Publications by authors named "Diana Murray"

60 Publications

Integrating 3D structural information into systems biology.

J Biol Chem 2021 Jan-Jun;296:100562. Epub 2021 Mar 18.

Department of Systems Biology, Department of Biochemistry and Molecular Biophysics, Department of Medicine, Zuckerman Mind Brain and Behavior Institute, Columbia University, New York, New York, USA. Electronic address:

Systems biology is a data-heavy field that focuses on systems-wide depictions of biological phenomena necessarily sacrificing a detailed characterization of individual components. As an example, genome-wide protein interaction networks are widely used in systems biology and continuously extended and refined as new sources of evidence become available. Despite the vast amount of information about individual protein structures and protein complexes that has accumulated in the past 50 years in the Protein Data Bank, the data, computational tools, and language of structural biology are not an integral part of systems biology. However, increasing effort has been devoted to this integration, and the related literature is reviewed here. Relationships between proteins that are detected via structural similarity offer a rich source of information not available from sequence similarity, and homology modeling can be used to leverage Protein Data Bank structures to produce 3D models for a significant fraction of many proteomes. A number of structure-informed genomic and cross-species (i.e., virus-host) interactomes will be described, and the unique information they provide will be illustrated with a number of examples. Tissue- and tumor-specific interactomes have also been developed through computational strategies that exploit patient information and through genetic interactions available from increasingly sensitive screens. Strategies to integrate structural information with these alternate data sources will be described. Finally, efforts to link protein structure space with chemical compound space offer novel sources of information in drug design, off-target identification, and the identification of targets for compounds found to be effective in phenotypic screens.
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http://dx.doi.org/10.1016/j.jbc.2021.100562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095114PMC
August 2021

Education and Outreach in Physical Sciences in Oncology.

Trends Cancer 2021 01 7;7(1):3-9. Epub 2020 Nov 7.

Department of Biochemistry and Molecular Biology, Mayo Clinic, Jacksonville, FL, USA; Department of Physiology and Biomedical Engineering, Mayo Clinic, Jacksonville, FL, USA; Department of Transplantation, Mayo Clinic, Jacksonville, FL, USA; Center for Immunotherapeutic Transport Oncophysics, Houston Methodist Research Institute, Houston, TX, USA. Electronic address:

Physical sciences are often overlooked in the field of cancer research. The Physical Sciences in Oncology Initiative was launched to integrate physics, mathematics, chemistry, and engineering with cancer research and clinical oncology through education, outreach, and collaboration. Here, we provide a framework for education and outreach in emerging transdisciplinary fields.
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http://dx.doi.org/10.1016/j.trecan.2020.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7895467PMC
January 2021

Oncoprotein-specific molecular interaction maps (SigMaps) for cancer network analyses.

Nat Biotechnol 2021 02 14;39(2):215-224. Epub 2020 Sep 14.

Department of Systems Biology, Columbia University Irving Medical Center, New York, NY, USA.

Tumor-specific elucidation of physical and functional oncoprotein interactions could improve tumorigenic mechanism characterization and therapeutic response prediction. Current interaction models and pathways, however, lack context specificity and are not oncoprotein specific. We introduce SigMaps as context-specific networks, comprising modulators, effectors and cognate binding-partners of a specific oncoprotein. SigMaps are reconstructed de novo by integrating diverse evidence sources-including protein structure, gene expression and mutational profiles-via the OncoSig machine learning framework. We first generated a KRAS-specific SigMap for lung adenocarcinoma, which recapitulated published KRAS biology, identified novel synthetic lethal proteins that were experimentally validated in three-dimensional spheroid models and established uncharacterized crosstalk with RAB/RHO. To show that OncoSig is generalizable, we first inferred SigMaps for the ten most mutated human oncoproteins and then for the full repertoire of 715 proteins in the COSMIC Cancer Gene Census. Taken together, these SigMaps show that the cell's regulatory and signaling architecture is highly tissue specific.
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http://dx.doi.org/10.1038/s41587-020-0652-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878435PMC
February 2021

Safety and tolerability of Vitamin D3 5000 IU/day in epilepsy.

Epilepsy Behav 2019 05 7;94:195-197. Epub 2019 Apr 7.

Department of Neurology, UCLA School of Medicine, Olive View-UCLA Medical Center, Sylmar, CA 91342, USA.

Purpose: Preclinical and early clinical research indicates that Vitamin D3 may reduce seizures in both animal models and open-label clinical trials.

Methods: This is an initial report of an ongoing pilot study of oral Vitamin D3 5000 IU/day in subjects with drug-resistant epilepsy. After Institutional Review Board (IRB) approval and informed consent, subjects with ;less than one focal onset or generalized tonic-clonic seizure per month were enrolled. Subjects entered a 4-week baseline, followed by a 12-week treatment period. Serum 25, OH Vitamin D3, Blood Urea Nitrogen (BUN), creatinine, and calcium levels were monitored at baseline and at 6 and 12 weeks.

Results: High-dose Vitamin D3 5000 IU/day was well tolerated. Serum 25, OH Vitamin D3 levels increased significantly at six and twelve weeks. Vitamin D insufficiency, defined as a 25, OH Vitamin D3 level of <20 ng/ml normalized in all subjects with insufficient vitamin D levels. Median seizure frequency declined from 5.18 seizures per month to 3.64 seizures per month at 6 weeks and to 4.2 seizures per month at 12 weeks. The median percent change in seizure frequency was -26.9% at six weeks, and -10.7% at 12 weeks (not significant, Wilcoxon Signed Rank Test, P > 0.34).

Conclusions: High-dose oral Vitamin D3, 5000 IU/day was safe and well tolerated in subjects with epilepsy. Vitamin D levels increased significantly at 6 and 12 weeks but never exceeded potentially toxic levels, defined as >100 ng/ml. To reduce variability, we will now recruit subjects who only have three or more seizures per month.
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http://dx.doi.org/10.1016/j.yebeh.2019.03.001DOI Listing
May 2019

A computational interactome and functional annotation for the human proteome.

Elife 2016 10 22;5. Epub 2016 Oct 22.

Center for Computational Biology and Bioinformatics, Department of Systems Biology, Columbia University, New York, United States.

We present a database, PrePPI (Predicting Protein-Protein Interactions), of more than 1.35 million predicted protein-protein interactions (PPIs). Of these at least 127,000 are expected to constitute direct physical interactions although the actual number may be much larger (~500,000). The current PrePPI, which contains predicted interactions for about 85% of the human proteome, is related to an earlier version but is based on additional sources of interaction evidence and is far larger in scope. The use of structural relationships allows PrePPI to infer numerous previously unreported interactions. PrePPI has been subjected to a series of validation tests including reproducing known interactions, recapitulating multi-protein complexes, analysis of disease associated SNPs, and identifying functional relationships between interacting proteins. We show, using Gene Set Enrichment Analysis (GSEA), that predicted interaction partners can be used to annotate a protein's function. We provide annotations for most human proteins, including many annotated as having unknown function.
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http://dx.doi.org/10.7554/eLife.18715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5115866PMC
October 2016

A prospective long-term study of external trigeminal nerve stimulation for drug-resistant epilepsy.

Epilepsy Behav 2015 Jan 11;42:44-7. Epub 2014 Dec 11.

Olive View/UCLA Medical Center, UCLA Department of Neurology, USA; NeuroSigma, Inc., USA. Electronic address:

Background: External trigeminal nerve stimulation (eTNS) is an emerging noninvasive therapy for drug-resistant epilepsy (DRE). We report the long-term safety and efficacy of eTNS after completion of a phase II randomized controlled clinical trial for drug-resistant epilepsy.

Methods: This was a prospective open-label long-term study. Subjects who completed the phase II randomized controlled trial of eTNS for DRE were offered long-term follow-up for 1year. Subjects who were originally randomized to control settings were crossed over to effective device parameters (30s on, 30s off, pulse duration of 250s, frequency of 120Hz). Efficacy was assessed using last observation carried forward or parametric imputation methods for missing data points. Outcomes included change in median seizure frequency, RRATIO, and 50% responder rate.

Results: Thirty-five of 50 subjects from the acute double-blind randomized controlled study continued in the long-term study. External trigeminal nerve stimulation was well tolerated. No serious device-related adverse events occurred through 12months of long-term treatment. At six and twelve months, the median seizure frequency for the original treatment group decreased by -2.39 seizures per month at 6 months (-27.4%) and -3.03 seizures per month at 12 months (-34.8%), respectively, from the initial baseline (p<0.05, signed-rank test). The 50% responder rates at three, six, and twelve months were 36.8% for the treatment group and 30.6% for all subjects.

Conclusion: The results provide long-term evidence that external trigeminal nerve stimulation is a safe and promising long-term treatment for drug-resistant epilepsy.
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http://dx.doi.org/10.1016/j.yebeh.2014.10.029DOI Listing
January 2015

Randomized controlled trial of trigeminal nerve stimulation for drug-resistant epilepsy.

Neurology 2013 Feb 30;80(9):786-91. Epub 2013 Jan 30.

Departments of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.

Objective: To explore the safety and efficacy of external trigeminal nerve stimulation (eTNS) in patients with drug-resistant epilepsy (DRE) using a double-blind randomized controlled trial design, and to test the suitability of treatment and control parameters in preparation for a phase III multicenter clinical trial.

Methods: This is a double-blind randomized active-control trial in DRE. Fifty subjects with 2 or more partial onset seizures per month (complex partial or tonic-clonic) entered a 6-week baseline period, and then were evaluated at 6, 12, and 18 weeks during the acute treatment period. Subjects were randomized to treatment (eTNS 120 Hz) or control (eTNS 2 Hz) parameters.

Results: At entry, subjects were highly drug-resistant, averaging 8.7 seizures per month (treatment group) and 4.8 seizures per month (active controls). On average, subjects failed 3.35 antiepileptic drugs prior to enrollment, with an average duration of epilepsy of 21.5 years (treatment group) and 23.7 years (active control group), respectively. eTNS was well-tolerated. Side effects included anxiety (4%), headache (4%), and skin irritation (14%). The responder rate, defined as >50% reduction in seizure frequency, was 30.2% for the treatment group vs 21.1% for the active control group for the 18-week treatment period (not significant, p = 0.31, generalized estimating equation [GEE] model). The treatment group experienced a significant within-group improvement in responder rate over the 18-week treatment period (from 17.8% at 6 weeks to 40.5% at 18 weeks, p = 0.01, GEE). Subjects in the treatment group were more likely to respond than patients randomized to control (odds ratio 1.73, confidence interval 0.59-0.51). eTNS was associated with reductions in seizure frequency as measured by the response ratio (p = 0.04, analysis of variance [ANOVA]), and improvements in mood on the Beck Depression Inventory (p = 0.02, ANOVA).

Conclusions: This study provides preliminary evidence that eTNS is safe and may be effective in subjects with DRE. Side effects were primarily limited to anxiety, headache, and skin irritation. These results will serve as a basis to inform and power a larger multicenter phase III clinical trial.

Classification Of Evidence: This phase II study provides Class II evidence that trigeminal nerve stimulation may be safe and effective in reducing seizures in people with DRE.
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http://dx.doi.org/10.1212/WNL.0b013e318285c11aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598453PMC
February 2013

Protein kinase Cθ C2 domain is a phosphotyrosine binding module that plays a key role in its activation.

J Biol Chem 2012 Aug 11;287(36):30518-28. Epub 2012 Jul 11.

Department of Chemistry, University of Illinois, Chicago, IL 60607, USA.

Protein kinase Cθ (PKCθ) is a novel PKC that plays a key role in T lymphocyte activation. To understand how PKCθ is regulated in T cells, we investigated the properties of its N-terminal C2 domain that functions as an autoinhibitory domain. Our measurements show that a Tyr(P)-containing peptide derived from CDCP1 binds the C2 domain of PKCθ with high affinity and activates the enzyme activity of the intact protein. The Tyr(P) peptide also binds the C2 domain of PKCδ tightly, but no enzyme activation was observed with PKCδ. Mutations of PKCθ-C2 residues involved in Tyr(P) binding abrogated the enzyme activation and association of PKCθ with Tyr-phosphorylated full-length CDCP1 and severely inhibited the T cell receptor/CD28-mediated activation of a PKCθ-dependent reporter gene in T cells. Collectively, these studies establish the C2 domain of PKCθ as a Tyr(P)-binding domain and suggest that the domain may play a major role in PKCθ activation via its Tyr(P) binding.
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http://dx.doi.org/10.1074/jbc.M112.391557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436300PMC
August 2012

Acute and long-term safety of external trigeminal nerve stimulation for drug-resistant epilepsy.

Epilepsy Behav 2011 Nov 29;22(3):574-6. Epub 2011 Sep 29.

Department of Neurology, Olive View Internal Medicine Program, UCLA School of Medicine, Los Angeles, CA 90095, USA.

Trigeminal nerve stimulation (TNS) is a novel therapy for drug-resistant epilepsy. We report in detail the safety of external TNS (eTNS), focusing on acute and long-term heart rate and systolic and diastolic blood pressure in response to TNS from the pilot feasibility study. The data indicate that eTNS of the infraorbital and supraorbital branches of the trigeminal nerve is safe and well tolerated.
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http://dx.doi.org/10.1016/j.yebeh.2011.06.024DOI Listing
November 2011

Genome-wide structural analysis reveals novel membrane binding properties of AP180 N-terminal homology (ANTH) domains.

J Biol Chem 2011 Sep 2;286(39):34155-63. Epub 2011 Aug 2.

Department of Pharmacology, Columbia University, New York, New York 11032, USA.

An increasing number of cytosolic proteins are shown to interact with membrane lipids during diverse cellular processes, but computational prediction of these proteins and their membrane binding behaviors remains challenging. Here, we introduce a new combinatorial computation protocol for systematic and robust functional prediction of membrane-binding proteins through high throughput homology modeling and in-depth calculation of biophysical properties. The approach was applied to the genomic scale identification of the AP180 N-terminal homology (ANTH) domain, one of the modular lipid binding domains, and prediction of their membrane binding properties. Our analysis yielded comprehensive coverage of the ANTH domain family and allowed classification and functional annotation of proteins based on the differences in local structural and biophysical features. Our analysis also identified a group of plant ANTH domains with unique structural features that may confer novel functionalities. Experimental characterization of a representative member of this subfamily confirmed its unique membrane binding mechanism and unprecedented membrane deforming activity. Collectively, these studies suggest that our new computational approach can be applied to genome-wide functional prediction of other lipid binding domains.
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http://dx.doi.org/10.1074/jbc.M111.265611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3190782PMC
September 2011

Enhancing medical students' conceptions of the CanMEDS Health Advocate Role through international service-learning and critical reflection: a phenomenological study.

Med Teach 2010 ;32(12):977-82

The University of British Columbia, Canada.

Background: Medical students are expressing increasing interest in international experiences in low-income countries where there are pronounced inequities in health and socio-economic development.

Aim: We carried out a detailed exploration of the international service-learning (ISL) experience of three medical students and the value of critical reflection as a pedagogical approach to enhance medical students' conceptions of the Canadian Medical Education Directions for Specialists (CanMEDS) Health Advocate Role.

Method: A phenomenological approach enabled us to study in considerable depth the students' experience from their perspective. Students kept reflective journals and wrote essays including detailed accounts of their experiences. The content of the students' journals and essays was analyzed using the critical incident technique.

Results: Students noted an increasingly meaningful sense of what it means to be vulnerable and marginalized, a heightened level of awareness of the social determinants of health and the related importance of community engagement, and a deeper appreciation of the health advocate role and key concepts embedded within it.

Conclusion: This in-depth phenomenological study focused on the detailed experiences of three students from whom we learned that social justice-oriented approaches to service-learning, coupled with critical reflection, provide potentially viable pedagogical approaches for learning the health advocate role. How this experience will affect the students' future medical practice is yet unknown.
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http://dx.doi.org/10.3109/01421590903394579DOI Listing
April 2011

High-throughput computational structure-based characterization of protein families: START domains and implications for structural genomics.

J Struct Funct Genomics 2010 Mar 11;11(1):51-9. Epub 2010 Apr 11.

Department of Pharmacology, College of Physicians and Surgeons of Columbia University, Center for Computational Biology and Bioinformatics, 630 West 168th St. PH 7W 313, New York, NY 10032, USA.

SkyLine, a high-throughput homology modeling pipeline tool, detects and models true sequence homologs to a given protein structure. Structures and models are stored in SkyBase with links to computational function annotation, as calculated by MarkUs. The SkyLine/SkyBase/MarkUs technology represents a novel structure-based approach that is more objective and versatile than other protein classification resources. This structure-centric strategy provides a multi-dimensional organization and coverage of protein space at the levels of family, function, and genome. The concept of "modelability", the ability to model sequences on related structures, provides a reliable criterion for membership in a protein family ("leverage") and underlies the unique success of this approach. The overall procedure is illustrated by its application to START domains, which comprise a Biomedical Theme for the Northeast Structural Genomics Consortium as part of the Protein Structure Initiative. START domains are typically involved in the non-vesicular transport of lipids. While 19 experimentally determined structures are available, the family, whose evolutionary hierarchy is not well determined, is highly sequence diverse, and the ligand-binding potential of many family members is unknown. The SkyLine/SkyBase/MarkUs approach provides significant insights and predicts: (1) many more family members (approximately 4,000) than any other resource; (2) the function for a large number of unannotated proteins; (3) instances of START domains in genomes from which they were thought to be absent; and (4) the existence of two types of novel proteins, those containing dual START domain and those containing N-terminal START domains.
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http://dx.doi.org/10.1007/s10969-010-9086-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2881152PMC
March 2010

Molecular dynamics simulations of PIP2 and PIP3 in lipid bilayers: determination of ring orientation, and the effects of surface roughness on a Poisson-Boltzmann description.

Biophys J 2009 Jul;97(1):155-63

Department of Pharmacology, Presbyterian Hospital, Columbia University, New York, New York, USA.

Molecular dynamics (MD) simulations of phosphatidylinositol (4,5)-bisphosphate (PIP2) and phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC) bilayers indicate that the inositol rings are tilted approximately 40 degrees with respect to the bilayer surface, as compared with 17 degrees for the P-N vector of POPC. Multiple minima were obtained for the ring twist (analogous to roll for an airplane). The phosphates at position 1 of PIP2 and PIP3 are within an Angström of the plane formed by the phosphates of POPC; lipids in the surrounding shell are depressed by 0.5-0.8 A, but otherwise the phosphoinositides do not substantially perturb the bilayer. Finite size artifacts for ion distributions are apparent for systems of approximately 26 waters/lipid, but, based on simulations with a fourfold increase of the aqueous phase, the phosphoinositide positions and orientations do not show significant size effects. Electrostatic potentials evaluated from Poisson-Boltzmann (PB) calculations show a strong dependence of potential height and ring orientation, with the maxima on the -25 mV surfaces (17.1 +/- 0.1 A for PIP2 and 19.4 +/- 0.3 A for PIP3) occurring near the most populated orientations from MD. These surfaces are well above the background height of 10 A estimated for negatively charged cell membranes, as would be expected for lipids involved in cellular signaling. PB calculations on microscopically flat bilayers yield similar maxima as the MD-based (microscopically rough) systems, but show less fine structure and do not clearly indicate the most probable regions. Electrostatic free energies of interaction with pentalysine are also similar for the rough and flat systems. These results support the utility of a rigid/flat bilayer model for PB-based studies of PIP2 and PIP3 as long as the orientations are judiciously chosen.
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http://dx.doi.org/10.1016/j.bpj.2009.04.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711355PMC
July 2009

Pilonidal sinus disease.

Authors:
Diana Murray

Nurs Stand 2009 Feb 18-24;23(24):51

Victoria Infirmary, Glasgow.

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http://dx.doi.org/10.7748/ns.23.24.51.s49DOI Listing
June 2009

Trigeminal nerve stimulation for epilepsy: long-term feasibility and efficacy.

Neurology 2009 Mar;72(10):936-8

Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.

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http://dx.doi.org/10.1212/01.wnl.0000344181.97126.b4DOI Listing
March 2009

Outcome of a workshop on applications of protein models in biomedical research.

Structure 2009 Feb;17(2):151-9

Swiss Institute of Bioinformatics, Biozentrum, University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland.

We describe the proceedings and conclusions from the "Workshop on Applications of Protein Models in Biomedical Research" (the Workshop) that was held at the University of California, San Francisco on 11 and 12 July, 2008. At the Workshop, international scientists involved with structure modeling explored (i) how models are currently used in biomedical research, (ii) the requirements and challenges for different applications, and (iii) how the interaction between the computational and experimental research communities could be strengthened to advance the field.
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http://dx.doi.org/10.1016/j.str.2008.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2739730PMC
February 2009

Structure of an acetyl-CoA binding protein from Staphylococcus aureus representing a novel subfamily of GCN5-related N-acetyltransferase-like proteins.

J Struct Funct Genomics 2008 Dec 16;9(1-4):7-20. Epub 2008 Aug 16.

Washington State University Tri-Cities, Richland, WA 99354, USA,

We have determined the solution NMR structure of SACOL2532, a putative GCN5-like N-acetyltransferase (GNAT) from Staphylococcus aureus. SACOL2532 was shown to bind both CoA and acetyl-CoA, and structures with and without bound CoA were determined. Based on analysis of the structure and sequence, a subfamily of small GCN5-related N-acetyltransferase (GNAT)-like proteins can be defined. Proteins from this subfamily, which is largely congruent with COG2388, are characterized by a cysteine residue in the acetyl-CoA binding site near the acetyl group, by their small size in relation to other GNATs, by a lack of obvious substrate binding site, and by a distinct conformation of bound CoA in relation to other GNATs. Subfamily members are found in many bacterial and eukaryotic genomes, and in some archaeal genomes. Whereas other GNATs transfer the acetyl group of acetyl-CoA directly to an aliphatic amine, the presence of the conserved cysteine residue suggests that proteins in the COG2388 GNAT-subfamily transfer an acetyl group from acetyl-CoA to one or more presently unidentified aliphatic amines via an acetyl (cysteine) enzyme intermediate. The apparent absence of a substrate-binding region suggests that the substrate is a macromolecule, such as another protein, or that a second protein subunit providing a substrate-binding region must combine with SACOL2532 to make a fully functional N-acetyl transferase.
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http://dx.doi.org/10.1007/s10969-008-9041-zDOI Listing
December 2008

The "electrostatic-switch" mechanism: Monte Carlo study of MARCKS-membrane interaction.

Biophys J 2008 Aug 23;95(4):1745-57. Epub 2008 May 23.

Department of Physical Chemistry and The Fritz Haber Research Center, Hebrew University of Jerusalem, Jerusalem 91904, Israel.

The binding of the myristoylated alanine-rich C kinase substrate (MARCKS) to mixed, fluid, phospholipid membranes is modeled with a recently developed Monte Carlo simulation scheme. The central domain of MARCKS is both basic (zeta = +13) and hydrophobic (five Phe residues), and is flanked with two long chains, one ending with the myristoylated N-terminus. This natively unfolded protein is modeled as a flexible chain of "beads" representing the amino acid residues. The membranes contain neutral (zeta = 0), monovalent (zeta = -1), and tetravalent (zeta = -4) lipids, all of which are laterally mobile. MARCKS-membrane interaction is modeled by Debye-Hückel electrostatic potentials and semiempirical hydrophobic energies. In agreement with experiment, we find that membrane binding is mediated by electrostatic attraction of the basic domain to acidic lipids and membrane penetration of its hydrophobic moieties. The binding is opposed by configurational entropy losses and electrostatic membrane repulsion of the two long chains, and by lipid demixing upon adsorption. The simulations provide a physical model for how membrane-adsorbed MARCKS attracts several PIP(2) lipids (zeta = -4) to its vicinity, and how phosphorylation of the central domain (zeta = +13 to zeta = +7) triggers an "electrostatic switch", which weakens both the membrane interaction and PIP(2) sequestration. This scheme captures the essence of "discreteness of charge" at membrane surfaces and can examine the formation of membrane-mediated multicomponent macromolecular complexes that function in many cellular processes.
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http://dx.doi.org/10.1529/biophysj.108.132522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2483737PMC
August 2008

A molecular switch required for retrovirus assembly participates in the hexagonal immature lattice.

EMBO J 2008 May 10;27(9):1411-20. Epub 2008 Apr 10.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

In the Rous sarcoma virus (RSV) Gag protein, the 25 amino-acid residues of the p10 domain immediately upstream of the CA domain are essential for immature particle formation. We performed systematic mutagenesis on this region and found excellent correlation between the amino-acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. We introduced exogenous cysteine residues that were predicted to form disulphide bonds across the dimer interface. Upon oxidation of immature particles, a disulphide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer indicates that the N-terminal domains of CA must expand relative to the murine leukaemia virus mature hexamer to accommodate the p10 contact; this expansion is strikingly similar to recent cryotomography results for immature human immunodeficiency virus particles.
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http://dx.doi.org/10.1038/emboj.2008.71DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374847PMC
May 2008

Measuring the quality of pediatric day surgery care.

J Healthc Qual 2007 Nov-Dec;29(6):36-44, 49

Department of Anesthesia, BC Children's Hospital, Vancouver, Canada.

This brief report describes the process of developing a valid and reliable questionnaire for quality measurement of pediatric day surgery care from the families' perspective. Questionnaire items were generated through a literature search and interviews with clinicians and parents. A computer-assisted telephone interview was used to administer the questionnaire to 448 parents within 72 hours of patient discharge. Tests of reliability and validity were administered, and questionnaire items were improved or omitted based on the results. The investigators plan to readminister the improved questionnaire to confirm its validity and reliability.
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http://dx.doi.org/10.1111/j.1945-1474.2007.tb00223.xDOI Listing
March 2008

The structural basis of novel endosome anchoring activity of KIF16B kinesin.

EMBO J 2007 Aug 19;26(15):3709-19. Epub 2007 Jul 19.

Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA.

KIF16B is a newly identified kinesin that regulates the intracellular motility of early endosomes. KIF16B is unique among kinesins in that its cargo binding is mediated primarily by the strong interaction of its PX domain with endosomal lipids. To elucidate the structural basis of this unique endosomal anchoring activity of KIF16B-PX, we determined the crystal structure of the PX domain and performed in vitro and cellular membrane binding measurements for KIF16B-PX and mutants. The most salient structural feature of KIF16B-PX is that two neighboring residues, L1248 and F1249, on the membrane-binding surface form a protruding hydrophobic stalk with a large solvent-accessible surface area. This unique structure, arising from the complementary stacking of the two side chains and the local conformation, allows strong hydrophobic membrane interactions and endosome tethering. The presence of similar hydrophobic pairs in the amino-acid sequences of other membrane-binding domains and proteins suggests that the same structural motif may be shared by other membrane-binding proteins, whose physiological functions depend on strong hydrophobic membrane interactions.
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http://dx.doi.org/10.1038/sj.emboj.7601800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1949010PMC
August 2007

Structural and membrane binding analysis of the Phox homology domain of Bem1p: basis of phosphatidylinositol 4-phosphate specificity.

J Biol Chem 2007 Aug 20;282(35):25737-47. Epub 2007 Jun 20.

Department of Chemistry, University of Illinois, Chicago, Illinois 60607-7061, USA.

Phox homology (PX) domains, which have been identified in a variety of proteins involved in cell signaling and membrane trafficking, have been shown to interact with phosphoinositides (PIs) with different affinities and specificities. To elucidate the structural origin of the diverse PI specificity of PX domains, we determined the crystal structure of the PX domain from Bem1p that has been reported to bind phosphatidylinositol 4-phosphate (PtdIns(4)P). We also measured the membrane binding properties of the PX domain and its mutants by surface plasmon resonance and monolayer techniques and calculated the electrostatic potentials for the PX domain in the absence and presence of bound PtdIns(4)P. The Bem1p PX domain contains a signature PI-binding site optimized for PtdIns(4)P binding and also harbors basic and hydrophobic residues on the membrane-binding surface. The membrane binding of the Bem1p PX domain is initiated by nonspecific electrostatic interactions between the cationic membrane-binding surface of the domain and anionic membrane surfaces, followed by the membrane penetration of hydrophobic residues. Unlike other PX domains, the Bem1p PX domain has high intrinsic membrane penetrating activity in the absence of PtdIns(4)P, suggesting that the partial membrane penetration may occur before specific PtdIns(4)P binding and last after the removal of PtdIns(4)P under certain conditions. This structural and functional study of the PtdIns(4)P-binding Bem1p PX domain provides new insight into the diverse PI specificities and membrane-binding mechanisms of PX domains.
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http://dx.doi.org/10.1074/jbc.M702861200DOI Listing
August 2007

Binding of phosphoinositide-specific phospholipase C-zeta (PLC-zeta) to phospholipid membranes: potential role of an unstructured cluster of basic residues.

J Biol Chem 2007 Jun 12;282(22):16644-53. Epub 2007 Apr 12.

Cell Signaling Laboratory, Wales Heart Research Institute, UK.

Phospholipase C-zeta (PLC-zeta) is a sperm-specific enzyme that initiates the Ca2+ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-delta1, but lacks the PH domain that anchors PLC-delta1 to phosphatidylinositol 4,5-bisphosphate, PIP2. Thus it is unclear how PLC-zeta interacts with membranes. The linker region between the X and Y catalytic domains of PLC-zeta, however, contains a cluster of basic residues not present in PLC-delta1. Application of electrostatic theory to a homology model of PLC-zeta suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC-zeta to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP2 into the 2:1 PC/PS vesicles increases K about 10-fold, to 5x10(3) M-1, a biologically relevant value. Expressed fragments corresponding to the PLC-zeta X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge=+7) within the linker region, PLC-zeta-(374-385), binds to PC/PS vesicles with higher affinity than PLC-zeta, but its binding is less sensitive to incorporating PIP2. The acidic residues flanking this basic cluster in PLC-zeta may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP2 adjacent to the catalytic domain.
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http://dx.doi.org/10.1074/jbc.M701072200DOI Listing
June 2007

Electrostatic interactions drive membrane association of the human immunodeficiency virus type 1 Gag MA domain.

J Virol 2007 Jun 28;81(12):6434-45. Epub 2007 Mar 28.

Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA.

The assembly of most retroviruses occurs at the plasma membrane. Membrane association is directed by MA, the N-terminal domain of the Gag structural protein. For human immunodeficiency virus type 1 (HIV-1), this association is mediated in part by a myristate fatty acid modification. Conflicting evidence has been presented on the relative importance of myristoylation, of ionic interactions between protein and membrane, and of Gag multimerization in membrane association in vivo. We addressed these questions biochemically by determining the affinity of purified myristoylated HIV-1 MA for liposomes of defined composition, both for monomeric and for dimeric forms of the protein. Myristoylation increases the barely detectable intrinsic affinity of the apo-protein for liposomes by only 10-fold, and the resulting affinity is still weak, similar to that of the naturally nonmyristoylated MA of Rous sarcoma virus. Membrane binding of HIV-1 MA is absolutely dependent on the presence of negatively charged lipid and is abrogated at high ionic strength. Forced dimerization of MA increases its membrane affinity by several orders of magnitude. When green fluorescent protein fusions of monomeric or dimeric MA are expressed in cells, the dimeric but not the monomeric protein becomes strongly membrane associated. Computational modeling supports these results and suggests a molecular mechanism for the modest effect of myristoylation on binding, wherein the membrane provides a hydrophobic environment for the myristate that is energetically similar to that provided by the protein. Overall, the results imply that the driving force for membrane association stems largely from ionic interactions between multimerized Gag and negatively charged phospholipids.
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http://dx.doi.org/10.1128/JVI.02757-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1900125PMC
June 2007

Strategies for high-throughput comparative modeling: applications to leverage analysis in structural genomics and protein family organization.

Proteins 2007 Mar;66(4):766-77

Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.

The technological breakthroughs in structural genomics were designed to facilitate the solution of a sufficient number of structures, so that as many protein sequences as possible can be structurally characterized with the aid of comparative modeling. The leverage of a solved structure is the number and quality of the models that can be produced using the structure as a template for modeling and may be viewed as the "currency" with which the success of a structural genomics endeavor can be measured. Moreover, the models obtained in this way should be valuable to all biologists. To this end, at the Northeast Structural Genomics Consortium (NESG), a modular computational pipeline for automated high-throughput leverage analysis was devised and used to assess the leverage of the 186 unique NESG structures solved during the first phase of the Protein Structure Initiative (January 2000 to July 2005). Here, the results of this analysis are presented. The number of sequences in the nonredundant protein sequence database covered by quality models produced by the pipeline is approximately 39,000, so that the average leverage is approximately 210 models per structure. Interestingly, only 7900 of these models fulfill the stringent modeling criterion of being at least 30% sequence-identical to the corresponding NESG structures. This study shows how high-throughput modeling increases the efficiency of structure determination efforts by providing enhanced coverage of protein structure space. In addition, the approach is useful in refining the boundaries of structural domains within larger protein sequences, subclassifying sequence diverse protein families, and defining structure-based strategies specific to a particular family.
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http://dx.doi.org/10.1002/prot.21191DOI Listing
March 2007

Successful implementation of the National Institutes of Health Stroke Scale on a stroke/neurovascular unit.

J Neurosci Nurs 2006 Sep;38(4 Suppl):309-15

Providence Stroke Center, Providence St. Vincent Medical Center, Portland, OR, USA.

The National Institutes of Health Stroke Scale (NIHSS) is accepted as the definitive clinical examination to assess stroke severity. This project examined barriers to implementation and NIHSS use by registered nurses on a stroke/neurovascular Unit. Staff members were surveyed to determine nurse-perceived barriers to the routine use of the NIHSS. Survey results were used to create interventions including staff education, emphasis on NIHSS assessment during interdisciplinary rounds, and use of pocket cards. When the survey was redistributed 9 months later to verify results of the quality improvement initiative and guide further interventions, NIHSS assessment had increased from 12% to 69%. NIHSS scores have been linked to an existing outcomes database to monitor acute stroke treatment and inpatient management outcomes.
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http://dx.doi.org/10.1097/01376517-200609000-00007DOI Listing
September 2006

Unique membrane interaction mode of group IIF phospholipase A2.

J Biol Chem 2006 Oct 23;281(43):32741-54. Epub 2006 Aug 23.

Department of Chemistry (M/C 111), University of Illinois, 845 West Taylor Street, Chicago, IL 60607, USA.

The mechanisms by which secretory phospholipases A(2) (PLA(2)s) exert cellular effects are not fully understood. Group IIF PLA(2) (gIIFPLA(2)) is a structurally unique secretory PLA(2) with a long C-terminal extension. Homology modeling suggests that the membrane-binding surface of this acidic PLA(2) contains hydrophobic residues clustered near the C-terminal extension. Vesicle leakage and monolayer penetration measurements showed that gIIFPLA(2) had a unique ability to penetrate and disrupt compactly packed monolayers and bilayers whose lipid composition recapitulates that of the outer plasma membrane of mammalian cells. Fluorescence imaging showed that gIIFPLA(2) could also readily enter and deform plasma membrane-mimicking giant unilamellar vesicles. Mutation analysis indicates that hydrophobic residues (Tyr(115), Phe(116), Val(118), and Tyr(119)) near the C-terminal extension are responsible for these activities. When gIIFPLA(2) was exogenously added to HEK293 cells, it initially bound to the plasma membrane and then rapidly entered the cells in an endocytosis-independent manner, but the cell entry did not lead to a significant degree of phospholipid hydrolysis. GIIFPLA(2) mRNA was detected endogenously in human CD4(+) helper T cells after in vitro stimulation and exogenously added gIIFPLA(2) inhibited the proliferation of a T cell line, which was not seen with group IIA PLA(2). Collectively, these data suggest that unique membrane-binding properties of gIIFPLA(2) may confer special functionality on this secretory PLA(2) under certain physiological conditions.
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http://dx.doi.org/10.1074/jbc.M606311200DOI Listing
October 2006

The role of electrostatics in protein-membrane interactions.

Biochim Biophys Acta 2006 Aug 14;1761(8):812-26. Epub 2006 Jul 14.

Weill Medical College of Cornell University, New York, NY 10021, USA.

Many experimental, structural and computational studies have established the importance of nonspecific electrostatics as a driving force for peripheral membrane association. Here we focus on this component of protein/membrane interactions by using examples ranging from phosphoinositide signaling to retroviral assembly. We stress the utility of the collaboration of experiment and theory in identifying and quantifying the role of electrostatics not only in contributing to membrane association, but also in affecting subcellular targeting, in the control of membrane binding, and in the organization of proteins and lipids at membrane surfaces.
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http://dx.doi.org/10.1016/j.bbalip.2006.07.002DOI Listing
August 2006

Computational analysis of the membrane association of group IIA secreted phospholipases A2: a differential role for electrostatics.

Biochemistry 2006 Feb;45(8):2584-98

Department of Microbiology and Immunology and the Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York 10021, USA.

Secreted phospholipases A2 (sPLA2's) are enzymes that hydrolyze glycerophospholipids at the sn-2 position, which leads to the production of lipid mediators of many cellular processes. These interfacial enzymes are regulated by their lipid specificity at two levels: membrane binding and substrate recognition. Different sPLA2's utilize different combinations of electrostatic and hydrophobic interactions to adsorb to membrane surfaces, which results in the wide range of membrane binding behaviors observed. Here, the finite difference Poisson Boltzmann (FDPB) method is used to quantitatively analyze the contribution of electrostatic interactions to the membrane association of two highly basic group II sPLA2's: Agkistrodon piscivorus piscivorus (AppD49) sPLA2 and nonpancreatic human group IIA (hGIIA) sPLA2. The calculations predict how membrane binding is affected by ionic strength, membrane composition, substitutions of residues in the enzymes, and the presence of calcium in the active site. In addition, the results provide molecular models for the membrane-associated forms of the enzymes. Furthermore, these models account for (1) changes in orientation and protonation state of both the native and charge reversal forms of the enzymes at the membrane surface and (2) the effect of protein/vesicle aggregation, as observed for hGIIA sPLA2. Importantly, the modeling quantitatively describes the complex membrane binding behaviors of these interfacial enzymes in terms of simple physical forces and provides structural information that is difficult to obtain experimentally. The computational analysis shows that nonspecific electrostatic interactions not only play a major role in recruiting these enzymes to membrane surfaces but also orient the enzymes for productive catalysis at the membrane interface.
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http://dx.doi.org/10.1021/bi051901tDOI Listing
February 2006

Plasma membrane phosphoinositide organization by protein electrostatics.

Nature 2005 Dec;438(7068):605-11

Department of Physiology and Biophysics, Health Sciences Center, Stony Brook University, Stony Brook, New York 11794, USA.

Phosphatidylinositol 4,5-bisphosphate (PIP2), which comprises only about 1% of the phospholipids in the cytoplasmic leaflet of the plasma membrane, is the source of three second messengers, activates many ion channels and enzymes, is involved in both endocytosis and exocytosis, anchors proteins to the membrane through several structured domains and has other roles. How can a single lipid in a fluid bilayer regulate so many distinct physiological processes? Spatial organization might be the key to this. Recent studies suggest that membrane proteins concentrate PIP2 and, in response to local increases in intracellular calcium concentration, release it to interact with other biologically important molecules.
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http://dx.doi.org/10.1038/nature04398DOI Listing
December 2005
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