Structure-Function Relations in the PI-PLC/GDPD Enzyme Superfamily

Phosphatidylinositol-specific phospholipases C (PI-PLC) are known to participate in many eukaryotic signal transduction pathways and act as virulence factors in lower organisms. Glycerophosphoryl diester phosphodiesterase (GDPD) enzymes are involved in phosphate homeostasis and phospholipid catabolism for energy production. Streptomyces antibioticus phosphatidylinositol-specific phospholipase C (SaPLC1) is a 38 kDa enzyme that displays characteristics of both enzyme superfamilies, representing an evolutionary link between these divergent enzyme classes. SaPLC1 also boasts a unique catalytic mechanism that involves a trans 1,6-cyclic inositol phosphate intermediate instead of the typical cis 1,2-cyclic inositol phosphate. The mechanism by which this occurs is still unclear. To attack this problem, we established a wide mutagenesis scan of the active site and measured activities of alanine mutants. A chemical rescue assay was developed to verify that the activity loss was due to the removal of the functional role of the mutated residue. 31P-NMR was employed in characterizing and quantifying intermediates in mutants that slowed the reaction sufficiently. We found that the H37A and H76A mutations support the hypothesis that these structurally conserved residues are also conserved in terms of their catalytic roles. H37 was found to be the general base (GB), while H76 plays the role of general acid (GA). K131 was identified as a semi-conserved key positive charge donor found at the entrance of the active site. By elucidating the SaPLC1 mechanism in relation to its active site architecture, we have increased our understanding of the structure-function relations that support catalysis in the PI-PLC/GDPD superfamily. These findings provide groundwork for in vivo studies of SaPLC1 function and its possible role in novel signaling or metabolism in Streptomyces.

Proteolytic enzymes, past and future

Today’s knowledge is based on yesterday’s research, which, for me, started some 60 years ago. In the introduction to this colloquium, the past history of proteolytic enzymes is briefly reviewed against the background of simultaneously developing concepts and methodologies in protein chemistry. This history is followed by a sketch of more recent developments of the role of proteolytic enzymes in physiological regulation and an outlook of future trends apparent from current research.

Bioinorganic and medicinal chemistry: aspects of gold(I)-protein complexes

Gold(I)-based drugs have been used successfully for the treatment of rheumatoid arthritis (RA) for several years. Although the exact mechanism of action of these gold(I) drugs for RA has not been clearly established, the interaction of these compounds with mammalian enzymes has been extensively studied. In this paper, we describe the interaction of therapeutic gold(I) compounds with mammalian proteins that contain cysteine (Cys) and selenocysteine (Sec) residues. Owing to the higher affinity of gold(I) towards sulfur and selenium, gold(I) drugs rapidly react with the activated cysteine or selenocysteine residues of the enzymes to form protein-gold(I)-thiolate or protein-gold(I)-selenolate complexes. The formation of stable gold(I)-thiolate/selenolate complexes generally lead to inhibition of the enzyme activity. The gold-thiolate/selenolate complexes undergo extensive ligand exchange reactions with other nucleophiles and such ligand exchange reactions alter the inhibitory effects of gold(I) complexes. Therefore, the effect of gold(I) compounds on the enzymatic activity of cysteine-or selenocysteine-containing proteins may play important roles in RA. The interaction of gold(I) compounds with different enzymes and the biochemical mechanism underlying the inhibition of enzymatic activities may have broad medicinal implications for the treatment of RA.

Computational chemistry for elucidating protein function: energetics and dynamics of myoglobin-ligand systems

State-of-the-art computational methodologies are used to investigate the energetics and dynamics of photodissociated CO and NO in myoglobin (Mb···CO and Mb···NO). This includes the combination of molecular dynamics, ab initio MD, free energy sampling, and effective dynamics methods to compare the results with studies using X-ray crystallography and ultrafast spectroscopy metho ds. It is shown that modern simulation techniques along with careful description of the intermolecular interactions can give quantitative agreement with experiments on complex molecular systems. Based on this agreement predictions for as yet uncharacterized species can be made.

Hematology, serum chemistry, and serum protein electrophoresis ranges for free-ranging roe deer (Capreolus capreolus) in Sweden.

We present the first reference ranges for hematology (n = 35 animals), serum biochemistry (n = 62), and serum protein electrophoresis (n = 32) in physically restrained free-ranging roe deer (Capreolus capreolus). Animals were captured in box traps and physically restrained for blood sampling during the winter in Sweden, 2011-13. No clinically significant sex or age differences were found.

Seawater carbonate chemistry and protein content, respiration, symbiodinium densities, survivorship of Pocillopora damicornis larvae in a laboratory experiment

Efforts to evaluate the response of coral larvae to global climate change (GCC) and ocean acidification (OA) typically employ short experiments of fixed length, yet it is unknown how the response is affected by exposure duration. In this study, we exposed larvae from the brooding coral Pocillopora damicornis to contrasts of temperature (24.00 °C [ambient] versus 30.49 °C) and pCO2 (49.4 Pa versus 86.2 Pa) for varying periods (1-5 days) to test the hypothesis that exposure duration had no effect on larval response as assessed by protein content, respiration, Symbiodinium density, and survivorship; exposure times were ecologically relevant compared to representative pelagic larval durations (PLD) for corals. Larvae differed among days for all response variables, and the effects of the treatment were relatively consistent regardless of exposure duration for three of the four response variables. Protein content and Symbiodinium density were unaffected by temperature and pCO2, but respiration increased with temperature (but not pCO2) with the effect intensifying as incubations lengthened. Survival, however, differed significantly among treatments at the end of the study, and by the 5th day, 78% of the larvae were alive and swimming under ambient temperature and ambient pCO2, but only 55-59% were alive in the other treatments. These results demonstrate that the physiological effects of temperature and pCO2 on coral larvae can reliably be detected within days, but effects on survival require > or = 5 days to detect. The detection of time-dependent effects on larval survivorship suggests that the influence of GCC and OA will be stronger for corals having long PLDs.

Seawater carbonate chemistry and protein and chlorophyll a content per nubbin of Acropora muricata during observations of La Saline fringing reef, La Reunion Island, western Indian Ocean, 2011

The effect of decreasing aragonite saturation state (Omega Arag) of seawater (elevated pCO2) on calcification rates of Acropora muricata was studied using nubbins prepared from parent colonies located at two sites of La Saline reef (La Réunion Island, western Indian Ocean): a back-reef site (BR) affected by nutrient-enriched groundwater discharge (mainly nitrate), and a reef flat site (RF) with low terrigenous inputs. Protein and chlorophyll a content of the nubbins, as well as zooxanthellae abundance, were lower at RF than BR. Nubbins were incubated at ~27°C over 2 h under sunlight, in filtered seawater manipulated to get differing initial pCO2 (1,440-340 µatm), Omega Arag (1.4-4.0), and dissolved inorganic carbon (DIC) concentrations (2,100-1,850 µmol/kg). Increasing DIC concentrations at constant total alkalinity (AT) resulted in a decrease in Omega Arag and an increase in pCO2. AT at the beginning of the incubations was kept at a natural level of 2,193 ± 6 µmol/kg (mean ± SD). Net photosynthesis (NP) and calcification were calculated from changes in pH and AT during the incubations. Calcification decrease in response to doubling pCO2 relative to preindustrial level was 22% for RF nubbins. When normalized to surface area of the nubbins, (1) NP and calcification were higher at BR than RF, (2) NP increased in high pCO2 treatments at BR compared to low pCO2 treatments, and (3) calcification was not related to Omega Arag at BR. When normalized to NP, calcification was linearly related to Omega Arag at both sites, and the slopes of the relationships were not significantly different. The increase in NP at BR in the high pCO2 treatments may have increased calcification and thus masked the negative effect of low Omega Arag on calcification. Removing the effect of NP variations at BR showed that calcification declined in a similar manner with decreased Omega Arag (increased pCO2) whatever the nutrient loading.

Seawater carbonate chemistry and protein sports of barnicle Balanus amphitrite during experiments, 2011

The majority of benthic marine invertebrates have a complex life cycle, during which the pelagic larvae select a suitable substrate, attach to it, and then metamorphose into benthic adults. Anthropogenic ocean acidification (OA) is postulated to affect larval metamorphic success through an altered protein expression pattern (proteome structure) and post-translational modifications. To test this hypothesis, larvae of an economically and ecologically important barnacle species Balanus amphitrite, were cultured from nauplius to the cyprid stage in the present (control) and in the projected elevated concentrations of CO2 for the year 2100 (the OA treatment). Cyprid response to OA was analyzed at the total proteome level as well as two protein post-translational modification (phosphorylation and glycosylation) levels using a 2-DE based proteomic approach. The cyprid proteome showed OA-driven changes. Proteins that were differentially up or down regulated by OA come from three major groups, namely those related to energy-metabolism, respiration, and molecular chaperones, illustrating a potential strategy that the barnacle larvae may employ to tolerate OA stress. The differentially expressed proteins were tentatively identified as OA-responsive, effectively creating unique protein expression signatures for OA scenario of 2100. This study showed the promise of using a sentinel and non-model species to examine the impact of OA at the proteome level.

Identification of protein targets of nevirapine reactive metabolites using click chemistry and mass spectrometry-based differential proteomics

Abstract : Adverse drug reactions (ADRs) are undesirable effects caused after administration of a single dose or prolonged administration of drug or result from the combination of two or more drugs. Idiosyncratic drug reaction (IDR) is an adverse reaction that does not occur in most patients treated with a drug and does not involve the therapeutic effect of the drug. IDRs are unpredictable and often life-threatening. Idiosyncratic reaction is dependent on drug chemical characteristics or individual immunological response. IDRs are a major problem for drug development because they are usually not detected during clinical trials. In this study we focused on IDRs of Nevirapine (NVP), which is a non-nucleoside reverse transcriptase inhibitor used for the treatment of Human Immunodeficiency Virus (HIV) infections. The use of NVP is limited by a relatively high incidence of skin rash. NVP also causes a rash in female Brown Norway (BN) rats, which we use as animal model for this study. Our hypothesis is that idiosyncratic skin reactions associated with NVP treatment are due to post-translational modifications of proteins (e.g., glutathionylation) detectable by MS. The main objective of this study was to identify the proteins that are targeted by a reactive metabolite of Nevirapine in the skin. The specific objectives derived from the general objective were as follow: 1) To implement the click chemistry approach to detect proteins modified by a reactive NVP-Alkyne (NVP-ALK) metabolite. The purpose of using NVP-ALK was to couple it with Biotin using cycloaddition Click Chemistry reaction. 2) To detect protein modification using Western blotting and Mass Spectrometry techniques, which is important to understand the mechanism of NVP induced toxicity. 3) To identify the proteins using MASCOT search engine for protein identification, by comparing obtained spectrum from Mass Spectrometry with theoretical spectrum to find a matching peptide sequence. 4) To test if the drug or drug metabolites can cause harmful effects, as the induction of oxidative stress in cells (via protein glutathionylation). Oxidative stress causes cell damage that mediates signals, which likely induces the immune response. The results showed that Nevirapine is metabolized to a reactive metabolite, which causes protein modification. The extracted protein from the treated BN rats matched 10% of keratin, which implies that keratin was the protein targeted by the NVP-ALK.

Correlations between designability and various structural characteristics of protein lattice models

Using six kinds of lattice types (4×4 ,5×5 , and6×6 square lattices;3×3×3 cubic lattice; and2+3+4+3+2 and4+5+6+5+4 triangular lattices), three different size alphabets (HP ,HNUP , and 20 letters), and two energy functions, the designability of proteinstructures is calculated based on random samplings of structures and common biased sampling (CBS) of proteinsequence space. Then three quantities stability (average energy gap),foldability, and partnum of the structure, which are defined to elucidate the designability, are calculated. The authors find that whatever the type of lattice, alphabet size, and energy function used, there will be an emergence of highly designable (preferred) structure. For all cases considered, the local interactions reduce degeneracy and make the designability higher. The designability is sensitive to the lattice type, alphabet size, energy function, and sampling method of the sequence space. Compared with the random sampling method, both the CBS and the Metropolis Monte Carlo sampling methods make the designability higher. The correlation coefficients between the designability, stability, and foldability are mostly larger than 0.5, which demonstrate that they have strong correlation relationship. But the correlation relationship between the designability and the partnum is not so strong because the partnum is independent of the energy. The results are useful in practical use of the designability principle, such as to predict the proteintertiary structure.