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The human optic nerve carries signals from the retina to the visual cortex of the brain. Each optic nerve is comprised of approximately one million nerve fibers that are organized into bundles of 800-1200 fibers surrounded by connective tissue and supportive glial cells. Damage to the optic nerve contributes to a number of blinding diseases including: glaucoma, neuromyelitis optica, optic neuritis, and neurofibromatosis; however, the molecular mechanisms of optic nerve damage and death are incompletely understood. Herein we present high spatial resolution MALDI imaging mass spectrometry (IMS) analysis of lipids and proteins to define the molecular anatomy of the human optic nerve. The localization of a number of lipids was observed in discrete anatomical regions corresponding to myelinated and unmyelinated nerve regions as well as to supporting connective tissue, glial cells, and blood vessels. A protein fragment from vimentin, a known intermediate filament marker for astrocytes, was observed surrounding nerved fiber bundles in the lamina cribrosa region. S100B was also found in supporting glial cell regions in the prelaminar region, and the hemoglobin alpha subunit was observed in blood vessel areas. The molecular anatomy of the optic nerve defined by MALDI IMS provides a firm foundation to study biochemical changes in blinding human diseases. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The article offers information on the metabolic serine hydrolases and their functions in mammalian physiology and disease. It describes intracellular and extracellular neutral lipases. It also describes phospholipase A2 Enzymes along with peptidases. Protein and glycan modifying hydrolases is also discussed in the article.

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly wrapped multilamellar structure around neurons enabling rapid and efficient signal propagation. The gene of oligodendrocyte lineage (golli) gives rise to a variety of developmentally regulated splice isoforms of myelin basic protein (MBP), denoted golli for early forms and classic for later ones. In mature myelin, the predominant splice isoform of classic MBP is 18.5 kDa; its central role is to maintain the structural integrity of the myelin sheath, by holding together the apposing cytoplasmic leaflets of the oligodendrocyte membrane in a tight, spiral, multilamellar arrangement. This protein's extreme physicochemical properties, net charge of +19 at neutral pH, low proportion of hydrophobic residues, alternating regions of predicted intrinsic disorder and order, induced folding upon association with membranes and other proteins, and diversification via combinatorial post-translational modifications, define not only its role as a molecular Velcro in compact myelin, but as a multifunctional hub that may also bind to a number of other proteins and small molecule ligands in myelinating oligodendrocytes. In particular, MBP may link the underlying cytoskeleton and proteins containing SH3 domains to the membrane, allowing it to transduce transmembrane signals to the cytosol. These associations are facilitated by MBP being an intrinsically disordered protein, creating a large effective protein surface, and by the formation of transient and/or induced ordered secondary structure elements for molecular recognition. These processes can be modulated by a molecular barcode of numerous post-translational modifications and interactions with proteins such as calmodulin. In the human demyelinating disease multiple sclerosis, an aberrant pattern of modifications may contribute to demyelination and confound inherent attempts at repair. The conformational dynamics of the various isoforms and modified variants of MBP and their interactions with other proteins-potentially allow them to participate in events coupling extracellular signals to cytoskeletal organization during myelination or remyelination. Various biophysical and cell biological approaches are beginning to elucidate these properties of MBP and are leading to a new understanding of the role of this protein as a linker and/or hub in structural and signaling networks in oligodendrocytes and myetin. [ABSTRACT FROM AUTHOR]

The Wilson disease protein or ATP7B is a P1B-type ATPase involved in human copper homeostasis. The extended N-terminus of ATP7B protrudes into the cytosol and contains six Cu(I) binding domains. This report presents the NMR structure of the polypeptide consisting of soluble Cu(I) binding domains 3 and 4. The two domains exhibit ferredoxin-like folds, are linked by a flexible loop, and act independently of one another. Domains 3 and 4 tend to aggregate in a concentration-dependent manner involving nonspecific intermolecular interactions. Both domains can be loaded with Cu(I) when provided as an acetonitrile complex or by the chaperone HAH1. HAH1 forms a 70% complex with domain 4 that is in fast exchange with the free protein in solution. The ability of HAH1 to form a complex only with some domains of ATP7B is an interesting property of this class of proteins and may have a signaling role in the function of the ATPases. [ABSTRACT FROM AUTHOR]

Constant pressure experiments were performed using track-etched polycarbonate membranes and rod-shaped bacteria (viz., Brevundimonas diminuta and Serratia marcescens) to study flux decline and backwashing during the early stages of microfiltration. The intermediate blocking law originally derived for spherical particles was modified to account for the approximate cylindrical shape of the selected bacteria. A deposition factor was introduced to empirically account for the morphology of bacterial deposits. The initial stages of flux decline prior to the secretion of new extracellular polymeric substances (EPS) was quantitatively described by the intermediate blocking law before transitioning to cake filtration at later times. Scanning electron microscopy (SEM) provided additional visual evidence that bacteria simultaneously deposited directly on the membrane and on each other during early stages of filtration as assumed by the intermediate blocking law. Empirical deposition factors decreased with initial permeate flux indicating its effect on bacteria deposition patterns, which was also confirmed by SEM. Bacteria were easily removed following short filtration times before significant secretion of new EPS by simply rinsing with ultra pure water, thereby completely restoring the clean membrane permeability. In contrast, this rinsing procedure did not completely recover the membrane permeability following longer durations when significant amounts of new EPS proteins and polysaccharides were secreted. Consequently, backwashing effectiveness during water and wastewater microfiltration will be high prior to EPS production whereas flux recovery may not be possible solely by hydrodynamic means once EPS are secreted. [ABSTRACT FROM AUTHOR]

An optimized analytical expression profiling strategy based on gel-free multidimensional protein identification technology (MudPIT) is reported for the systematic investigation of biochemical (mal)-adaptations associated with healthy and diseased heart tissue. Enhanced shotgun proteomic detection coverage and improved biological inference is achieved by pre-fractionation of excised mouse cardiac muscle into subcellular components, with each organellar fraction investigated exhaustively using multiple repeat MudPIT analyses. Functional-enrichment, high-confidence identification, and relative quantification of hundreds of organelle- and tissue-specific proteins are achieved readily, including detection of low abundance transcriptional regulators, signaling factors, and proteins linked to cardiac disease. Important technical issues relating to data validation, including minimization of artifacts stemming from biased under-sampling and spurious false discovery, together with suggestions for further fine-tuning of sample preparation, are discussed. A framework for follow-up bioinformatic examination, pattern recognition, and data mining is also presented in the context of a stringent application of MudPIT for probing fundamental aspects of heart muscle physiology as well as the discovery of perturbations associated with heart failure. [Copyright &y& Elsevier]

The metabolism of long-chain fatty acids in brain and their incorporation into signaling molecules such as diacylglycerol and LPA and into structural components of membranes, including myelin, requires activation by long-chain acyl-CoA synthetase (ACSL). Because ACSL3 and ACSL6 are the predominant ACSL isoforms in brain, we cloned and characterized these isoforms from rat brain and identified a novel ACSL6 clone (ACSL6H_v2). ACSL6H_v2 and the previously reported ACSL6H_v1 represent splice variants that include exon 13 or 14, respectively. Homologue sequences of both of these variants are present in the human and mouse databases. ACSL3, ACSL6H_v1, and ACSL6H_v2 with Flag-epitopes at the C-termini were expressed in Escherichia coli and purified on Flag-affinity columns. The three recombinant proteins were characterized. Compared to ACSL4, another brain isoform, ACSL3, ACSL6H_v1, and ACSL6_v2 showed similarities in kinetic values for CoA, palmitate, and arachidonate, but their apparent Km values for oleate were 4- to 6-fold lower than for ACSL4. In a direct competition assay with palmitate, all the polyunsaturated fatty acids tested were strong competitors only for ACSL4 with IC50 values of 0.5 to 5 μM. DHA was also strongly preferred by ACSL6H_v2. The apparent Km value for ATP of ACSL6H_v1 was 8-fold higher than that of ACSL6H_v2. ACSL3 and the two variants of ACSL6 were more resistant than ACSL4 to heat inactivation. Despite the high amino acid identity between ACSL3 and ACSL4, rosiglitazone inhibited only ACSL4. Triacsin C, an inhibitor of ACSL1 and ACSL4, also inhibited ACSL3, but did not inhibit the ACSL6 variants. These data further document important differences in the closely related ACSL isoforms and show that amino acid changes near the consensus nucleotide binding site alter function in the two splice variants of ACSL6. [ABSTRACT FROM AUTHOR]

A PsbQ homologue has been found associated with photo system II complexes in Synechocystis sp. PCC 6803 where it is involved in optimal photoautotrophic growth and water splitting under CaCI2- depleted conditions [Thornton, L. B., Ohkawa, H., Roose, J. L., Kashino, Y., Keren, N., and Pakrasi, H. B. (2004) Plant Cell 16, 2164-2175]. By inactivating psbQ in strains carrying photosystem 11-specific mutations, we have identified stringent requirements for PsbQ in vivo. Whereas under nutrient-replete conditions the ΔPsbQ mutant was similar to wild type, a strain lacking PsbQ and PsbV was not photoautotrophic, exhibiting decreased oxygen evolution and decreased photosystem II assembly compared to the APsbV mutant. Combining the removal of PsbU and PsbQ introduced an altered requirement for Ca2+ and Cl-, and photoautotrophic growth of the ΔPsbQ strain was prevented in nutrient-limiting media depleted in Ca2+ Cl-, and iron. Unlike other photosystem II extrinsic proteins PsbQ did not participate in the acquisition of thermotolerance; however, photoautotrophic growth at elevated temperatures was impaired in this mutant. Growth of the ΔPsbV:ΔPsbQ mutant was restored at pH 10.0: in contrast, an additional deletion between Arg-384 and Val-392 in the CP47 protein of photosy stem II prevented recovery at alkaline pH. When conditions prevented photoautotrophy in strains lacking PsbQ, photoheterotrophic growth was indistinguishable to wild type, indicating that photosystem II had been inactivated. These data substantiate a role for PsbQ in optimizing photosystem II activity in Synechocystis sp. PCC 6803 and establish an absolute requirement for the subunit under specific biochemical and physiological conditions. [ABSTRACT FROM AUTHOR]

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet. [ABSTRACT FROM AUTHOR]

The left-handed polyproline II (PII) helix is a structure that has been given a great deal of attention lately because of its role in a wide variety of physiologically important processes and potential significance in protein unfolded states. Recent work by several authors has shown that residues besides proline can adopt this structure. A scale of relative PII-helix-forming propensities has been generated but only for single guest residues in a proline-based host system. Here, we present multiple guest residues in a proline-based host system. Using circular dichroism spectroscopy, we have shown that not only single residues, but also short sequences of non-proline residues can adopt the PII conformation. [ABSTRACT FROM AUTHOR]

Tocopherol transfer protein (TTP) is a key regulator of vitamin E homeostasis. TTP is presumed to function by transporting the hydrophobic vitamin between cellular compartments, thus facilitating its secretion to the extracellular space. Indeed, recombinant TTP demonstrates marked ability to facilitate tocopherol transfer between lipid bilayers. We report the biochemical characterization of six missense mutations TTP that are found in human AVED patients. We expressed the H101Q, A120T, R192H, R59W, E141K, and R221W TTP mutants in Escherichia coli, and purified the proteins to homogeneity. We the characterized TTP and its mutant counterparts with respect to their affinity for RRR-α-tocopherol and to their ability to catalyze tocopherol transfer between membranes. We observe the R59W, E141K, and R211W mutations, associated with the severe, early-onset version of AVED, are impaired in tocopherol binding and transfer activity. Surprisingly, despite the profound clinical effect of the R59W, E141W, and R221W mutations in vivo, their impact on TTP activity in vitro is quite benign (2-3 fold reduction in transfer kinetics). Furthermore, mutations associated with milder forms of the AVED disease, while causing pronounced perturbations in tocopherol homeostasis in vivo, are remarkably similar to the wild-type protein in the tocopherol transfer assays in vitro. Our data indicate that tocopherol transfer activity in vitro does not properly recapitulate the physiological functions of TTP. These findings suggest the possibility that the AVED syndrome may not arise from and inability of TTP to bind or to transfer alpha tocopherol, but rather from defects in other activities of the protein. [ABSTRACT FROM AUTHOR]

G-Protein-coupled receptors (GPCRs) are an important superfamily of transmembrane proteins involved in cellular communication. Recently, it has been shown that dimerization is a widely occurring phenomenon in the GPCR superfamily, with likely important physiological roles. Here we use a novel hidden-site class model of evolution as a sequence analysis tool to predict possible dimerization interfaces in GPCRs. This model aims to simulate the evolution of proteins at the amino acid level, allowing the analysis of their sequences in an explicitly evolutionary context. Applying this model to aminergic GPCR sequences, we first validate the general reasoning behind the model. We then use the model to perform a family specific analysis of GPCRs. Accounting for the family structure of these proteins, this approach detects different evolutionarily conserved and accessible patches on transmembrane (TM) helices 4-6 in different families. On the basis of these findings, we propose an experimentally testable dimerization mechanism, involving interactions among different combinations of these helices in different families of aminergic GPCRs. [ABSTRACT FROM AUTHOR]

Fluorescent-labeled derivatives of the Antennapedia-derived cell-penetating peptide penetratin, and of the simpler but similarly charged peptides R[SUB6]GC-NH[SUB2] and K[SUB6]GC-NH[SUB2], are shown to be able to translocate into large unilamellar lipid vesicles in the presence of a transbilayer potential (inside negative). Vesicles with diverse lipid compositions, and combining physiological proportions of neutral and anionic lipids, are able to support substantial potential-dependent uptake of all three cationic peptides. The efficiency of peptide uptake under these conditions is strongly modulated by the vesicle lipid composition, in a manner that suggests that more than one mechanism of peptide uptake may operate in different systems. Remarkably, peptide uptake is accompanied by only minor perturbations of the overall bather function of the lipid bilayer, as assessed by assays of vesicle leakiness under the same conditions. Fluorescence microscopy of living CV- 1 and HeLa cells incubated with the labeled peptides shows that the peptides accumulate in peripheral vesicular structures at early times of incubation, consistent with an initial endosomal localization as recently reported, but gradually accumulate in the cytoplasm and nucleus during more extended incubations (several hours). Our findings indicate that these relatively hydrophilic, polybasic cell-penetrating peptides can translocate through lipid bilayers by a potential- and composition-dependent pathway that causes only minimal perturbation to the overall integrity and barrier function of the bilayer. [ABSTRACT FROM AUTHOR]

Investigates the mechanism by which nitrogen monoxide (NO) affects IRP1 (iron-regulatory protein 1) RNA activity. Mobilization of iron from the cells by NO; Increase in RNA-binding activity of IRP1 by NO generators in cell lysates.

Investigates the mechanism of Escherichia coli enoyl (acyl carrier protein) reductase (EACPR) inhibition by antibacterial agent triclosan. Kinetic characteristics of EACPR; Triclosan as a stoichiometric inhibitor in the potency assay; Characterization of inhibition by triclosan in the steady state.

Investigates the biological function of 14-3-3 proteins by identifying its high-affinity antagonists. Identification of peptides which binds to the protein from a random phage display technology; Isolation and characterization of the high-affinity nonphosphorylated peptide ligand.

Presents evidence for altered ion transport in Saccharomyces cerevisiae overexpressing human MDR 1 protein. Overexpression of full length, functional, and properly localized human MDR 1 protein in independently isolated 9.3/pFF1 colonies; Role of human MDR 1 in catalysis of H+ transport and its impact on membrane potential in yeast.

Studies the structure of Xenopus Rad51 protein, a eukaryotic homologue of RecA protein, in solution by small-angle neutron scattering (SANS). Effect of nucleotides on SANS patterns for Xenopus Rad51.1 protein; Effect of DNA binding on the Xenopus Rad51.1 protein filament.

Illustrates the chromophore formation in green fluorescent protein (GFP) from the jellyfish Aequorea victoria. Usage of polymerase chain reaction for GFP gene expression; Fluorescence measurements; Description of the chromophore formation process; Rate of protein folding occurrence; Involvement of cyclized chromophore oxidation.

Identifies the critical amino acid sequence divergence underlying functional differences between proteins folate receptor(FR)-alpha and FR-beta from both human and murine sources. Functional distinction between FR-beta and FR-alpha; Disruption of proper folding of FR-beta by peptide 39-59 of FR-alpha.

Investigates the nuclear accumulation of high mobility group 2 (HMG2) protein within the nucleus. Distribution of HMG2 in the nucleus of higher eukaryotic cells; Active transport of HMG2 protein into the nucleus after translation in the cytoplasm; Function of the close configuration of basic regions as the nuclear localization signal.

Studies the source of transport site asymmetry in the human red blood cell band 3 anion exchange protein determined by nuclear magnetic resonance (NMR) measurements of external Cl- affinity. Cell preparation; Leaky ghost membranes; Treatment with inhibitors and nystatin.

Suggests that Mr 18 000 protein may mediate sperm-egg fusion during fertilization. Immunofluorescence and preferential association of the Mr 18 000 protein with acrosome-reacted sperm; Influence of Mr 18 000 protein on liposome activity.

Presents a detailed analysis of the DNA condensing properties of transition protein-2 (TP2) with various polynucleotides using circular dichroism spectroscopy. Isoation of rat oligonucleosomal DNA; Ellipticity values; Zinc-binding sites; Biological significance.

Examines the interaction of amphiphilic peptide pardaxin and its analogues with large unilamellar vesicles composed of phosphatydilserine. Analysis of the peptide to induce fusion and leakage; Structure and function relationship of the peptides.

Identifies the reactive cysteine in flavin protein domain of DNA photolyase of the bacterium Escherichia coli. Reaction of 8-(Methylsulfonyl)FAD protein with single cysteine residue (Cys293) in the flavin domain; Covalent incorporation of 8-(Methylsulfonyl)FAD in the flavin domain.

Examines the effects of calcium and ethylenediaminetetraacetic acid (EDTA) on the molecular conformation of ventricular protein VLC2 within myosin. Use of monoclonal antibodies directed against VLC2; Measurement of the association constants; Immunofluorescence study; Spatial arrangements and binding mechanisms between HC and VLAC2 proteins.

The article presents a chemical analysis on the relaxation rate for an ultrafast folding protein. It discusses the connection between free-energy surfaces and chevron plots in a kinetic study of an ultrafast folding protein and the 35-residue subdomain from the villin headpiece. It is seen in the analysis that a nearly denaturant-invariant, unfolding/refolding relaxation rate can be produced by a large movement of a small free-energy barrier along with a denaturant diffusion coefficient.

Light, oxygen, or voltage (LOV) domains constitute a new class of chromoprotein modules. They form the blue-light-sensitive loci of the phototropins, a recently discovered class of plant photoreceptors that regulate a variety of responses. LOV domains consist of approximately 100 amino acids and noncovalently bind a single flavin. Blue-light absorption initiates a photochemical reaction which results in the formation of a covalent adduct between a conserved cysteine and the flavin.5'6 It is believed that this species, referred to as S390 given its absorption band in the near-UV, corresponds to the signaling state of the protein. The lifetime of the adduct in various LOV domains ranges from minutes to hours,5'79 which implies that even under physiological illumination, there is a high probability for absorption of a second, near-UV photon.