Biomedical subjects

Alexander V Vener

Publications and source records attributed to Alexander V Vener.

At least 19 recordsLinked to original sources

PubMed2007

The Arabidopsis PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction centre D1 protein.

The extrinsic photosystem II (PSII) protein of 33 kDa (PsbO), which stabilizes the water-oxidizing complex, is represented in Arabidopsis thaliana (Arabidopsis) by two isoforms. Two T-DNA insertion mutant lines deficient in either the PsbO1 or the PsbO2 protein were retarded in growth in comparison with the wild type, while differing from each other phenotypically. Both PsbO proteins were able to support the oxygen evolution activity of PSII, although PsbO2 was less efficient than PsbO1 under photoinhibitory conditions. Prolonged high light stress led to reduced growth and fitness of the mutant lacking PsbO2 as compared with the wild type and the mutant lacking PsbO1. During a short period of treatment of detached leaves or isolated thylakoids at high light levels, inactivation of PSII electron transport in the PsbO2-deficient mutant was slowed down, and the subsequent degradation of the D1 protein was totally inhibited. The steady-state levels of in vivo phosphorylation of the PSII reaction centre proteins D1 and D2 were specifically reduced in the mutant containing only PsbO2, in comparison with the mutant containing only PsbO1 or with wild-type plants. Phosphorylation of PSII proteins in vitro proceeded similarly in thylakoid membranes from both mutants and wild-type plants. However, dephosphorylation of the D1 protein occurred much faster in the thylakoids containing only PsbO2. We conclude that the function of PsbO1 in Arabidopsis is mostly in support of PSII activity, whereas the interaction of PsbO2 with PSII regulates the turnover of the D1 protein, increasing its accessibility to the phosphatases and proteases involved in its degradation.

Arabidopsis↗

PubMed2007

Identification of phosphorylated proteins.

Reversible protein phosphorylation is crucially involved in all aspects of plant cell physiology. The highly challenging task of revealing and characterizing the dynamic protein phosphorylation networks in plants has only recently begun to become feasible, owing to application of dedicated proteomics and mass spectrometry techniques. The experimental methodology that identified most of the presently known proteins phosphorylated in vivo is based on protein cleavage with trypsin, following chromatographic enrichment of phosphorylated peptides and mass spectrometric fragmentation and sequencing of these phosphopeptides. This procedure is most efficient when it is limited to the tryptic digestion of proteins in distinct isolated fractions or compartments of plant cells. Immobilized metal affinity chromatography (IMAC) is most useful for phosphopeptide enrichment after methylation of the peptides in the complex protein digests. The following tandem mass spectrometry of the isolated phosphopeptides results in both identification of phosphorylated proteins and mapping of the in vivo phosphorylation sites. The relative quantitation of the extent of phosphorylation at individual protein modification sites may be accomplished by either stable isotope labeling technique or dedicated liquid chromatography-mass spectrometry measurements.

Cell Fractionation↗

PubMed2006

Micelle-induced folding of spinach thylakoid soluble phosphoprotein of 9 kDa and its functional implications.

Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al. (2003) Proc. Natl. Acad. Sci. 100, 757-762). Nonphosphorylated TSP9 is associated with the membrane, whereas, after light-induced phosphorylation, a fraction of the phosphorylated TSP9 is released into the aqueous stroma. By NMR spectroscopy, we have determined the structural features of nonphosphorylated TSP9 both in aqueous solution and in membrane mimetic micelles. The results show that both wild type nonphosphorylated TSP9 and a triple-mutant (T46E + T53E + T60E) mimic of the triphosphorylated form of TSP9 are disordered under aqueous conditions, but adopt an ordered conformation in the presence of detergent micelles. The micelle-induced structural features, which are similar in micelles either of SDS or dodecylphosphocholine (DPC), consist of an N-terminal alpha-helix, which may represent the primary site of interaction between TSP9 and binding partners, and a less structured helical turn near the C-terminus. These structured elements contain mainly hydrophobic residues. NMR relaxation data for nonphosphorylated TSP9 in SDS micelles indicated that the molecule is highly flexible with the highest order in the N-terminal alpha-helix. Intermolecular NOE signals, as well as spin probe-induced broadening of NMR signals, demonstrated that the SDS micelles contact both the structured and a portion of the unstructured regions of TSP9, in particular, those containing the three phosphorylation sites (T46, T53, and T60). This interaction may explain the selective dissociation of phosphorylated TSP9 from the membrane. Our study presents a structural model for the role played by the structured and unstructured regions of TSP9 in its membrane association and biological function.

Crystallography, X-Ray↗

PubMed2006

Environmentally modulated phosphorylation and dynamics of proteins in photosynthetic membranes.

Recent advances in vectorial proteomics of protein domains exposed to the surface of photosynthetic thylakoid membranes of plants and the green alga Chlamydomonas reinhardtii allowed mapping of in vivo phosphorylation sites in integral and peripheral membrane proteins. In plants, significant changes of thylakoid protein phosphorylation are observed in response to stress, particularly in photosystem II under high light or high temperature stress. Thylakoid protein phosphorylation in the algae is much more responsive to the ambient redox and light conditions, as well as to CO(2) availability. The light-dependent multiple and differential phosphorylation of CP29 linker protein in the green algae is suggested to control photosynthetic state transitions and uncoupling of light harvesting proteins from photosystem II under high light. The similar role for regulation of the dynamic distribution of light harvesting proteins in plants is proposed for the TSP9 protein, which together with other recently discovered peripheral proteins undergoes specific environment- and redox-dependent phosphorylation at the thylakoid surface. This review focuses on the environmentally modulated reversible phosphorylation of thylakoid proteins related to their membrane dynamics and affinity towards particular photosynthetic protein complexes.

Algal Proteins↗

PubMed2006

Association and insulin regulated translocation of hormone-sensitive lipase with PTRF.

Polymerase I and transcript release factor (PTRF) is in human adipocytes mainly localized at the plasma membrane. This localization was under control of insulin, which translocated PTRF to the cytosol and nucleus, indicating a novel role for PTRF in insulin transcriptional control. In the plasma membrane PTRF was specifically bound to a triacylglycerol-metabolizing subclass of caveolae containing hormone-sensitive lipase (HSL). In response to insulin PTRF was translocated to the cytosol in parallel with HSL. PTRF and HSL were quantitatively immunoprecipitated from the cytosol by antibodies against either PTRF or HSL. The findings indicate also a novel extranuclear function for PTRF in the control of lipolysis.

Adipocytes↗

PubMed2006

State transitions revisited-a buffering system for dynamic low light acclimation of Arabidopsis.

The mobile part of the light-harvesting chlorophyll (chl) a/b protein complex (LHCII), composed of the Lhcb1 and Lhcb2 proteins, is the basic unit of chloroplast state transitions--the short term tuning system in balancing the excitation energy between Photosystem (PS) II and PSI. State transitions are catalysed by the thylakoid associated STN7 kinase, and we show here that besides the phosphorylation of the Lhcb1 and Lhcb2 proteins, also the phosphorylation of Lhcb4.2 (CP29) is under the control of the STN7 kinase. Upon growth of Arabidopsis WT and stn7 mutant plants under low and moderate light conditions, the WT plants favoured state 2 whereas stn7 was locked in state 1. The lack of the STN7 kinase and state transitions in stn7 also modified the thylakoid protein contents upon long-term low light acclimation resulting, for example, in low Lhcb1 and in elevated Lhca1 and Lhca2 protein amounts as compared to WT. Adjustments of thylakoid protein contents probably occurred at post-transcriptional level since the DNA microarray experiments from each growth condition did not reveal any significant differences between stn7 and WT transcriptomes. The resulting high Lhcb2/Lhcb1 ratio in stn7 upon growth at low light was accompanied by lower capacity for NPQ than in WT. On the contrary, higher amounts of PsbS in stn7 under moderate and high light growth conditions resulted in higher NPQ compared to WT and consequently also in a protection of PSII against photoinhibition. STN7 kinase and the state transitions are suggested to have a physiological significance for dynamic acclimation to low but fluctuating growth light conditions. They are shown to function as a buffering system upon short high light illumination peaks by shifting the thylakoids from state 2 to state 1 and thereby down regulating the induction of stress-responsive genes, a likely result from transient over-reduction of PSI acceptors.

Adaptation, Physiological↗

PubMed2006

Profound redox sensitivity of peptidyl-prolyl isomerase activity in Arabidopsis thylakoid lumen.

Proteomic, enzymatic, and mutant analyses revealed that peptidyl-prolyl isomerase (PPIase) activity in the chloroplast thylakoid lumen of Arabidopsis is determined by two immunophilins: AtCYP20-2 and AtFKBP13. These two enzymes are responsible for PPIase activity in both soluble and membrane-associated fractions of thylakoid lumen suggesting that other lumenal immunophilins are not active towards the peptide substrates. In thiol-reducing conditions PPIase activity of the isolated AtFKBP13 and of the total thylakoid lumen is suppressed several fold. Profound redox-dependence of PPIase activity implies oxidative activation of protein folding catalysis under oxidative stress and photosynthetic oxygen production in the thylakoid lumen of plant chloroplasts.

Amino Acid Sequence↗

PubMed2006

Environmentally modulated phosphoproteome of photosynthetic membranes in the green alga Chlamydomonas reinhardtii.

Mapping of in vivo protein phosphorylation sites in photosynthetic membranes of the green alga Chlamydomonas reinhardtii revealed that the major environmentally dependent changes in phosphorylation are clustered at the interface between the photosystem II (PSII) core and its light-harvesting antennae (LHCII). The photosynthetic membranes that were isolated form the algal cells exposed to four distinct environmental conditions affecting photosynthesis: (i) dark aerobic, corresponding to photosynthetic State 1; (ii) dark under nitrogen atmosphere, corresponding to photosynthetic State 2; (iii) moderate light; and (iv) high light. The surface-exposed phosphorylated peptides were cleaved from the membrane by trypsin, methyl-esterified, enriched by immobilized metal affinity chromatography, and sequenced by nanospray-quadrupole time-of-flight mass spectrometry. A total of 19 in vivo phosphorylation sites were mapped in the proteins corresponding to 15 genes in C. reinhardtii. Amino-terminal acetylation of seven proteins was concomitantly determined. Sequenced amino termini of six mature LHCII proteins differed from the predicted ones. The State 1-to-State 2 transition induced phosphorylation of the PSII core components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10. Exposure of the algal cells to either moderate or high light caused additional phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to specific hyperphosphorylation of CP29 at seven distinct residues, phosphorylation of another minor LHCII constituent, CP26, at a single threonine, and double phosphorylation of additional subunits of a major LHCII complex including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Environmentally induced protein phosphorylation at the interface of PSII core and the associated antenna proteins, particularly multiple differential phosphorylations of CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions and for LHCII uncoupling from PSII under high light stress to allow thermal energy dissipation.

Algal Proteins↗

PubMed2006

Hormonal control of reversible translocation of perilipin B to the plasma membrane in primary human adipocytes.

In adipocytes, perilipin coats and protects the central lipid droplet, which stores triacylglycerol. Alternative mRNA splicing gives rise to perilipin A and B. Hormones such as catecholamines and insulin regulate triacylglycerol metabolism through reversible serine phosphorylation of perilipin A. It was recently shown that perilipin was also located in triacylglycerol-synthesizing caveolae of the plasma membrane. We now report that perilipin at the plasma membrane of primary human adipocytes was phosphorylated on a cluster of threonine residues (299, 301, and 306) within an acidic domain that forms part of the lipid targeting domain. Perilipin B comprised <10% of total perilipin but was the major isoform associated with the plasma membrane of human adipocytes. This association was controlled by insulin and catecholamine: perilipin B was specifically depleted from the plasma membrane in response to the catecholamine isoproterenol, while insulin increased the amount of threonine phosphorylated perilipin at the plasma membrane. The reversible translocation of perilipin B to and from the plasma membrane in response to insulin and isoproterenol, respectively, suggests a specific function for perilipin B to protect newly synthesized triacylglycerol in the plasma membrane.

Adipocytes↗

PubMed2006

CO2 limitation induces specific redox-dependent protein phosphorylation in Chlamydomonas reinhardtii.

Acclimation of the green alga Chlamydomonas reinhardtii to limiting environmental CO2 induced specific protein phosphorylation at the surface of photosynthetic thylakoid membranes. Four phosphopeptides were identified and sequenced by nanospray quadrupole TOF MS from the cells acclimating to limiting CO2. One phosphopeptide originated from a protein that has not been annotated. We found that this unknown expressed protein (UEP) was encoded in the genome of C. reinhardtii. Three other phosphorylated peptides belonged to Lci5 protein encoded by the low-CO2-inducible gene 5 (lci5). The phosphorylation sites were mapped in the tandem repeats of Lci5 ensuring phosphorylation of four serine and three threonine residues in the protein. Immunoblotting with Lci5-specific antibodies revealed that Lci5 was localized in chloroplast and confined to the stromal side of the thylakoid membranes. Phosphorylation of Lci5 and UEP occurred strictly at limiting CO2; it required reduction of electron carriers in the thylakoid membrane, but was not induced by light. Both proteins were phosphorylated in the low-CO2-exposed algal mutant deficient in the light-activated protein kinase Stt7. Phosphorylation of previously unknown basic proteins UEP and Lci5 by specific redox-dependent protein kinase(s) in the photosynthetic membranes reveals the early response of green algae to limitation in the environmental inorganic carbon.

Acclimatization↗

PubMed2005

A previously found thylakoid membrane protein of 14kDa (TMP14) is a novel subunit of plant photosystem I and is designated PSI-P.

We show that the thylakoid membrane phosphoprotein TMP14 is a novel subunit of plant photosystem I (PSI). Blue native/SDS-PAGE and sucrose gradient fractionation demonstrated the association of the protein exclusively with PSI. We designate the protein PSI-P. The presence of PSI-P subunit in Arabidopsis mutants lacking other PSI subunits was analyzed and suggested a location in the proximity of PSI-L, -H and -O subunits. The PSI-P protein was not differentially phosphorylated in state 1 and state 2.

Arabidopsis↗

PubMed2005

STN8 protein kinase in Arabidopsis thaliana is specific in phosphorylation of photosystem II core proteins.

Combination of reversed genetics with analyses of in vivo protein phosphorylation in Arabidopsis thaliana revealed that STN8 protein kinase is specific in phosphorylation of N-terminal threonine residues in D1, D2, and CP43 proteins, and Thr-4 in the PsbH protein of photosystem II. Phosphorylation of D1, D2, and CP43 in the light-exposed leaves of two Arabidopsis lines with T-DNA insertions in the stn8 gene was found significantly reduced in the assays with anti-phosphothreonine antibodies. Protein phosphorylation in each of the mutants was quantified comparatively to the wild type by mass spectrometric analyses of phosphopeptides released from the photosynthetic membranes and differentially labeled with stable isotopes. The lack of STN8 caused 50-60% reduction in D1 and D2 phosphorylation, but did not change the phosphorylation level of two peptides that could correspond to light-harvesting proteins encoded by seven different genes in Arabidopsis. Phosphorylation of the PsbH protein at Thr-4 was completely abolished in the plants lacking STN8. Phosphorylation of Thr-4 in the wild type required both light and prior phosphorylation at Thr-2, indicating that STN8 is a light-activated kinase that phosphorylates Thr-4 only after another kinase phosphorylates Thr-2. Analysis of the STN8 catalytic domain suggests that selectivity of STN8 in phosphorylation of the very N-terminal residues in D1, D2, and CP43, and Thr-4 in PsbH pre-phosphorylated at Thr-2 may be explained by the long loops obstructing entrance into the kinase active site and seven additional basic residues in the vicinity of the catalytic site, as compared with the homologous STN7 kinase responsible for phosphorylation of light-harvesting proteins.

Amino Acid Sequence↗

PubMed2005

Vectorial proteomics.

Vectorial proteomics is a methodology for the differential identification and characterization of proteins and their domains exposed to the opposite sides of biological membranes. Proteomics of membrane vesicles from defined isolated membranes automatically determine cellular localization of the identified proteins and reduce complexity of protein characterizations. The enzymatic shaving of naturally-oriented, or specifically-inverted sealed membrane vesicles, release the surface-exposed peptides from membrane proteins. These soluble peptides are amenable to various chromatographic separations and to sequencing by mass spectrometry, which provides information on the topology of membrane proteins and on their posttranslational modifications. The membrane shaving techniques have made a breakthrough in the identification of in vivo protein phosphorylation sites in membrane proteins form plant photosynthetic and plasma membranes, and from caveolae membrane vesicles of human fat cells. This approach has also allowed investigation of dynamics for in vivo protein phosphorylation in membranes from cells exposed to different conditions. Vectorial proteomics of membrane vesicles with retained peripheral proteins identify extrinsic proteins associated with distinct membrane surfaces, as well as a variety of posttranslational modifications in these proteins. The rapid integration of versatile vectorial proteomics techniques in the functional characterization of biological membranes is anticipated to bring significant insights in cell biology.

Animals↗

PubMed2005

Light-harvesting complex II protein CP29 binds to photosystem I of Chlamydomonas reinhardtii under State 2 conditions.

The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI). We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4). MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected. The LHCI-PSI supercomplex isolated from the alga in State 2 was found to contain strongly associated CP29 in phosphorylated form. Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein. It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems.

Animals↗

PubMed2004

Vectorial proteomics reveal targeting, phosphorylation and specific fragmentation of polymerase I and transcript release factor (PTRF) at the surface of caveolae in human adipocytes.

Caveolae, the specialized invaginations of plasma membranes, formed sealed vesicles with outwards-orientated cytosolic surface after isolation from primary human adipocytes. This morphology allowed differential, vectorial identification of proteins at the opposite membrane surfaces by proteolysis and MS. Extracellular-exposed caveolae-specific proteins CD36 and copper-containing amine oxidase were concealed inside the vesicles and resisted trypsin treatment. The cytosol-orientated caveolins were efficiently digested by trypsin, producing peptides amenable to direct MS sequencing. Isolation of peripheral proteins associated with the cytosolic surface of caveolae revealed a set of proteins that contained nuclear localization signals, leucine-zipper domains and PEST (amino acid sequence enriched in proline, glutamic acid, serine and threonine) domains implicated in regulation by proteolysis. In particular, PTRF (polymerase I and transcript release factor) was found as a major caveolae-associated protein and its co-localization with caveolin was confirmed by immunofluorescence confocal microscopy. PTRF was present at the surface of caveolae in the intact form and in five different truncated forms. Peptides (44 and 45 amino acids long) comprising both the PEST domains were sequenced by nanospray-quadrupole-time-of-flight MS from the full-length PTRF, but were not found in the truncated forms of the protein. Two endogenous cleavage sites corresponding to calpain specificity were identified in PTRF; one of them was in a PEST domain. Both cleavage sites were flanked by mono- or diphosphorylated sequences. The phosphorylation sites were localized to Ser-36, Ser-40, Ser-365 and Ser-366 in PTRF. Caveolae of human adipocytes are proposed to function in targeting, relocation and proteolytic control of PTRF and other PEST-domain-containing signalling proteins.

Adipocytes↗

PubMed2004

N-terminal processing and modifications of caveolin-1 in caveolae from human adipocytes.

Caveolin, the principal structural protein of caveolae membrane domains, has a cytosol-exposed N-terminal part that was cleaved off by trypsin treatment of caveolae vesicles isolated from primary human adipocytes. Sequencing of the released tryptic peptides by nanospray quadrupole time-of-flight mass spectrometry revealed that both caveolin-1alpha and caveolin-1beta were processed by excision of the starting methionines. The N-terminus of the mature caveolin-1alpha was acetylated, while caveolin-1beta was found in acetylated as well as in non-acetylated forms. Fractional phosphorylation of serine-36 in the mature caveolin-1alpha and of the homologous serine-5 in caveolin-1beta was identified. This is the first experimental evidence for in vivo phosphorylation of caveolin-1 at the consensus site for phosphorylation by protein kinase C. The phosphorylation was found in both the acetylated and non-acetylated variants of caveolin-1beta. This variability in modifications is consistent with critical involvement of the N-terminal domain of caveolin in the regulation of caveolae.

Acetylation↗

PubMed2004

The transit peptide of CP29 thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes acetylation and phosphorylation.

The surface-exposed peptides were cleaved by trypsin from the photosynthetic thylakoid membranes isolated from the green alga Chlamydomonas reinhardtii. Two phosphorylated peptides, enriched from the peptide mixture and sequenced by nanospray quadrupole time-of-flight mass spectrometry, revealed overlapping sequences corresponding to the N-terminus of a nuclear-encoded chlorophyll a/b-binding protein CP29. In contrast to all known nuclear-encoded thylakoid proteins, the transit peptide in the mature algal CP29 was not removed but processed by methionine excision, N-terminal acetylation and phosphorylation on threonine 6. The importance of this phosphorylation site is proposed as the reason of the unique transit peptide retention.

Acetylation↗

PubMed2004

Rates of elementary catalytic steps for different metal forms of the family II pyrophosphatase from Streptococcus gordonii.

Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn(2+) rather than Mg(2+), the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and II PPases. We measured rates of PP(i) hydrolysis, PP(i) synthesis, and P(i)/water oxygen exchange catalyzed by sgPPase with Mn(2+), Mg(2+), or Co(2+) in the high-affinity metal-binding site and Mg(2+) in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P(i)). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PP(i) complex isomerization known to occur in family I PPases; (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PP(i) hydrolysis and product release; (c) the marked acceleration of sgPPase catalysis in the presence of Mn(2+) and Co(2+) results from a combined effect of these ions on bound PP(i) hydrolysis and P(i) release; (d) sgPPase exhibits lower affinity for both PP(i) and P(i); and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K(m), which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PP(i) concentrations).

Binding Sites↗