The physiology of protein S-acylation

Protein S-acylation, the only fully reversible posttranslational lipid modification of proteins, is emerging as a ubiquitous mechanism to control the properties and function of a diverse array of proteins and consequently physiological processes. S-acylation results from the enzymatic addition of long-chain lipids, most typically palmitate, onto intracellular cysteine residues of soluble and transmembrane proteins via a labile thioester linkage. Addition of lipid results in increases in protein hydrophobicity that can impact on protein structure, assembly, maturation, trafficking, and function. The recent explosion in global S-acylation (palmitoyl) proteomic profiling as a result of improved biochemical tools to assay S-acylation, in conjunction with the recent identification of enzymes that control protein S-acylation and de-acylation, has opened a new vista into the physiological function of S-acylation. This review introduces key features of S-acylation and tools to interrogate this process, and highlights the eclectic array of proteins regulated including membrane receptors, ion channels and transporters, enzymes and kinases, signaling adapters and chaperones, cell adhesion, and structural proteins. We highlight recent findings correlating disruption of S-acylation to pathophysiology and disease and discuss some of the major challenges and opportunities in this rapidly expanding field

Multiscale Simulations of Biological Membranes : The Challenge To Understand Biological Phenomena in a Living Substance

Biological membranes are tricky to investigate. They are complex in terms of molecular composition and structure, functional over a wide range of time scales, and characterized by nonequilibrium conditions. Because of all of these features, simulations are a great technique to study biomembrane behavior. A significant part of the functional processes in biological membranes takes place at the molecular level; thus computer simulations are the method of choice to explore how their properties emerge from specific molecular features and how the interplay among the numerous molecules gives rise to function over spatial and time scales larger than the molecular ones. In this review, we focus on this broad theme. We discuss the current state-of-the-art of biomembrane simulations that, until now, have largely focused on a rather narrow picture of the complexity of the membranes. Given this, we also discuss the challenges that we should unravel in the foreseeable future. Numerous features such as the actin-cytoskeleton network, the glycocalyx network, and nonequilibrium transport under ATP-driven conditions have so far received very little attention; however, the potential of simulations to solve them would be exceptionally high. A major milestone for this research would be that one day we could say that computer simulations genuinely research biological membranes, not just lipid bilayers.Peer reviewe

Investigation of biological macromolecules using atomic force microscope-based techniques

The atomic force microscope (AFM) provides a powerful instrument for investigating and manipulating biological samples down to the subnanometer scale. In contrast to other microscopy methods, AFM does not require labeling, staining, nor fixation of samples and allows the specimen to be fully hydrated in buffer solution during the experiments. Moreover, AFM clearly compares in resolution to other techniques. In general, the AFM can be operated in an imaging or a force spectroscopy mode. In the present work, advantage was taken of this versatility to investigate single biomolecules and biomolecular assemblies. A novel approach to investigate the visco-elastic behavior of biomolecules under force was established, using dextran as an example. While a molecule tethered between a solid support and the cantilever tip was stretched at a constant velocity, the thermally driven oscillation of the cantilever was recorded. Analysis of the cantilever Brownian noise provided information about the visco-elastic properties of dextran that corresponded well to parameters obtained by alternative methods. However, the approach presented here was easier to implement and less time-consuming than previously used methods. A computer controlled force-clamp system was set up, circumventing the need for custom built analogue electronics. A commercial PicoForce AFM was extended by two computers which hosted data acquisition hardware. While the first computer recorded data, the second computer drove the AFM bypassing the manufacturer's microscope control software. To do so, a software-based proportional-integral-differential (PID) controller was implemented on the second computer. It allowed the force applied to a molecule to be held constant over time. After tuning of the PID controller, response times obtained using that force-clamp setup were comparable to those of the recently reported analogue systems. The performance of the setup was demonstrated by force-clamp unfolding of a pentameric Ig25 construct and the membrane protein NhaA. In the latter case, short-lived unfolding intermediates that were populated for less than 10 ms, could be revealed. Conventional single-molecule dynamic force spectroscopy was used to unfold the serine:threonine antiporter SteT from Bacillus subtilis, an integral membrane protein. Unfolding force patterns revealed the unfolding barriers stabilizing structural segments of SteT. Ligand binding did not induce new unfolding barriers suggesting that weak interactions with multiple structural segments were involved. In contrast, ligand binding caused changes in the energy landscape of all structural segments, thus turning the protein from a brittle, rigid into a more stable, structurally flexible conformation. Functionally, rigidity in the ligand-free state was thought to facilitate specific ligand binding, while flexibility and increased stability were required for conformational changes associated with substrate translocation. These results support the working model for transmembrane transport proteins that provide alternate access of the binding site to either face of the membrane. Finally, high-resolution imaging was exploited to visualize the extracellular surface of Cx26 gap junction hemichannels (connexons). AFM topographs reveal pH-dependent structural changes of the extracellular connexon surface in presence of HEPES, an aminosulfonate compound. At low pH (< 6.5), connexons showed a narrow and shallow channel entrance, which represented the closed pore. Increasing pH values resulted in a gradual opening of the pore, which was reflected by increasing channel entrance widths and depths. At pH > 7.6 the pore was fully opened and the pore diameter and depth did not increase further. Importantly, coinciding with pore gating a slight rotation of the subunits was observed. In the absence of aminosulfonate compounds, such as HEPES, acidification did not affect pore diameters and depths, retaining the open state. Thus, the intracellular concentration of taurine, a naturally abundant aminosulfonate compound, might be used to tune gap junction sensitivity at low pH

Structural studies of fragments of G-protein coupled receptors and their ligands by NMR

In the course of my doctoral studies I characterized the structure and dynamics of G-protein coupled receptor (GPCRs) fragments and their ligands by high-resolution NMR. The receptors of the GPCR family are transmembrane proteins of prime biological importance. All members of this family possess similar architecture of seven membrane-spanning α-helices and are involved in various signal transduction processes. First part of my work is devoted to the investigation of the structural determinants of the GPCR ligand peptide YY and monitoring the folding process of this peptide in solution. PYY is a 36- residue C-terminally amidated polypeptide that belongs to the neuropeptide Y family of peptide hormones. These molecules are involved in the regulation of a variety of physiological processes, such as for example food uptake. In the second part of my thesis I directed my efforts towards elucidation of the structure and probing the dynamic properties of the transmembrane fragments of the GPCRs in native-like environments. The subject of my studies was the -factor G-protein coupled Ste2p receptor, which is involved in sensing pheromones in yeast. Two large polypeptide fragments including the first and the second (peptide TM1TM2) and the seventh (peptide TM7) transmembrane domains of the Ste2p receptor were structurally characterized in micellar solution. The obtained results provide important insights into the GPCR architecture in a membrane bilayer. In the first part of my work I focused on the structural determinants and the folding process of the peptide YY (PYY) in solution. Some of the peptides from neuropeptide Y family adopt a well-defined hairpin structure in water that was first shown for avian pancreatic peptide (aPP) using X-ray crystallography. This helical hairpin is commonly referred to as PP-fold and is characterized by a N-terminal polyproline helix, which is back-folded via a-turn onto a C-terminal -helix. The solution structure of the PYY displayed a highly similar helical hairpin, however in the highly homologous neuropeptide Y we were surprised by the absence of the tertiary structure. To investigate the significance of the tertiary contacts, Tyr and Pro residues at the hydrophobic interface of the hairpin- type structure of PYY were replaced by Ala residues, and the conformational and dynamical properties of the resulting peptides were analyzed by high-resolution NMR spectroscopy. Previously we established the 15N{1H}-NOE as a convenient method to quantify the extent of back-folding. A comparison of the data from different Ala mutant peptides to those of native PYY nicely reflected the differences in backbone rigidity of the N-terminus. Most of the Pro->Ala or the Tyr->Ala mutants possessed increased backbone dynamics, and the differences in N-terminal mobility among them reflected various degrees to which they sample conformations close to the PP-fold. By varying temperature or the methanol content of the aqueous solvent and monitoring chemical shifts we followed the residue-specific formation of tertiary contacts while changing the physical or chemical environment. The PYY peptide in methanol solution was characterized both by determining its solution structure as well as by its internal backbone dynamics as derived from 15N relaxation data. The latter is characterized by a complete loss of tertiary structure. Chemical shifts of Cα in the heat-denaturation experiments displayed sigmoidal curves with very similar points of inflection indicating that both secondary, as well as tertiary structure in the heat denaturation, was lost synchronously. The results suggest that helical hairpin formation in PYY peptide is both reversible and cooperative and that specific N- and C-terminal tertiary hydrophobic contacts between the polyproline and the -helix promote the folding process. In addition, structural analysis of substitutions in the turn region indicates that the loop does not constrain the hairpin structure. The results may also have implications for our understanding of the binding of these peptides to their receptors. In the second part of the thesis the structure and dynamics of two large fragments of Ste2p the G-protein coupled -factor receptor from yeast were investigated. Both GPCR fragments were expressed and purified by our colleagues from the group of Prof. Fred Naider (College of Staten Island, NY). At first I investigated the 73-residue (Ste2p(267-339)) peptide TM7 consisting of the 3rd extracellular loop, the 7th transmembrane helix and 40 residues from the cytosolic C-terminal domain in dodecylphosphocholine micelles using solution NMR spectroscopy. The structure revealed the presence of an -helix in the segment encompassing residues 10 to 30, which was perturbed around the internal Pro24 residue. 15N-relaxation and RDC data supported a rather stable fold for the transmembrane part of TM7, whereas the exposed segments were more flexible. Spin-label data indicated that the TM7 helix was integrated into dodecylphosphocholine micelles, but displayed flexibility around the internal Pro24 site, exposing residues 22 to 26 to solution and revealed a second site of interaction with the micelle within a region comprising residues 43-58, which formed part of a less well- defined nascent helix. Further I extended my work on a single membrane-spanning TM7 fragment to a longer 80-residue (Ste2p(31-110)) double membrane-spanning peptide TM1TM2, consisting of 19 residues from the N-terminal domain, the 1st transmembrane helix, the first cytoplasmic loop, the second transmembrane helix and 7 residues from the first extracellular loop of the Ste2p receptor. Because of the larger complexity of a double membrane-spanning fragment different isotope labeling patterns were utilized including [15N], [15N, 13C], [15N, 13C, 2H]-labeled and selectively [15N]-labeled at specific amino acid residues or protonated only at selected methyl groups peptides. The structure of TM1TM2 peptide in lyso-palmitoylphosphatidylglycerol micelles revealed the presence of three-helices encompassing residues 39-47, 49-72 and 80-103, with higher flexibility around the internal Arg58 site of the first transmembrane domain. Several long-range interhelical NOE connectivities supported the folding of TM1TM2 into a tertiary structure forming a crossed helix that splays apart toward the extracellular regions and contains considerable flexibility in the G56VRSG60 region. 15N-relaxation and hydrogen-deuterium exchange data support a stable fold for the transmembrane parts of TM1TM2, whereas the solvent-exposed segments were more flexible. Interestingly the NMR structure was consistent with the results of biochemical experiments that identified the ligand-binding site within this region of the receptor. The results obtained during my Ph.D. studies reveal important aspects of the GPCR ligand peptide PYY structure and folding in solution so as shed light on the structure of large fragments of yeast pheromone receptor Ste2p in native-like micellar environment. Zusammenfassung Im Laufe meiner Promotion habe ich die Struktur und Dynamik von G-Protein- gekoppelte Rezeptor-(GPCRs) Fragmenten und ihren Liganden mittels hochauflösender NMR charakterisiert. Die Rezeptoren der GPCR-Familie sind Transmembran-Proteine von zentraler biologischer Bedeutung. Alle Mitglieder dieser Familie besitzen eine ähnliche Architektur mit sieben transmembranären α-Helices, und nehmen in verschiedenen Signaltransduktionsprozessen teil. Der erste Teil meiner Arbeit widmet sich der Untersuchung der strukturellen Determinanten des GPCR Liganden Peptid YY und der Verfolgung des Faltungsprozesses dieses Peptids in Lösung. PYY ist ein Polypeptid mit 36 Aminosäuren und C-terminaler Amidierung, das zu der Neuropeptid Y-Familie von Peptid-Hormonen gehört. Diese Moleküle sind in der Regulation einer Vielzahl physiologischer Prozesse involviert, wie zum Beispiel bei der Lebensmittelaufnahme. Im zweiten Teil meiner Arbeit richtete ich meine Bemühungen auf die Aufklärung der Struktur und die dynamischen Eigenschaften der Transmembran-Fragmente der GPCRs in nativen Bedingungen. Das Thema meiner Studien war der α-Faktor G-Protein-gekoppelter Rezeptor Ste2p, der involviert in der Pheromonerkennung in Hefe ist. Zwei große Polypeptid-Fragmente, bestehend aus der ersten und zweiten (Peptid TM1TM2) und der siebten Transmembran-Domän (Peptid TM7) des Ste2p-Rezeptors, wurden in micellärer Lösung strukturell charakterisiert. Die Ergebnisse liefern wichtige Einblicke in die GPCR-Architektur in einem Membran-Bilayer. Im ersten Teil meiner Arbeit konzentrierte ich mich auf die strukturellen Faktoren und den Faltungsprozess des Peptid YY (PYY) in Lösung. Einige der Peptide aus Neuropeptid Y-Familie haben eine klar definierte hairpin-Struktur in Wasser; diese wurde zum ersten Mal gezeigt für das Avian Pankreas-Peptid mittels Röntgenstrahl-Kristallographie. Dieser helikale ‘hairpin’ wird gemeinhin als PP-fold bezeichnet und besteht aus einer N-terminalen Polyprolin-Helix, die zurückfaltet über einen β-turn auf eine C-terminale α-Helix. Die Lösungsstruktur des PYY zeigt einen sehr ähnlichen helikalen ‘hairpin’, jedoch im hoch-homologen Neuropeptid Y beobachteten wir zu unserem Erstauenen keine Tertiärstruktur. Um die Bedeutung der tertiären Kontakte zu untersuchen, wurden Tyr- und Pro-Reste an der hydrophoben Oberfläche der ‘hairpin’- Struktur von PYY ersetzt durch Alanin und die konformationellen und dynamischen Eigenschaften der resultierenden Peptide wurden analysiert mittels hochauflösender NMR-Spektroskopie. Zuvor haben wir die 15N{1H}-NOE als eine passende Methode zur Quantifizierung des Umfangs der Rückfaltung etabliert. Ein Vergleich der Daten aus unterschiedlichen Ala-Peptid- Mutanten mit dem nativen PYY spiegelt schön die Unterschiede in der Steifheit des ‘backbones’ des N-Terminus wieder. Die meisten der Pro-> Ala oder der Tyr-> Ala Mutanten besaßen eine erhöhte ‘backbone’-Dynamik, und die Unterschiede in der N-terminalen Mobilität unter ihnen spiegelt verschiedene Grade wieder, zu dem sie Probe Konformationen annimmt, die dem ‘PP-fold’ ähneln. Durch Variation der Temperatur oder des Methanolgehalts des wässrigen Lösungsmittels und Verfolgung des ‘chemical shift’ konnten wir die aminosäure-spezifische Bildung der Tertiärkontakte während der Änderung der physikalischen oder chemischen Umgebung verfolgen. Das PYY Peptid in Methanollösung wurde charakterisiert sowohl durch die Bestimmung seiner Lösungsstruktur als auch durch ihre interne ‘backbone’-Dynamik mittels 15N-relaxation-Daten. Die ‘backbone’-Dynamik zeichnet sich durch einen vollständigen Verlust der tertiären Struktur aus. Die ‘Chemical shifts’ der Cα in den Hitze-Denaturierungs-Experimenten zeigten sigmoidale Kurven mit sehr ähnliche Wendepunkten, was darauf hinweist, dass sowohl Sekundär- als auch Tertiärstruktur in der Hitzedenaturierung synchron verloren werden. Die Ergebnisse deuten darauf hin, dass die Bildung des helikalen ‘hairpin’ im PYY Peptid reversibel und kooperativ ist und dass spezifische N-und C-terminale hydrophobe Tertiärkontakte zwischen der Polyprolinhelix und der α-Helix den Faltungsprozess fördern. Darüber hinaus deutet die Strukturanalyse von Substitutionen in der ‘turn’-Region darauf hin, dass der ‘loop’ die ‘hairpin’-Struktur nicht hemmt. Die Ergebnisse können auch Auswirkungen für unser Verständnis der Bindung dieser Peptide auf ihren Rezeptoren haben. Im zweiten Teil der Dissertation wurde die Struktur und Dynamik von zwei großen Fragmenten von Ste2p, dem G-Protein-gekoppelten α-Faktor-Rezeptor von Hefe untersucht. Beide GPCR-Fragmente wurden exprimiert und aufgereinigt von unseren Kollegen aus der Arbeitsgruppe von Prof. Fred Naider (College of Staten Island, NY). Zuerst untersuchte ich das 73-aminosäure-Peptid TM7 (Ste2p (267-339)) bestehend aus dem dritten extrazellulären ‘loop’, der siebten Transmembran-Helix und 40 Aminosäuren aus der zytosolische C-terminalen Domäne in Dodecylphosphocholin- Micellen mittels NMR-Spektroskopie. Die Struktur offenbarte die Anwesenheit einer α-Helix im Segment von Aminosäurerest 10 bis 30, die um das interne Pro24 gestört wird. 15N-relaxation und RDC-Daten unterstützten einen recht stabilen ‘fold’ für den Transmembran-Anteil des TM7, hingegen die ausgesetzten Segmente waren flexibler. Die Spin-Label-Daten weisten darauf hin, dass die TM7-Helix in die Dodecylphosphocholin-Micellen integriert wurde, aber zeigten Flexibilität rund um das interne Pro24, da die Aminosäuren 22 bis 26 in die Lösung zeigen, desweiteren zeigten sie einen zweiten Interaktionsort mit der Micelle innerhalb der Region von Aminosäurerest 43 bis 58, die einen Teil einer weniger gut definierten im Entstehen begriffenen Helix bildet. Im weiteren verlängerte ich meine Arbeit an einem einfachen Transmembran-Fragment TM7 zu einem längeren 80-Aminosäure-Doppel-Transmembran-Peptid TM1TM2 (Ste2p (31-110)), bestehend vom 19 Aminosäuren aus der N-terminalen Domäne, die erste Transmembran-Helix, der erste zytoplasmatische ‘loop’, die zweite Transmembran-Helix und 7 Aminosäuren aus dem ersten extrazellulären ‘loop’ des Ste2p-Rezeptors. Aufgrund der größeren Komplexität des doppelten Transmembran-Fragments wurden verschiedene Isotopen-Labeling-Muster genutzt: [15N], [15N, 13C], [15N, 13C, 2H]-markiert und selektiv [15N]- markiert an bestimmten Aminosäuren oder protoniert nur an ausgewählten Methyl-Gruppen-Peptiden. Die Struktur des TM 1 TM 2-Peptids in LYSO-palmitoylphosphatidylglycerol-Micellen zeigte das Vorhandensein von drei α-Helices, von Aminosäure 39-47, 49-72 und 80-103, mit einer größeren Flexibilität rund um das interne Arg58 der ersten Transmembran-Domäne. Mehrere ‘long range-interhelical NOE’ Verbindungen unterstützen die Faltung von TM1TM2 in eine Tertiärstruktur, die eine gekreuzte Helix bildet, die sich ausdehnt in Richtung der extrazellulären Regionen und die erhebliche Flexibilität in der G56VRSG60 Region enthält. 15N-relaxation- und Wasserstoff-Deuterium-Austausch-Daten unterstützten einen stabilen ‘fold’ für die Transmembran-Teile von TM1TM2, während die lösungsmittel-exponierten Segmente flexibler waren. Interessanterweise ist die NMR-Struktur im Einklang mit den Ergebnissen der biochemischen Experimente, die die Ligandenbindungsort in dieser Region des Rezeptors identifizierten. Die erzielten Ergebnisse während meiner Promotionsstudien zeigen wichtige Aspekte der GPCR-Peptid-Liganden PYY-Struktur und seiner Faltung in der Lösung, sowie geben sie Aufschluss über die Struktur der großen Fragmente des Hefe- Pheromon-Rezeptor Ste2p in nativer Micellenumgebung

Membrane bending is critical for assessing the thermodynamic stability of proteins in the membrane

The ability of biological membranes to bend is critical to understanding the interaction between proteins and the lipid bilayer. Experimental and computational studies have shown that the membrane can bend to expose charged and polar residues to the lipid headgroups and water, greatly reducing the cost of protein insertion. However, current computational approaches are poorly equipped to accurately model such deformation; atomistic simulations often do not reach the time-scale necessary to observe large-scale rearrangement, and continuum approaches assume a flat, rigid bilayer. In this thesis we present an efficient computational model of a deformable membrane for probing these interactions with elasticity theory and continuum electrostatics. To validate the model, we first investigate the insertion of three membrane proteins and three aqueous proteins. The model finds the membrane proteins and aqueous proteins stable and unstable in the membrane, respectively. We also investigate the sensitivity of these predictions to changes in several key parameters. The model is then applied to interactions between the membrane and the voltage sensor segments of voltage-gated potassium channels. Despite their high numbers of basic residues, experiments have shown that voltage sensors can be stably accommodated in the membrane. For simple continuum electrostatics approaches that assume a flat membrane, the penalty of inserting these charged residues would seem to prohibit voltage sensor insertion. However, in our method the membrane deforms to enable interaction between solvent and the charged residues. Our calculations predict that the highly charged S4 helices of several potassium channels are in fact stable in the membrane, in accord with experimental observations. Experimental and computational evidence has shown that the cost for inserting multiple charged amino acids into the membrane is not additive; it is not as costly to insert a second charge once a first has already been inserted. Our model reflects this phenomenon and provides a simple mechanical explanation linked to membrane deformation. We additionally consider the energetics of passive ion penetration into the membrane from bulk solvent. We use coarse-grained molecular dynamics to guide our input parameters and show that ion permeation energy profiles agree with atomistic simulations when membrane bending is included

Molecular cloning and characterization of a glucose transporter-related gene from Trypanosoma brucei

Available from British Library Document Supply Centre- DSC:DX97425 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Palmitoylation and regulation of divalent cation transport by TRPM7 and TRPM6

Magnesium regulates numerous cellular functions and enzymatic reactions, and abnormal magnesium homeostasis contributes to vascular dysfunction and the development of hypertension. The transient receptor potential melastatin 7 (TRPM7) is ubiquitously expressed and regulates embryonic development and pathogenesis of several common diseases. It is also a key player in cardiovascular magnesium homeostasis, cardiac fibrosis, and angiotensin II-induced hypertension. The TRPM7 integral membrane ion channel domain regulates transmembrane movement of divalent cations, primarily Ca, Mg and Zn, and its kinase domain controls gene expression via histone phosphorylation. Mechanisms regulating TRPM7 are elusive. TRPM7 not only localizes on the cell surface where it controls divalent cation fluxes but also exists in intracellular vesicles where it controls zinc uptake and release. Palmitoylation is a dynamic reversible posttranslational modification, which regulates ion channel activity, stability, and subcellular localization. We found TRPM7 is palmitoylated at a cluster of cysteines (Cys1143, Cys1144 and Cys1146) at the C terminal end of its TRP domain in multiple cell types. Palmitoylation controls the exit of TRPM7 from the endoplasmic reticulum and the distribution of TRPM7 between cell surface and intracellular pools. Using the Retention Using Selective Hooks (RUSH) system, we arrested TRPM7 in the Golgi and manipulated its palmitoylation with 2-boromopalmitate (2-BP). Pharmacological reduction of TRPM7 palmitoylation reduced its delivery to the cell surface membrane when it was released from the Golgi. we discovered that palmitoylated TRPM7 traffics from the Golgi to the surface membrane whereas non-palmitoylated TRPM7 is sequestered in intracellular vesicles. In addition, we engineered chimeric forms of TRPM7 in which the palmitoylation sites were replaced with the analogous region of TRPM2 or TRPM5, which do not contain cysteines. It also concludes that inhibiting palmitoylation of TRPM7 results in reduced TRPM7 abundance on cell surface. We identified the Golgi-resident enzyme zDHHC17 as responsible for palmitoylating TRPM7 and find that TRPM7 is de-palmitoylated by some acyl-thioesterases post-Golgi and re-palmitoylated by plasma-membrane-resident zDHHC5. The close homologue TRPM6 is also palmitoylated on the C-terminal side of its TRP domain. To investigate the impact of palmitoylation on TRPM7 ion transport activity, we attempted to measure Mg influx at cell surface and Zn influx in intracellular vesicles, but these two assays were unsuccessful. Using fluo4 to measure intracellular Ca we determined that TRPM7 mediated transmembrane calcium uptake is significantly reduced when TRPM7 is not palmitoylated. In addition, we also measured the relationship between phosphorylation/cleavage of TRPM7 and Its palmitoylation. Phosphorylation and palmitoylation are two independent post-translational modifications of TRPM7. Phosphorylated TRPM7 was palmitoylated to the same extent as total TRPM7. Non-palmitoylated TRPM7 chimeras were less phosphorylated, probably as a result of their reduced abundance at the cell surface. Quantitative proteomic analysis of the protein partners of wild type and non-palmitoylated TRPM7 identified vesicular proteins as more enriched with non-palmitoylated TRPM7, and nuclear proteins more enriched with palmitoylated TRPM7, suggesting cleavage and nuclear translocation of the TRPM7 kinase domain may be influenced by palmitoylation. Our findings illustrate palmitoylation controls ion channel activity of TRPM7 and that TRPM7 trafficking is dependent on its palmitoylation. Palmitoylation of TRPM7 might implicated in control of gene transcription by altering nuclear localization of its cleaved kinase domain. In conclusion, we defined palmitoylation as a new mechanism for post translational modification and regulation of TRPM7 other TRPs

Characterization of ARV1-Mediated Sterol Transport in Yeast and Mammalian Systems

Saccharomyces cerevisiae Arv1p (ARE2 required for viability 1) is an endoplasmic reticulum (ER)-localized, functionally conserved protein that was initially observed to mediate subcellular sterol distribution, and has since been implicated in the movement of multiple lipid species. In this thesis, we examined the role of ARV1 in S. cerevisiae and mammalian systems by two approaches. In yeast, we used gene deletion to access loss of Arv1p function. In mammalian cells we utilized antisense oligonucleotides (ASOs) to decrease ARV1 expression in vitro and in vivo. In the yeast model, loss of Arv1p function results in sensitivity to modulators of sphingolipid homeostasis and aberrant accumulation of exogenous sterols. Transcription microarrays demonstrated that ARV1 deletion impacts ER homeostasis and activates the transcription factor HAC1, a component of the unfolded protein response (UPR) signaling cascade in yeast. Moreover, arv1Δ strains exhibited constitutive UPR induction, mediated by the unfolded protein sensor Ire1p. Genetic interaction studies revealed that the arv1Δ ire1Δ homozygous haploid strain is inviable, suggesting the UPR protects the cell from arv1Δ-mediated stress. In order to assess the stimulus for arv1Δ-mediated UPR induction, arv1Δ ire1Δ heterozygous diploids were transformed with mutated Ire1p core luminal domains (cLDs) that are sufficient to transmit the signal for UPR induction but are defective in sensing unfolded proteins in the ER lumen. The mutant cLDs were able to rescue the lethality of the arv1Δ ire1Δ haploid. These strains exhibited increased UPR induction that was independent and additive with protein misfolding. Furthermore, ARV1 deficiency in murine macrophages activated PERK-mediated UPR induction, particularly an upregulation of the cell death effector, CHOP. ARV1 deficiency also caused apoptosis, likely due to prolonged UPR induction, a phenomenon that was exacerbated by inhibiting cholesterol esterification at the ER. In murine and human models, ARV1 is implicated in intracellular cholesterol homeostasis and bile acid metabolism. ASO-mediated decreases in ARV1 expression in vivo occurred primarily in the liver and adipose. ARV1 ASO-treated animals did not exhibit UPR activation but were hypercholesterolemic and had increased levels of hepatic and plasma bile acids. Consequently, accumulating bile acids transiently activated FXR-regulatory pathways, including target genes SHP, CYP7α1, NTCP and ABCB11. Furthermore, knockdown of ARV1 expression in hepatocytes established a role for human ARV1 in intracellular cholesterol distribution. ARV1 ASO-treated HepG2 cells exhibited accumulation of ER cholesterol, decreased SREBP processing and decreased expression of SREBP targets, suggesting that human ARV1 may mediate cholesterol export from the ER. In summation, loss of ARV1 has a profound impact on lipid homeostasis in yeast and metazoans. Various sterol detoxification pathways are activated in order to offset the loss of ARV1. In a hepatocyte, cholesterol biosynthesis is decreased and bile acid secretion is increased, in response to ARV1 deficiency. In yeast and macrophage models, where conversion of excess sterols into bile acids is not possible, the UPR is activated in order to compensate for loss of ARV1 function. Taken as a whole, these studies reflect the role of ARV1 in ER sterol distribution and trafficking, and the profound impact of decreased ARV1 expression on intracellular sterol homeostasis

Emerging Diversity in Lipid-Protein Interactions

Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions