation of other substrates. Aggregation of the EGFR kinase domain by Peptide C likely underlies activation, and Peptide C precipitates several other proteins. Peptide C was found to form fibrils independent of the presence of EGFR, and these fibrils may facilitate aggregation and activation of the kinase domain. These results establish that a peptide substrate of EGFR may increase catalytic activity by promoting kinase domain dimerization by an aggregation-mediated mechanism.
doi:10.1042/BJ20130537
PMCID: PMC4048812  PMID: 23734957
EGFR; dimer; kinetics; aggregation; activation; fibril
15.  The structure of a Burkholderia pseudomallei immunophilin–inhibitor complex reveals new approaches to antimicrobial development 
The Biochemical journal  2011;437(3):413-422.
Mips (macrophage infectivity potentiators) are a subset of immunophilins associated with virulence in a range of micro-organisms. These proteins possess peptidylprolyl isomerase activity and are inhibited by drugs including rapamycin and tacrolimus. We determined the structure of the Mip homologue [BpML1 (Burkholderia pseudomallei Mip-like protein 1)] from the human pathogen and biowarfare threat B. pseudomallei by NMR and X-ray crystallography. The crystal structure suggests that key catalytic residues in the BpML1 active site have unexpected conformational flexibility consistent with a role in catalysis. The structure further revealed BpML1 binding to a helical peptide, in a manner resembling the physiological interaction of human TGFβRI (transforming growth factor β receptor I) with the human immunophilin FKBP12 (FK506-binding protein 12). Furthermore, the structure of BpML1 bound to the class inhibitor cycloheximide N-ethylethanoate showed that this inhibitor mimics such a helical peptide, in contrast with the extended prolyl-peptide mimicking shown by inhibitors such as tacrolimus. We suggest that Mips, and potentially other bacterial immunophilins, participate in protein–protein interactions in addition to their peptidylprolyl isomerase activity, and that some roles of Mip proteins in virulence are independent of their peptidylprolyl isomerase activity.
doi:10.1042/BJ20110345
PMCID: PMC4045482  PMID: 21574961
Burkholderia pseudomallei; NMR; peptidylprolyl isomerase; small-molecule inhibitor; X-ray crystallography
16.  PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress 
The Biochemical journal  2013;451(3):375-388.
SYNOPSIS
Vascular injury and chronic arterial diseases result in exposure of vascular smooth muscle cells (VSMCs) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with platelet-derived growth factor (PDGF)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and upregulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1, or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 led to a remarkable stabilization α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix and abrogated VSMC hyperproliferation and migration. Interestingly, treatment of cells with PDGF prevented protein damage and cell death due to exposure to the lipid peroxidation product, 4-hydroxynonenal. These results demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under conditions of high oxidative stress found to occur in vascular lesions.
doi:10.1042/BJ20121344
PMCID: PMC4040966  PMID: 23421427
autophagy; atherosclerosis; restenosis; 4-hydroxynonenal; growth factor; spautin
17.  FLIPL induces caspase-8 activity in the absence of interdomain caspase-8 cleavage and alters substrate specificity 
The Biochemical journal  2011;433(3):447-457.
Caspase-8 is an initiator caspase that is activated by death receptors to initiate the extrinsic pathway of apoptosis. Caspase-8 activation involves dimerization and subsequent interdomain autoprocessing of caspase-8 zymogens, and recently published work has established that elimination of the autoprocessing site of caspase-8 abrogates its pro-apoptotic function while leaving its proliferative function intact. The observation that the developmental abnormalities of caspase-8 deficient mice are shared by mice lacking the dimerization adapter FADD or the caspase paralog FLIPL has led to the hypothesis that FADD-dependent formation of heterodimers between caspase-8 and FLIPL could mediate the developmental role of caspase-8. Using an inducible dimerization system we demonstrate that cleavage of the catalytic domain of caspase-8 is crucial for its activity in the context of activation by homodimerization. However, we find that use of FLIPL as a partner for caspase-8 in dimerization-induced activation rescues the requirement for intersubunit linker proteolysis in both protomers. Moreover, before processing, caspase-8 in complex with FLIPL does not generate a fully active enzyme, but an attenuated species able to process only select natural substrates. Based on these results we propose a mechanism of caspase-8 activation by dimerization in the presence of FLIPL, as well as a mechanism of caspase-8 functional divergence in apoptotic and non-apoptotic pathways.
doi:10.1042/BJ20101738
PMCID: PMC4024219  PMID: 21235526
apoptosis; activation mechanism; protein dimerization
18.  Proteomic Analysis and Molecular Modeling Characterize the Iron-Regulatory Protein, Hemojuvelin/Repulsive Guidance Molecule c 
The Biochemical journal  2013;452(1):87-95.
Hemojuvelin (HJV) plays a key role in iron metabolism in mammals by regulating expression of the liver-derived hormone, hepcidin, which controls systemic iron uptake and release. Mutations in HJV cause juvenile hemochromatosis, a rapidly progressing iron overload disorder in humans. HJV, also known as repulsive guidance molecule c (RGMc), is a member of the three-protein RGM family. RGMs are GPI-linked glycoproteins that share ~50% amino acid identity and several structural motifs, including the presence of 14 cysteines in analogous locations. Unlike RGMa and RGMb, HJV/RGMc is composed of both single-chain and two-chain isoforms. To date there is no structural information for any member of the RGM family. Here we have mapped the disulfide bonds in mouse HJV/RGMc using a proteomics strategy combining sequential mass spectrometry (MS) steps composed of electron transfer dissociation (ETD) and collision-induced dissociation (CID), in which ETD induces cleavage of disulfide linkages, and CID establishes disulfide bond assignments between liberated peptides. Our results identify an HJV/RGMc molecular species containing 4 disulfide linkages. We predict using ab initio modeling that this molecule is a single-chain HJV/RGMc isoform. Our observations outline a general approach using tandem MS and ab initio molecular modeling to define unknown structural features in proteins.
doi:10.1042/BJ20121845
PMCID: PMC3890427  PMID: 23464809
hemojuvelin; repulsive guidance molecule; iron metabolism; mass spectrometry; molecular modeling; disulfide bonds; protein structure
19.  Frataxin-bypassing Isu1: characterization of the bypass activity in cells and mitochondria 
The Biochemical journal  2014;459(1):71-81.
Frataxin is a conserved mitochondrial protein, and deficiency underlies the neurodegenerative disease Friedreich’s ataxia. Frataxin interacts with the core machinery for Fe–S cluster assembly in mitochondria. Recently we reported that in frataxin-deleted yeast strains, a spontaneously occurring mutation in one of two genes encoding redundant Isu scaffold proteins, bypassed the mutant phenotypes. In the present study we created strains expressing a single scaffold protein, either Isu1 or the bypass mutant M107I Isu1. Our results show that in the frataxin-deletion strain expressing the bypass mutant Isu1, cell growth, Fe–S cluster protein activities, haem proteins and iron homoeostasis were restored to normal or close to normal. The bypass effects were not mediated by changes in Isu1 expression level. The persulfide-forming activity of the cysteine desulfurase was diminished in the frataxin deletion (Δyfh1 ISU1) and was improved by expression of the bypass Isu1 (Δyfh1 M107I ISU1). The addition of purified bypass M107I Isu1 protein to a Δyfh1 lysate conferred similar enhancement of cysteine desulfurase as did frataxin, suggesting that this effect contributed to the bypass mechanism. Fe–S cluster-forming activity in isolated mitochondria was stimulated by the bypass Isu1, albeit at a lower rate. The rescuing effects of the bypass Isu1 point to ways that the core defects in Friedreich’s ataxia mitochondria can be restored.
doi:10.1042/BJ20131273
PMCID: PMC4021491  PMID: 24433162
cysteine desulfurase; eukaryote; frataxin; iron–sulfur; Isu1 scaffold; mitochondrion
20.  Proteasomal interaction as a critical activity modulator of the human constitutive androstane receptor 
The Biochemical journal  2014;458(1):95-107.
The CAR (constitutive androstane receptor; NR1I3) is a critical xenobiotic sensor that regulates xenobiotic metabolism, drug clearance, energy and lipid homoeostasis, cell proliferation and development. Although constitutively active, in hepatocytes CAR is normally held quiescent through a tethering mechanism in the cytosol, anchored to a protein complex that includes several components, including heat-shock protein 90. Release and subsequent nuclear translocation of CAR is triggered through either direct binding to ligand activators such as CITCO {6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime} or through indirect chemical activation, such as with PB (phenobarbital). In the present study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR’s interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with either PB or CITCO. Paradoxically, these effects occur following accumulation of ubiquitinated hCAR (human CAR). Furthermore, a non-proteolytic function was indicated by its interaction with a SUG1 (suppressor for Gal1), a subunit of the 26S proteasome. Taken together, these data demonstrate that the proteasome complex functions at multiple levels to regulate the functional biology of hCAR activity.
doi:10.1042/BJ20130685
PMCID: PMC4019979  PMID: 24224465
constitutive androstane receptor; hepatocyte; human; lactacystin; MG-132; NR1I3; proteasome
21.  Mutation in the Fe–S scaffold protein Isu bypasses frataxin deletion 
The Biochemical journal  2012;441(1):473-480.
Frataxin is a conserved mitochondrial protein deficient in patients with Friedreich’s ataxia. Frataxin has been implicated in control of iron homoeostasis and Fe–S cluster assembly. In yeast or human mitochondria, frataxin interacts with components of the Fe–S cluster synthesis machinery, including the cysteine desulfurase Nfs1, accessory protein Isd11 and scaffold protein Isu. In the present paper, we report that a single amino acid substitution (methionine to isoleucine) at position 107 in the mature form of Isu1 restored many deficient functions in Δyfh1 or frataxin-depleted yeast cells. Iron homoeostasis was improved such that soluble/usable mitochondrial iron was increased and accumulation of insoluble/non-usable iron within mitochondria was largely prevented. Cytochromes were returned to normal and haem synthesis was restored. In mitochondria carrying the mutant Isu1 and no frataxin, Fe–S cluster enzyme activities were improved. The efficiency of newFe–S cluster synthesis in isolated mitochondria was markedly increased compared with frataxin-negative cells, although the response to added iron was minimal. The M107I substitution in the highly conserved Isu scaffold protein is typically found in bacterial orthologues, suggesting that a unique feature of the bacterial Fe–S cluster machinery may be involved. The mechanism by which the mutant Isu bypasses the absence of frataxin remains to be determined, but could be related to direct effects on Fe–S cluster assembly and/or indirect effects on mitochondrial iron availability.
doi:10.1042/BJ20111637
PMCID: PMC4018837  PMID: 21936771
frataxin; haem; iron; iron–sulfur cluster (Fe–S cluster); mitochondrion
22.  Myoplasmic resting Ca2+ regulation by ryanodine receptors is under the control of a novel Ca2+-binding region of the receptor 
Biochemical Journal  2014;460(Pt 2):261-271.
Passive SR (sarcoplasmic reticulum) Ca2+ leak through the RyR (ryanodine receptor) plays a critical role in the mechanisms that regulate [Ca2+]rest (intracellular resting myoplasmic free Ca2+ concentration) in muscle. This process appears to be isoform-specific as expression of either RyR1 or RyR3 confers on myotubes different [Ca2+]rest. Using chimaeric RyR3–RyR1 receptors expressed in dyspedic myotubes, we show that isoform-dependent regulation of [Ca2+]rest is primarily defined by a small region of the receptor encompassing amino acids 3770–4007 of RyR1 (amino acids 3620–3859 of RyR3) named as the CLR (Ca2+ leak regulatory) region. [Ca2+]rest regulation by the CLR region was associated with alteration of RyRs’ Ca2+-activation profile and changes in SR Ca2+-leak rates. Biochemical analysis using Tb3+-binding assays and intrinsic tryptophan fluorescence spectroscopy of purified CLR domains revealed that this determinant of RyRs holds a novel Ca2+-binding domain with conformational properties that are distinctive to each isoform. Our data suggest that the CLR region provides channels with unique functional properties that modulate the rate of passive SR Ca2+ leak and confer on RyR1 and RyR3 distinctive [Ca2+]rest regulatory properties. The identification of a new Ca2+-binding domain of RyRs with a key modulatory role in [Ca2+]rest regulation provides new insights into Ca2+-mediated regulation of RyRs.
This paper reports the finding of a new class of Ca2+-binding domain of intracellular Ca2+ channels from muscle cells. This domain provides channels with distinctive properties that result in channel-specific modulation of the intracellular resting Ca2+ concentration.
doi:10.1042/BJ20131553
PMCID: PMC4019983  PMID: 24635445
calcium-binding site; calcium leak; myotube; skeletal muscle; terbium fluorescence; tryptophan fluorescence; [Ca2+]rest, intracellular resting myoplasmic free Ca2+ concentration; CLR, Ca2+ leak regulatory; DHPR, dihydropyridine receptor; fura 2/AM, fura 2 acetoxymethyl ester; HEK, human embryonic kidney; MHS, malignant hyperthermia syndrome; RyR, ryanodine receptor; SERCA1, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1; SR, sarcoplasmic reticulum
23.  Kinase and channel activity of TRPM6 are co-ordinated by a dimerization motif and pocket interaction 
Biochemical Journal  2014;460(Pt 2):165-175.
Mutations in the gene that encodes the atypical channel-kinase TRPM6 (transient receptor potential melastatin 6) cause HSH (hypomagnesaemia with secondary hypocalcaemia), a disorder characterized by defective intestinal Mg2+ transport and impaired renal Mg2+ reabsorption. TRPM6, together with its homologue TRPM7, are unique proteins as they combine an ion channel domain with a C-terminally fused protein kinase domain. How TRPM6 channel and kinase activity are linked is unknown. Previous structural analysis revealed that TRPM7 possesses a non-catalytic dimerization motif preceding the kinase domain. This interacts with a dimerization pocket lying within the kinase domain. In the present study, we provide evidence that the dimerization motif in TRPM6 plays a critical role in regulating kinase activity as well as ion channel activity. We identify mutations within the TRPM6 dimerization motif (Leu1718 and Leu1721) or dimerization pocket (L1743A, Q1832K, A1836N, L1840A and L1919Q) that abolish dimerization and establish that these mutations inhibit protein kinase activity. We also demonstrate that kinase activity of a dimerization motif mutant can be restored by addition of a peptide encompassing the dimerization motif. Moreover, we observe that mutations that disrupt the dimerization motif and dimerization pocket interaction greatly diminish TRPM6 ion channel activity, in a manner that is independent of kinase activity. Finally, we analyse the impact on kinase activity of ten disease-causing missense mutations that lie outwith the protein kinase domain of TRPM6. This revealed that one mutation lying nearby the dimerization motif (S1754N), found previously to inhibit channel activity, abolished kinase activity. These results provide the first evidence that there is structural co-ordination between channel and kinase activity, which is mediated by the dimerization motif and pocket interaction. We discuss that modulation of this interaction could comprise a major regulatory mechanism by which TRPM6 function is controlled.
We show that TRPM6 kinase activity is linked to channel activity. This occurs through a kinase-independent mechanism involving the dimerization motif binding to a pocket within the kinase domain. A disease-causing mutation (S1754N) lying nearby the dimerization pocket inactivates kinase activity.
doi:10.1042/BJ20131639
PMCID: PMC4019984  PMID: 24650431
dimerization motif; hypomagnesaemia; ion channel; phosphorylation; protein kinase; transient receptor potential melastatin (TRPM); E, embryonic day; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; HRP, horseradish peroxidase; HSH, hypomagnesaemia with secondary hypocalcaemia; LDS, lithium dodecyl sulfate; MBP, myelin basic protein; TBST, TBS containing Tween 20; TRPM, transient receptor potential melastatin
24.  Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles 
Biochemical Journal  2014;460(Pt 2):199-210.
Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30–Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130–Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33–Cys130′ and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33–Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1.
Erv1 is a sulfydryl oxidase, an essential component of mitochondrial MIA pathway. The present study shows that both shuttle cysteine residues of Erv1 are required for its function, they play complementary, but distinct, roles to ensure rapid turnover of active enzyme.
doi:10.1042/BJ20131540
PMCID: PMC4019985  PMID: 24625320
CXXC motif; mitochondrial import; mitochondrial import and assembly pathway (MIA pathway); thiol oxidase; ALR, augmenter of liver regeneration; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; AtErv1, Arabidopsis thaliana Erv1; Cox17, cytochrome c oxidase 17; CTC, charge-transfer complex; Erv, essential for respiration and viability; hMia40, human Mia40; IAM, iodoacetamide; IMS, intermembrane space; LtErv, Leishmania tarentolae Erv; MIA, mitochondrial import and assembly; mBBr, monobromobimane; mmPEG24, methyl-PEG24-maleimide; NEM, N-ethylmaleimide; PfErv, Plasmodium falciparum Erv; TCA, trichloroacetic acid; TCEP, tris-(2-carboxyethyl)phosphine; Tim, translocase of the inner membrane; TPI1, triose phosphate isomerase 1; TRP1, tryptophan requiring 1; WT, wild-type
25.  Structural and biochemical characterization of the KLHL3–WNK kinase interaction important in blood pressure regulation 
Biochemical Journal  2014;460(Pt 2):237-246.
WNK1 [with no lysine (K)] and WNK4 regulate blood pressure by controlling the activity of ion co-transporters in the kidney. Groundbreaking work has revealed that the ubiquitylation and hence levels of WNK isoforms are controlled by a Cullin-RING E3 ubiquitin ligase complex (CRL3KLHL3) that utilizes CUL3 (Cullin3) and its substrate adaptor, KLHL3 (Kelch-like protein 3). Loss-of-function mutations in either CUL3 or KLHL3 cause the hereditary high blood pressure disease Gordon's syndrome by stabilizing WNK isoforms. KLHL3 binds to a highly conserved degron motif located within the C-terminal non-catalytic domain of WNK isoforms. This interaction is essential for ubiquitylation by CRL3KLHL3 and disease-causing mutations in WNK4 and KLHL3 exert their effects on blood pressure by disrupting this interaction. In the present study, we report on the crystal structure of the KLHL3 Kelch domain in complex with the WNK4 degron motif. This reveals an intricate web of interactions between conserved residues on the surface of the Kelch domain β-propeller and the WNK4 degron motif. Importantly, many of the disease-causing mutations inhibit binding by disrupting critical interface contacts. We also present the structure of the WNK4 degron motif in complex with KLHL2 that has also been reported to bind WNK4. This confirms that KLHL2 interacts with WNK kinases in a similar manner to KLHL3, but strikingly different to how another KLHL protein, KEAP1 (Kelch-like enoyl-CoA hydratase-associated protein 1), binds to its substrate NRF2 (nuclear factor-erythroid 2-related factor 2). The present study provides further insights into how Kelch-like adaptor proteins recognize their substrates and provides a structural basis for how mutations in WNK4 and KLHL3 lead to hypertension.
WNK kinases regulate mammalian blood pressure. The level of WNK protein in a cell is regulated by the KLHL3–CUL3 ubiquitin ligase. We define the interaction between KLHL3 and WNK, identifying the WNK degron, and present the crystal structure of the KLHL3–WNK degron complex.
doi:10.1042/BJ20140153
PMCID: PMC4019986  PMID: 24641320
Bric-a-brac; Tramtrack; and Broad complex (BTB domain); Cullin; hypertension; Kelch-like protein (KLHL); Kelch-like protein 2 (KLHL2); ubiquitin; BTB, Bric-a-brac, Tramtrack, and Broad complex; CRL3KLHL3, Cullin3-RING ligase in complex with KLHL; CUL3, Cullin3; KEAP1, Kelch-like enoyl-CoA hydratase-associated protein 1; KLHL, Kelch-like protein; NCC, Na+/Cl− ion co-transporter; NKCC2, Na+/K+/2Cl− co-transporter 2; NRF2, nuclear factor-erythroid 2-related factor 2; OSR1, oxidative stress-responsive kinase 1; rTEV, recombinant tobacco etch virus; RT-PCR, reverse transcription–PCR; SPAK, SPS1-related proline/alanine-rich kinase; TCEP, tris-(2-carboxyethyl)phosphine; TEV, tobacco etch virus; WNK, with no lysine (K)

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