ABSTRACT
Potassium ion (K+) plays a critical role as an essential electrolyte in all biological systems. Genetically-encoded fluorescent K+ biosensors are promising tools to further improve our understanding of K+-dependent processes under normal and pathological conditions. Here, we report the crystal structure of a previously reported genetically-encoded fluorescent K+ biosensor, GINKO1, in the K+-bound state. Using structure-guided optimization and directed evolution, we have engineered an improved K+ biosensor, designated GINKO2, with higher sensitivity and specificity. We have demonstrated the utility of GINKO2 for in vivo detection and imaging of K+ dynamics in multiple model organisms, including bacteria, plants, and mice.
Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer , Animals , Biosensing Techniques/methods , Fluorescence Resonance Energy Transfer/methods , Ions , Mice , PotassiumABSTRACT
Interactions between glycan-binding proteins (GBPs) and glycosphingolipids (GSLs) present in cell membranes are implicated in a wide range of biological processes. However, studying GSL binding is hindered by the paucity of purified GSLs and the weak affinities typical of monovalent GBP-GSL interactions. Native mass spectrometry (nMS) performed using soluble model membranes is a promising approach for the discovery of GBP ligands, but the detection of weak interactions remains challenging. The present work introduces MEmbrane ANchor-assisted nMS (MEAN-nMS) for the detection of low-affinity GBP-GSL complexes. The assay utilizes a membrane anchor, produced by covalent cross-linking of the GBP and a lipid in the membrane, to localize the GBP on the surface and promote GSL binding. Ligands are identified by nMS detection of intact GBP-GSL complexes (MEAN-nMS) or using a catch-and-release (CaR) strategy, wherein GSLs are released from GBP-GSL complexes upon collisional activation and detected (MEAN-CaR-nMS). To establish reliability, a library of purified gangliosides incorporated into nanodiscs was screened against human immune lectins, and the results compared with affinities of the corresponding ganglioside oligosaccharides. Without a membrane anchor, nMS analysis yielded predominantly false negatives. In contrast, all ligands were identified by MEAN-(CaR)-nMS, with no false positives. To highlight the potential of MEAN-CaR-nMS for ligand discovery, a natural library of GSLs was incorporated into nanodiscs and screened against human and viral proteins to uncover elusive ligands. Finally, nMS-based detection of GSL ligands directly from cells is demonstrated. This breakthrough paves the way for shotgun glycomics screening using intact cells.
Subject(s)
Glycosphingolipids , Mass Spectrometry , Glycosphingolipids/chemistry , Glycosphingolipids/metabolism , Mass Spectrometry/methods , Humans , Cell Membrane/metabolism , Cell Membrane/chemistry , Ligands , Protein BindingABSTRACT
Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.
Subject(s)
Calcium , Sarcoplasmic Reticulum , Animals , Calcium/metabolism , Calcium Signaling , Calcium-Binding Proteins/chemistry , Humans , Mammals/metabolism , Molecular Dynamics Simulation , Muscle Proteins , Peptides/metabolism , Peptides/pharmacology , Proteolipids/chemistry , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistryABSTRACT
The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show that PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min-1. In contrast, PGAM5 is cleaved with the highest efficiency (kcat/KM) compared with PINK1 and Smac/Diablo. In proteoliposomes, a truncated ß-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5, and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design.
Subject(s)
Metalloproteases/metabolism , Metalloproteases/physiology , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/physiology , Apoptosis Regulatory Proteins/metabolism , Catalytic Domain , Endopeptidases/metabolism , HEK293 Cells , HeLa Cells , Humans , Metalloproteases/genetics , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Proteins/genetics , Peptide Hydrolases/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , ProteolysisABSTRACT
Membranes surrounding the biological cell and its internal compartments host proteins that catalyze chemical reactions essential for the functioning of the cell. Rather than being a passive structural matrix that holds membrane-embedded proteins in place, the membrane can largely shape the conformational energy landscape of membrane proteins and impact the energetics of their chemical reaction. Here, we highlight the challenges in understanding how lipids impact the conformational energy landscape of macromolecular membrane complexes whose functioning involves chemical reactions including proton transfer. We review here advances in our understanding of how chemical reactions occur at membrane interfaces gleaned with both theoretical and experimental advances using simple protein systems as guides. Our perspective is that of bridging experiments with theory to understand general physicochemical principles of membrane reactions, with a long term goal of furthering our understanding of the role of the lipids on the functioning of complex macromolecular assemblies at the membrane interface.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/metabolism , Membranes/chemistry , Membranes/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Models, Molecular , Molecular Dynamics SimulationABSTRACT
Proteolysis is an integral component of life and has been implicated in many disease processes. To improve our understanding of peptidase function, it is imperative to develop tools to uncover substrate specificity and cleavage efficiency. Here, we combine the quantitative power of tandem mass tags (TMTs) with an established peptide cleavage assay to yield quantitative Multiplex Substrate Profiling by Mass Spectrometry (qMSP-MS). This assay was validated with papain, a well-characterized cysteine peptidase, to generate cleavage efficiency values for hydrolysis of 275 unique peptide bonds in parallel. To demonstrate the breath of this assay, we show that qMSP-MS can uncover the substrate specificity of minimally characterized intramembrane rhomboid peptidases, as well as define hundreds of proteolytic activities in complex biological samples, including secretions from lung cancer cell lines. Importantly, our qMSP-MS library uses synthetic peptides whose termini are unmodified, allowing us to characterize not only endo- but also exo-peptidase activity. Each cleaved peptide sequence can be ranked by turnover rate, and the amino acid sequence of the best substrates can be used for designing fluorescent reporter substrates. Discovery of peptide substrates that are selectively cleaved by peptidases which are active at the site of disease highlights the potential for qMSP-MS to guide the development of peptidase-activating drugs for cancer and infectious disease.
Subject(s)
Mass Spectrometry/methods , Peptide Hydrolases/metabolism , Aspergillus/metabolism , Cell Line, Tumor , Fluorescence , Humans , Lung Neoplasms/metabolism , Papain/metabolism , Proteolysis , Reproducibility of Results , Substrate SpecificityABSTRACT
Staphylococcus aureus ArlRS is a key two-component regulatory system necessary for adhesion, biofilm formation, and virulence. The response regulator ArlR consists of a C-terminal DNA-binding effector domain and an N-terminal receiver domain that is phosphorylated by ArlS, the cognate transmembrane sensor histidine kinase. We demonstrate that the receiver domain of ArlR adopts the canonical α5ß5 response regulator assembly, which dimerizes upon activation, using beryllium trifluoride as an aspartate phosphorylation mimic. Activated ArlR recognizes a 20-bp imperfect inverted repeat sequence in the ica operon, which is involved in intercellular adhesion polysaccharide production. Crystal structures of the inactive and activated forms reveal that activation induces a significant conformational change in the ß4-α4 and ß5-α5-connecting loops, in which the α4 and α5 helices constitute the homodimerization interface. Crystal structures of the DNA-binding ArlR effector domain indicate that it is able to dimerize via a non-canonical ß1-ß2 hairpin domain swapping, raising the possibility of a new mechanism for signal transduction from the receiver domain to effector domain. Taken together, the current study provides structural insights into the activation of ArlR and its recognition, adding to the diversity of response regulation mechanisms that may inspire novel antimicrobial strategies specifically targeting Staphylococcus.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Staphylococcus aureus , Anti-Infective Agents/chemistry , Anti-Infective Agents/therapeutic use , Bacterial Proteins/genetics , Crystallography, X-Ray , Methicillin Resistance , Models, Molecular , Phosphorylation , Protein Binding , Protein Domains , Protein Multimerization , Protein Structure, Secondary , Staphylococcal Infections/drug therapy , Staphylococcus aureus/geneticsABSTRACT
The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn4-Ala and Thr5-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.
Subject(s)
Muscle Proteins/chemistry , Muscle Proteins/metabolism , Protein Multimerization , Proteolipids/chemistry , Proteolipids/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistry , Amino Acid Sequence , Humans , Models, Molecular , Protein Binding , Protein Structure, QuaternaryABSTRACT
Modulation of T-cell immune functions by blocking key immune checkpoint protein interactions using monoclonal antibodies (mAbs) has been an innovative immunotherapeutic strategy. T-cells are regulated by different checkpoint proteins at the immunological synapse including the B7 ligands (B7-1 or CD80 and B7-2 or CD86), which is discussed in this review. These ligands are typically expressed on antigen presenting cells and interact with CD28 and cytotoxic T lymphocyte antigen-4 (CTLA-4) receptors on T-cells. Their interactions with CD28 trigger a costimulatory signal that potentiates T-cell activation, function and survival in response to cognate antigen. In addition, their interactions with CTLA-4 can also inhibit certain effector T-cell responses, particularly in response to sustained antigen stimulation. Through these mechanisms, the balance between T-cell activation and suppression is maintained, preventing the occurrence of immunopathology. Given their crucial roles in immune regulation, targeting B7 ligands has been an attractive strategy in cancer and autoimmunity. This review presents an overview of the essential roles of B7-1, highlighting the therapeutic benefits of modulating this protein in immunotherapy, and reviewing earlier and state-of-the-art efforts in developing anti-B7-1 inhibitors. Finally, we discuss the challenges facing the design of selective B7-1 inhibitors and present our perspectives for future developments.
Subject(s)
B7-1 Antigen/metabolism , Immunotherapy , Animals , Autoimmunity , B7-1 Antigen/chemistry , CTLA-4 Antigen/chemistry , CTLA-4 Antigen/metabolism , Drug Discovery , Humans , LigandsABSTRACT
KEY POINTS: 25-Hydroxyvitamin D (25OHD) is a partial agonist of TRPV1 whereby 25OHD can weakly activate TRPV1 yet antagonize the stimulatory effects of the full TRPV1 agonists capsaicin and oleoyl dopamine. 25OHD binds to TRPV1 within the same vanilloid binding pocket as capsaicin. 25OHD inhibits the potentiating effects of PKC-mediated TRPV1 activity. 25OHD reduces T-cell activation and trigeminal neuron calcium signalling mediated by TRPV1 activity. These results provide evidence that TRPV1 is a novel receptor for the biological actions of vitamin D in addition to the well-documented effects of vitamin D upon the nuclear vitamin D receptor. The results may have important implications for our current understanding of certain diseases where TRPV1 and vitamin D deficiency have been implicated, such as chronic pain and autoimmune diseases, such as type 1 diabetes. ABSTRACT: The capsaicin receptor TRPV1 plays an important role in nociception, inflammation and immunity and its activity is regulated by exogenous and endogenous lipophilic ligands. As vitamin D is lipophilic and involved in similar biological processes as TRPV1, we hypothesized that it directly regulates TRPV1 activity and function. Our calcium imaging and electrophysiological data demonstrate that vitamin D (25-hydroxyvitamin D (25OHD) and 1,25-hydroxyvitamin D (1,25OHD)) can weakly activate TRPV1 at physiologically relevant concentrations (100 nM). Furthermore, both 25OHD and 1,25OHD can inhibit capsaicin-induced TRPV1 activity (IC50 = 34.3 ± 0.2 and 11.5 ± 0.9 nM, respectively), but not pH-induced TRPV1 activity, suggesting that vitamin D interacts with TRPV1 in the same region as the TRPV1 agonist capsaicin. This hypothesis is supported by our in silico TRPV1 structural modelling studies, which place 25OHD in the same binding region as capsaicin. 25OHD also attenuates PKC-dependent TRPV1 potentiation via interactions with a known PKC phospho-acceptor residue in TRPV1. To provide evidence for a physiological role for the interaction of vitamin D with TRPV1, we employed two different cellular models known to express TRPV1: mouse CD4+ T-cells and trigeminal neurons. Our results indicate that 25OHD reduces TRPV1-induced cytokine release from T-cells and capsaicin-induced calcium activity in trigeminal neurons. In summary, we provide evidence that vitamin D is a novel endogenous regulator of TRPV1 channel activity that may play an important physiological role in addition to its known effects through the canonical nuclear vitamin D receptor pathway.
Subject(s)
Transient Receptor Potential Channels , Animals , Capsaicin/pharmacology , Mice , Neurons , Rats, Sprague-Dawley , TRPV Cation Channels , Vitamin D/pharmacologyABSTRACT
The interaction of phospholamban (PLN) with the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric PLN and storage of PLN as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that PLN remains associated with SERCA and that the PLN pentamer is required for the regulation of cardiac contractility. We have previously used two-dimensional (2D) crystallization and electron microscopy to study the interaction between SERCA and PLN. To further understand this interaction, we compared small helical crystals and large 2D crystals of SERCA in the absence and presence of PLN. In both crystal forms, SERCA molecules are organized into identical antiparallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large 2D crystals, which allow PLN differential access to potential sites of interaction with SERCA. Nonetheless, we show that a PLN oligomer interacts with SERCA in a similar manner in both crystal forms. In the 2D crystals, a PLN pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of PLN also interacts with M3 of SERCA, though the PLN oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of PLN interacts with M3 of SERCA and that it plays a distinct structural and functional role in SERCA regulation. The interaction of the pentamer places the cytoplasmic domains of PLN at the membrane surface proximal to the calcium entry funnel of SERCA. This interaction may cause localized perturbation of the membrane bilayer as a mechanism for increasing the turnover rate of SERCA.
Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/metabolism , Protein Multimerization , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Structure, Quaternary , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistryABSTRACT
Decades of work have contributed to our in-depth mechanistic understanding of soluble proteases, but much less is known about the catalytic mechanism of intramembrane proteolysis due to inherent difficulties in both preparing and analyzing integral membrane enzymes and transmembrane substrates. New work from Naing et al. tackles this challenge by examining the catalytic parameters of an aspartyl intramembrane protease homologous to the enzyme that cleaves amyloid precursor protein, finding that both chemistry and register contribute to specificity in substrate cleavage.
Subject(s)
Amyloid/metabolism , Aspartic Acid Proteases/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Proteolysis , Amyloid/chemistry , Aspartic Acid Proteases/chemistry , Catalysis , Cell Membrane/chemistry , Membrane Proteins/chemistryABSTRACT
Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final and committed step in the Kennedy pathway for triacylglycerol (TAG) biosynthesis and, as such, elucidating its mode of regulation is critical to understand the fundamental aspects of carbon metabolism in oleaginous crops. In this study, purified Brassica napus diacylglycerol acyltransferase 1 (BnaDGAT1) in n-dodecyl-ß-d-maltopyranoside micelles was lipidated to form mixed micelles and subjected to detailed biochemical analysis. The degree of mixed micelle fluidity appeared to influence acyltransferase activity. BnaDGAT1 exhibited a sigmoidal response and eventual substrate inhibition with respect to increasing concentrations of oleoyl-CoA. Phosphatidate (PA) was identified as a feed-forward activator of BnaDGAT1, enabling the final enzyme in the Kennedy pathway to adjust to the incoming flow of carbon leading to TAG. In the presence of PA, the oleoyl-CoA saturation plot became more hyperbolic and desensitized to substrate inhibition indicating that PA facilitates the transition of the enzyme into the more active state. PA may also relieve possible autoinhibition of BnaDGAT1 brought about by the N-terminal regulatory domain, which was shown to interact with PA. Indeed, PA is a key effector modulating lipid homeostasis, in addition to its well recognized role in lipid signaling. BnaDGAT1 was also shown to be a substrate of the sucrose non-fermenting-1-related kinase 1 (SnRK1), which catalyzed phosphorylation of the enzyme and converted it to a less active form. Thus, this known regulator of carbon metabolism directly influences TAG biosynthesis.
Subject(s)
Brassica napus/enzymology , Diacylglycerol O-Acyltransferase/metabolism , Phosphatidic Acids/metabolism , Triglycerides/biosynthesis , Acyl Coenzyme A/metabolism , Brassica napus/genetics , Carbohydrate Metabolism , Catalysis , Diacylglycerol O-Acyltransferase/genetics , Energy Metabolism , Homeostasis , Lipids/physiology , Phosphorylation , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolismABSTRACT
The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.
Subject(s)
Calcium-Binding Proteins/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Crystallography, X-Ray/methods , Humans , Hydrolysis , Ion Transport , Membrane Proteins/metabolism , Molecular Conformation , Muscle Proteins/metabolism , Muscle Relaxation/physiology , Muscle, Skeletal/metabolism , Myocardium/metabolism , Protein Conformation , Proteolipids/metabolism , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/chemistryABSTRACT
The apparent bottleneck in the accumulation of oil during seed development in some oleaginous plant species is the formation of triacylglycerol (TAG) by the acyl-CoA-dependent acylation of sn-1,2-diacylglycerol catalyzed by diacylglycerol acyltransferase (DGAT, EC 2.3.1.20). Improving DGAT activity using protein engineering could lead to improvements in seed oil yield (e.g. in canola-type Brassica napus). Directed evolution of B. napus DGAT1 (BnaDGAT1) previously revealed that one of the regions where amino acid residue substitutions lead to higher performance in BnaDGAT1 is in the ninth predicted transmembrane domain (PTMD9). In this study, several BnaDGAT1 variants with amino acid residue substitutions in PTMD9 were characterized. Among these enzyme variants, the extent of yeast TAG production was affected by different mechanisms, including increased enzyme activity, increased polypeptide accumulation, and possibly reduced substrate inhibition. The kinetic properties of the BnaDGAT1 variants were affected by the amino acid residue substitutions, and a new kinetic model based on substrate inhibition and sigmoidicity was generated. Based on sequence alignment and further biochemical analysis, the amino acid residue substitutions that conferred increased TAG accumulation were shown to be present in the DGAT1-PTMD9 region of other higher plant species. When amino acid residue substitutions that increased BnaDGAT1 enzyme activity were introduced into recombinant Camelina sativa DGAT1, they also improved enzyme performance. Thus, the knowledge generated from directed evolution of DGAT1 in one plant species can be transferred to other plant species and has potentially broad applications in genetic engineering of oleaginous crops and microorganisms.
Subject(s)
Brassica napus/genetics , Diacylglycerol O-Acyltransferase , Lipid Metabolism , Plant Proteins , Saccharomyces cerevisiae/enzymology , Amino Acid Substitution , Brassica napus/enzymology , Diacylglycerol O-Acyltransferase/biosynthesis , Diacylglycerol O-Acyltransferase/genetics , Mutation, Missense , Organisms, Genetically Modified/genetics , Organisms, Genetically Modified/metabolism , Plant Proteins/biosynthesis , Plant Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Saccharomyces cerevisiae/geneticsABSTRACT
Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases.
Subject(s)
Allosteric Regulation , Cell Membrane/enzymology , Escherichia coli/enzymology , Haemophilus influenzae/enzymology , Membrane Proteins/metabolism , Providencia/enzymology , Serine Proteases/metabolism , Cell Membrane/metabolism , Escherichia coli/metabolism , Fluorescence Resonance Energy Transfer , Haemophilus influenzae/metabolism , Kinetics , Protein Binding , Proteolysis , Providencia/metabolismABSTRACT
Obesity often leads non-alcoholic fatty liver disease, insulin resistance and hyperlipidemia. Expression of carboxylesterase CES1 is positively correlated with increased lipid storage and plasma lipid concentration. Here we investigated structural and metabolic consequences of a single nucleotide polymorphism in CES1 gene that results in p.Gly143Glu amino acid substitution. We generated a humanized mouse model expressing CES1WT (control), CES1G143E and catalytically dead CES1S221A (negative control) in the liver in the absence of endogenous expression of the mouse orthologous gene. We show that the CES1G143E variant exhibits only 20% of the wild-type lipolytic activity. High-fat diet fed mice expressing CES1G143E had reduced liver and plasma triacylglycerol levels. The mechanism by which decreased CES1 activity exerts this hypolipidemic phenotype was determined to include decreased very-low density lipoprotein secretion, decreased expression of hepatic lipogenic genes and increased fatty acid oxidation as determined by increased plasma ketone bodies and hepatic mitochondrial electron transport chain protein abundance. We conclude that attenuation of human CES1 activity provides a beneficial effect on hepatic lipid metabolism. These studies also suggest that CES1 is a potential therapeutic target for non-alcoholic fatty liver disease management.
Subject(s)
Carboxylic Ester Hydrolases/genetics , Genetic Predisposition to Disease , Liver/pathology , Non-alcoholic Fatty Liver Disease/genetics , Obesity/metabolism , Animals , Carboxylic Ester Hydrolases/metabolism , Chimera/genetics , Diet, High-Fat/adverse effects , Disease Models, Animal , Female , Humans , Lipid Metabolism/genetics , Lipids/blood , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Non-alcoholic Fatty Liver Disease/blood , Non-alcoholic Fatty Liver Disease/metabolism , Non-alcoholic Fatty Liver Disease/pathology , Obesity/blood , Obesity/etiology , Polymorphism, Single NucleotideABSTRACT
Rhomboids are ubiquitous intramembrane serine proteases that cleave transmembrane substrates. Their functions include growth factor signaling, mitochondrial homeostasis, and parasite invasion. A recent study revealed that the Escherichia coli rhomboid protease EcGlpG is essential for its extraintestinal pathogenic colonization within the gut. Crystal structures of EcGlpG and the Haemophilus influenzae rhomboid protease HiGlpG have deciphered an active site that is buried within the lipid bilayer but exposed to the aqueous environment via a cavity at the periplasmic face. A lack of physiological transmembrane substrates has hampered progression for understanding their catalytic mechanism and screening inhibitor libraries. To identify a soluble substrate for use in the study of rhomboid proteases, an array of internally quenched peptides were assayed with HiGlpG, EcGlpG and PsAarA from Providencia stuartti. One substrate was identified that was cleaved by all three rhomboid proteases, with HiGlpG having the highest cleavage efficiency. Mass spectrometry analysis determined that all enzymes hydrolyze this substrate between norvaline and tryptophan. Kinetic analysis in both detergent and bicellular systems demonstrated that this substrate can be cleaved in solution and in the lipid environment. The substrate was subsequently used to screen a panel of benzoxazin-4-one inhibitors to validate its use in inhibitor discovery.
Subject(s)
Escherichia coli Proteins/antagonists & inhibitors , Peptide Hydrolases/metabolism , Peptides/pharmacology , Chromatography, Liquid , Escherichia coli/enzymology , Escherichia coli Proteins/metabolism , Haemophilus influenzae/enzymology , Kinetics , Mass Spectrometry , Protease Inhibitors/pharmacology , Recombinant Proteins/metabolismABSTRACT
This is a perspective article entitled "Frontiers in computational biophysics: understanding conformational dynamics of complex lipid mixtures relevant to biology" which is following a CECAM meeting with the same name.
Subject(s)
Cell Membrane/chemistry , Cell Membrane/metabolism , Computational Biology/methods , Lipid Bilayers/chemistry , Molecular Dynamics Simulation , Protein Binding , ThermodynamicsABSTRACT
Diacylglycerol acyltransferase 1 (DGAT1) is an integral membrane enzyme catalyzing the final and committed step in the acyl-coenzyme A (CoA)-dependent biosynthesis of triacylglycerol (TAG). The biochemical regulation of TAG assembly remains one of the least understood areas of primary metabolism to date. Here, we report that the hydrophilic N-terminal domain of Brassica napus DGAT1 (BnaDGAT11-113) regulates activity based on acyl-CoA/CoA levels. The N-terminal domain is not necessary for acyltransferase activity and is composed of an intrinsically disordered region and a folded segment. We show that the disordered region has an autoinhibitory function and a dimerization interface, which appears to mediate positive cooperativity, whereas the folded segment of the cytosolic region was found to have an allosteric site for acyl-CoA/CoA. Under increasing acyl-CoA levels, the binding of acyl-CoA with this noncatalytic site facilitates homotropic allosteric activation. Enzyme activation, on the other hand, is prevented under limiting acyl-CoA conditions (low acyl-CoA-to-CoA ratio), whereby CoA acts as a noncompetitive feedback inhibitor through interaction with the same folded segment. The three-dimensional NMR solution structure of the allosteric site revealed an α-helix with a loop connecting a coil fragment. The conserved amino acid residues in the loop interacting with CoA were identified, revealing details of this important regulatory element for allosteric regulation. Based on these results, a model is proposed illustrating the role of the N-terminal domain of BnaDGAT1 as a positive and negative modulator of TAG biosynthesis.