The Open Biochemistry Journal[JOURNAL]
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130 papers
The Hepatitis C Virus NS5A Stimulates NS5B During In Vitro RNA Synthesis in a Template Specific Manner.
Quezada EM, Kane CM
Open Biochem J · 2009 Apr · PMID 19590581 · Full text
The hepatitis C virus (HCV) NS5B protein contains the RNA dependent RNA polymerase (RdRp) activity that catalyzes the synthesis of the viral genome with other host and viral factors. NS5A is an HCV-encoded protein previo...
The hepatitis C virus (HCV) NS5B protein contains the RNA dependent RNA polymerase (RdRp) activity that catalyzes the synthesis of the viral genome with other host and viral factors. NS5A is an HCV-encoded protein previously shown to localize to the replisome and be necessary for viral replication. However, its role in replication has not been defined. Using an in vitro biochemical assay, we detected a stimulatory effect of NS5A on the NS5B replication reaction with minimal natural templates. NS5A stimulates replication by NS5B on two templates derived from the 3' end of the RNA genome (4 fold +/- 1.3 fold). A pre-incubation step with the two proteins prior to the replication reaction and substoichiometric levels of NS5A are required for detecting stimulation. With a template derived from the 3'end complementary to the RNA genome (the negative strand) no stimulation was observed. Furthermore, with a synthetic template that allows studying different phases of replication, NS5A stimulates NS5B during elongation. These findings suggest that NS5A stimulates NS5B during synthesis of the complementary (i.e., negative) strand of the RNA genome.
Analysis of endothelial protein C receptor functionality on living cells'.
Ducros E, Mirshahi S, Bermot C … +1 more , Mirshahi M
Open Biochem J · 2009 May · PMID 19572027 · Full text
Activated protein C (APC) is a major control system of blood coagulation. APC prevents coagulation pathway by degrading Va and VIIIa plasma's coagulation factors. Protein C activation requires its binding to specific end...
Activated protein C (APC) is a major control system of blood coagulation. APC prevents coagulation pathway by degrading Va and VIIIa plasma's coagulation factors. Protein C activation requires its binding to specific endothelial cell receptor (EPCR). APC binding to EPCR also activates a wide range of defense mechanisms (anti-inflammatory, antiapoptosis...). EPCR expression by cells can be detected by various methods, including immunoanalysis and molecular biology. However, no assays evaluate its functionality. A method, inspired of a standard fibrinoformation time assay, was developed to estimate EPCR ability to bind APC on living cell surface in vitro. Endothelial cells were incubated with APC and fibrinoformation on cells was followed by spectrophotometry (plasma absorbance increases with fibrin polymerization). Membrane-bound EPCR retain APC, thus prolonging fibrinoformation time in a dose-dependent manner. Control was realized with EPCR-negative cells. This new method can be used on any cell type to study the expression of other coagulation receptors.
Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry.
Baouz S, Schmitter JM, Chenoune L … +4 more , Beauvallet C, Blanquet S, Woisard A, Hountondji C
Open Biochem J · 2009 Mar · PMID 19557155 · Full text
The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). There...
The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNA(Ile), the 2',3'-dialdehyde derivative of tRNA(Ile) was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNA(Ile) aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNA(Ile)ox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNA(Ile) and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence (601)KMSKS(605). Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [(32)P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.
Neurobiology of pain in children: an overview.
Loizzo A, Loizzo S, Capasso A
Open Biochem J · 2009 Feb · PMID 19543535 · Full text
The evaluation of pain in the newborn and the infant is difficult because pain is mainly a subjective phenomenon. Until a few years ago, several myths persisted. First, the myth that children, especially infants, do not...
The evaluation of pain in the newborn and the infant is difficult because pain is mainly a subjective phenomenon. Until a few years ago, several myths persisted. First, the myth that children, especially infants, do not feel pain the way adults do, therefore there is no untoward consequences for them. Second, lack of assessment and reassessment for the presence of pain. Third, misunderstanding of how to conceptualise and quantify a subjective experience. Fourth, lack of knowledge of pain treatment. Fifth, the notion that addressing pain in children takes too much time and effort, in ultimate analysis resulting in wasting time. Sixth, fears of hidden -and not easy to diagnose or prevent- adverse effects of analgesic medications, including respiratory depression and addiction. Finally, from a conceptual point of view, high thresholds of pain in neonates and infants were considered to be present by natural character, and useful in protecting infant from pain during birth and transit through the narrow vaginal channel.The present review is focused on the description of different theories on the pain pathogenesis in children.
Does phenoloxidase contributed to the resistance? Selection with butane-fipronil enhanced its activities from diamondback moths.
Liu S, Niu H, Xiao T … +3 more , Xue C, Liu Z, Luo W
Open Biochem J · 2009 · PMID 19401784 · Full text
Using microtitration method, the relationship between Phenoloxidase activity and the resistance of the diamondback moth Plutella xylostella (Linnaeus) to the novel insecticide butane-fipronil was determined in vitro. Aft...
Using microtitration method, the relationship between Phenoloxidase activity and the resistance of the diamondback moth Plutella xylostella (Linnaeus) to the novel insecticide butane-fipronil was determined in vitro. After selection of the tenth-generation by butane-fipronil, the resistance of the fourth instar larvae was increased 83.80-fold as compared to the susceptible strain. Phenoloxidase activity of the resistant strain (PO(r)) was 1.29-fold higher than the susceptible one (PO(s)). However, the Km and optimum pH values were similar in resistant and susceptible strains, which were 1.11 mM and 6.5, respectively. Both PO(r) and PO(s) have maximum stability at pH values less than 7.0, although PO(s) was less stable at lower pH values than PO(r). In addition, the thermal stabilities of the two phenoloxidase were very similar. It is suggested that PO may play an important role in the increasing resistance of pests to pesticides.
Calcium-induced conformational transition of trout ependymins monitored by tryptophan fluorescence.
Ganss B, Hoffmann W
Open Biochem J · 2009 · PMID 19401757 · Full text
Ependymins are secretory, calcium-binding sialoproteins which are the predominant constituents of the cerebrospinal fluid of many teleost fish. A bound form of these regeneration-responsive glycoproteins is associated wi...
Ependymins are secretory, calcium-binding sialoproteins which are the predominant constituents of the cerebrospinal fluid of many teleost fish. A bound form of these regeneration-responsive glycoproteins is associated with collagen fibrils of the extracellular matrix. Here, the tryptophan fluorescence of ependymins was monitored at various Ca(2+) concentrations. Two distinct states were identified with a relatively sharp transition at about 1 mM Ca(2+). In agreement with previous circular dichroism measurements, this strongly supports the hypothesis that a calcium-induced conformational change is important for the interaction of ependymins with components of the extracellular matrix. Such interactions with constituents of various basal laminae would also explain the important roles of piscine ependymins as well as invertebrate and mammalian ependymin-related proteins for cell adhesion processes and cell migration.
Protein domain boundary predictions: a structural biology perspective.
Kirillova S, Kumar S, Carugo O
Open Biochem J · 2009 · PMID 19401756 · Full text
One of the important fields to apply computational tools for domain boundaries prediction is structural biology. They can be used to design protein constructs that must be expressed in a stable and functional form and mu...
One of the important fields to apply computational tools for domain boundaries prediction is structural biology. They can be used to design protein constructs that must be expressed in a stable and functional form and must produce diffraction-quality crystals. However, prediction of protein domain boundaries on the basis of amino acid sequences is still very problematical. In present study the performance of several computational approaches are compared. It is observed that the statistical significance of most of the predictions is rather poor. Nevertheless, when the right number of domains is correctly predicted, domain boundaries are predicted within very few residues from their real location. It can be concluded that prediction methods cannot be used yet as routine tools in structural biology, though some of them are rather promising.
Characterization of the Cdc6 Homologues from the Euryarchaeon Thermoplasma acidophilum.
Haugland GT, Innselset M, Madern D … +1 more , Birkeland NK
Open Biochem J · 2008 · PMID 19238189 · Full text
Archaeal cell division cycle protein 6 (Cdc6) homologues are thought to be involved in the initiation process of DNA replication. In the present study, a biochemical characterization of the two Cdc6 proteins from the arc...
Archaeal cell division cycle protein 6 (Cdc6) homologues are thought to be involved in the initiation process of DNA replication. In the present study, a biochemical characterization of the two Cdc6 proteins from the archaeon Thermoplasma acidophilum has been performed. Both TaCdc6-1 and TaCdc6-2 behave as monomers in solution and both are abundantly expressed in vivo. Further, TaCdc6-1 shows strong ability to undergo autophosphorylation compared to TaCdc6-2 and the autophosphorylation activity is not affected by DNA or MCM.
Influence of aromatic and aliphatic moieties on thrombin inhibitors potency.
Poyarkov A, Rocabayera X, Poyarkova S … +1 more , Kukhar V
Open Biochem J · 2008 · PMID 19238188 · Full text
Thrombin is a plasma serine protease that plays a key role in coagulation and hemostasis but also in thromboembolic diseases. Direct thrombin inhibitors could be beneficial for future anticoagulant therapy in the prophyl...
Thrombin is a plasma serine protease that plays a key role in coagulation and hemostasis but also in thromboembolic diseases. Direct thrombin inhibitors could be beneficial for future anticoagulant therapy in the prophylaxis of venous and arterial thrombosis as well as myocardial infarction. To design the efficient thrombin inhibitors we have synthesized and studied peptide-based inhibitors resistant to enzymatic degradation. Compounds with general formula X-DArg-D-Phe-OMe, where X = residue of 3-[6-ethyl-7-hydroxy-3-(4-methyl-thiazol-2-yl)-4-oxo-4H-chromen-2-yl]-propionic acid (chromone) and lauric acid were synthesized by classic methods of peptides synthesis in solution. The comparative inhibitory analysis of prepared compounds in relation to thrombin was conducted. The analysis of the inhibition effect of the peptide with retro-D-sequence modified by residues of natural organic compounds (chromone or fatty acid moiety) has demonstrated that modification with the fatty acid residue appeared to be the most successful one. Introduction of lauric acid residue (Ki = 1,76 muM) maximally increased the inhibition effect. These findings establish an important role of fatty moiety in structure of inhibitors in preferential binding and inhibition of thrombin active side.
Biosynthesis of natural and hyperelongated chondroitin sulfate glycosaminoglycans: new insights into an elusive process.
Little PJ, Ballinger ML, Burch ML … +1 more , Osman N
Open Biochem J · 2008 · PMID 19238187 · Full text
Proteoglycans are important components of the extracellular matrix of all tissues. Proteoglycans are comprised of a core protein and one or more covalently attached glycosaminoglycan (GAG) chains. The major chondroitin s...
Proteoglycans are important components of the extracellular matrix of all tissues. Proteoglycans are comprised of a core protein and one or more covalently attached glycosaminoglycan (GAG) chains. The major chondroitin sulfate (CS) and dermatan sulfate (DS) proteoglycans are aggrecan, versican, biglycan and decorin. Cells synthesize GAGs of natural or basal lengths and the GAG chains are subject to considerable growth factor, hormonal and metabolic regulation to yield longer GAG chains with altered structure and function. The mechanism by which the CS/DS GAG chains are polymerized is unknown. Recent work has identified several monosaccharide transferases which when co-expressed yield GAG polymers and the length of the polymers depends upon the pair of enzymes coexpressed. The further extension of these chains is regulated by signaling pathways. Inhibition of these latter pathways may be a therapeutic target to prevent the elongation which is associated with increased binding of atherogenic lipids and the disease process of atherosclerosis.
Differential activity of caspase-3 regulates susceptibility of lung and breast tumor cell lines to Paclitaxel.
Odonkor CA, Achilefu S
Open Biochem J · 2008 · PMID 19238186 · Full text
Recent development of tumor resistance to paclitaxel presents a major problem to cancer treatment. An unsettled controversy in the cancer chemotherapy field, however, is whether caspases play a prominent role in paclitax...
Recent development of tumor resistance to paclitaxel presents a major problem to cancer treatment. An unsettled controversy in the cancer chemotherapy field, however, is whether caspases play a prominent role in paclitaxel-induced death in tumors. Previous studies suggest that cleavage of caspase-3 is not instrumental for the execution of death in tumors treated with paclitaxel, while other reports indicate that caspase-dependent pathways may be critical for paclitaxel cytotoxicity. In this study, we investigated the role of caspase-3 in breast and lung tumor cell line sensitivity to paclitaxel. Clonogenic survival and live/dead viability-assays, together with enzymatic activity and cell proliferation assays, reveal that the levels of paclitaxel-induced caspase-3 enzymatic activity in tumor cells correlate directly with tumor sensitivity to the drug.We observed a 2-fold increase in caspase-3 activity in 4T1-Luc breast tumor cells, but a 3-fold and 4-fold decrease in A549 and A427 lung tumor cell lines, respectively. Together, our results suggest that caspase-activation and activity levels are not only key determinants of paclitaxel-induced death in tumors but also serve as good indicators for tumor susceptibility to paclitaxel therapy. Our studies also indicate that within clinically relevant doses of paclitaxel, the ability to rid tumor populations of dormant tumor cells controls the rate of tumor recurrence.
Subunit Mobility and the Chaperone Activity of Recombinant alphaB-Crystallin.
Krushelnitsky A, Mukhametshina N, Gogolev Y … +5 more , Tarasova N, Faizullin D, Zinkevich T, Gnezdilov O, Fedotov V
Open Biochem J · 2008 · PMID 18949083 · Full text
The comparison of the chaperone-like activity of native and covalently cross-linked human alphaB-crystallins has confirmed the important role of the subunit mobility in the chaperoning mechanism. Our data clearly demonst...
The comparison of the chaperone-like activity of native and covalently cross-linked human alphaB-crystallins has confirmed the important role of the subunit mobility in the chaperoning mechanism. Our data clearly demonstrate that the chaperone-like activity of alpha-crystallin is not only a surface phenomenon as was suggested by some researchers.
Impact of the L-arginine-Nitric Oxide Pathway and Oxidative Stress on the Pathogenesis of the Metabolic Syndrome.
C R A, T M C B, C M … +2 more , A C R, A C MR
Open Biochem J · 2008 · PMID 18949082 · Full text
The discovery of the physiological roles of nitric oxide has revolutionized the understanding of regulation of vascular tone, platelet adhesion and aggregation, and immune activation. Perhaps the most intriguing aspect o...
The discovery of the physiological roles of nitric oxide has revolutionized the understanding of regulation of vascular tone, platelet adhesion and aggregation, and immune activation. Perhaps the most intriguing aspect of nitric oxide (NO) is that it is a gas that, in the absence of receptors, can regulate both normal physiological events and mediate cytotoxicity under pathological conditions. NO is produced from L-arginine by NO synthases (NOS), yielding L-citrulline and NO. The regulation of L-arginine pathway activity occurs at the level of NO production. The metabolic syndrome is a cluster of insulin resistance, elevated blood pressure, and atherogenic dyslipidemia, a common basis of cardiovascular disease. It occurs in genetically susceptible individuals with environmental influences and has serious economic and social consequences. Pharmacological and non-pharmacological therapies should be individualized and targeted to normalize its alterations of blood pressure, HDL cholesterol, triglycerides and glucose values. Despite the increasing prevalence of the metabolic syndrome in the last decades, there has been little progress in the understanding of the precise mechanisms involved in the pathogenesis of this syndrome and its complications. Emerging evidence is available that NO, inflammation and oxidative stress play important roles in the physiopathology of this syndrome. This review summarizes and evaluates the participation of the L-arginine-NO pathway and oxidative stress in the physiopathology of the metabolic syndrome and cardiovascular events at the systemic level, as well as the effects of exercise on this syndrome.
Identification of Metastasis Associated Antigen 1 (MTA1) by Serological Screening of Prostate Cancer cDNA Libraries.
Geng L, Deepak P A, Aija L … +4 more , Fuming C, Amanda M, Robert C R, Stephanie E B M
Open Biochem J · 2008 · PMID 18949081 · Full text
Over the past 10 years the serological analysis of recombinant cDNA expression libraries (SEREX) has proved to be an effective method for the identification of tumour antigens. In the present study, two prostate cancer l...
Over the past 10 years the serological analysis of recombinant cDNA expression libraries (SEREX) has proved to be an effective method for the identification of tumour antigens. In the present study, two prostate cancer libraries were constructed and screened using autologous sera. Fifty five genes were isolated, including 46 known genes and 9 previously uncharacterised genes. Among the known genes, a metastasis-associated gene, MTA1, previously identified by differential cDNA hybridisation, was preferentially expressed in a panel of malignant tissues compared with normal tissues, as analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). MTA1 transcripts were observed to be over-expressed in normal human testes as well as various cancer tissues when compared to the panel of normal tissues. MTA1 antigen reacted with 2 of 13 allogeneic prostate cancer patient sera tested, but no sera reactivity was observed to any of the normal adult sera tested. Furthermore, a similar distribution and expression level of MTA-1 was observed in murine tissues and cancer cell lines. Based on these findings and previous reports on the literature on this gene, MTA-1 can be considered not only as a "biomarker" of aggressive disease but also as a potential therapeutic target.
Properties of the SR Ca-ATPase in an Open Microsomal Membrane Preparation.
A F, C J, H-J A
Open Biochem J · 2008 · PMID 18949080 · Full text
SR vesicles isolated from rabbit muscle were treated by a SDS incubation and subsequent dialysis to obtain open membrane fragments that allow a direct access to the luminal membrane surface and especially to the ion-bind...
SR vesicles isolated from rabbit muscle were treated by a SDS incubation and subsequent dialysis to obtain open membrane fragments that allow a direct access to the luminal membrane surface and especially to the ion-binding sites in the P-E(2) conformation of the Ca-ATPase. The open membrane fragments showed about 80% of the enzyme activity in the untreated membranes. Pump function was investigated by using electrochromic styryl dyes. The kinetic properties of cytoplasmic ion binding showed no significant differences between the Ca-ATPases in SR vesicles and in membrane fragments. From pH-dependent Ca(2+) binding it could be deduced that due to the SDS treatment the density of negatively charged lipid was increased by one elementary charge per 12 lipid molecules. Major differences between Ca-ATPase from SR vesicles and membrane fragments were the respective fluorescence amplitudes. This effect is, however, produced by dye-lipid interaction and not by pump function. It was demonstrated that time-resolved kinetics may be study by the use of caged compounds such as caged ATP or caged calcium also in the case of the membrane fragments.
Nitrergic response to cyclophosphamide treatment in blood and bone marrow.
G A K, N Kh A, N H M … +4 more , H L H, A G G, R M O, S S D
Open Biochem J · 2008 · PMID 18949079 · Full text
Daily intraperitoneal injection of cyclophosphamide (CPA) (50 mgkg(-1) of body weight) for 5 days resulted in reduced levels of marrow and blood cellularity, which was most pronounced in 18 days post-treatment (pt). On d...
Daily intraperitoneal injection of cyclophosphamide (CPA) (50 mgkg(-1) of body weight) for 5 days resulted in reduced levels of marrow and blood cellularity, which was most pronounced in 18 days post-treatment (pt). On day 18 after CPA treatment the enhancedlevels of nitric oxide (NO) precursors and metabolites (L-arginine, L-citrulline, reactive nitrogen species (RNS)) of marrow and blood cells (platelet, neutrophil, lymphocyte and monocyte) resulted from up-regulation of Ca(II)/calmodulin(CaM)-independent "inducible" NO synthase (iNOS), with a lessercontribution of Ca(II)/CaM-dependent "constitutive" cNOS isoforms to systemic NO.Biphasic response to CPA of marrow nitrergic system, i.e. both iNOS and cNOS showed significantly depressed activities, as well as diminished levels of NO metabolites on day 9 pt, suggested that signals in addition to NO might be involved in CPA-induced inhibition of hematopoesis, while a gradual increase of neutrophil and platelet NOS activity appeared to be contributed to a CPA-induced development of granulopenia, thrombocytopenia and hemorrhage.
Prediction of Residue Status to Be Protected or Not Protected From Hy-drogen Exchange Using Amino Acid Sequence Only.
Nikita V D, Oxana V G
Open Biochem J · 2008 · PMID 18949078 · Full text
We have outlined here some structural aspects of local flexibility. Important functional properties are related to flexible segments. We try to predict regions that have been shown to exhibit the highest probability of b...
We have outlined here some structural aspects of local flexibility. Important functional properties are related to flexible segments. We try to predict regions that have been shown to exhibit the highest probability of being folded in the equilibrium intermediate or native state and will be protected from hydrogen exchange using amino acid sequence only. Our approach FoldUnfold for the prediction of unstructured regions has been applied to seven different proteins. For 80% of the residues considered in this paper we can predict correctly their status: will they be protected or not from hydrogen exchange. An additional goal of our study is to assess whether properties inferred using the bioinformatics approach are easily applicable to predict behavior of proteins in solution.
Regulation of glycogen synthase kinase 3beta functions by modification of the small ubiquitin-like modifier.
Eun Jeoung L, Sung Hee H, Jaesun C … +5 more , Sung Hwa S, Kwang Hum Y, Min Kyoung K, Tae Yoon P, Sang Sun K
Open Biochem J · 2008 · PMID 18949077 · Full text
Modification of the Small Ubiquitin-like Modifier (SUMO) (SUMOylation) appears to regulate diverse cellular processes, including nuclear transport, signal transduction, apoptosis, autophagy, cell cycle control, ubiquitin...
Modification of the Small Ubiquitin-like Modifier (SUMO) (SUMOylation) appears to regulate diverse cellular processes, including nuclear transport, signal transduction, apoptosis, autophagy, cell cycle control, ubiquitin-dependent degradation and gene transcription. Glycogen synthase kinase 3beta (GSK 3beta) is a serine/threonine kinase that is thought to contribute to a variety of biological events, including embryonic development, metabolism, tumorigenesis, and cell death. GSK 3beta is a constitutively active kinase that regulates many intracellular signaling pathways by phosphorylating substrates such as beta-catenin. We noticed that the putative SUMOylation sites are localized on K(292 )residueof (291)FKFPQ(295) in GSK 3beta based on analysis of the SUMOylation consensus sequence. In this report, we showed that the SUMOylation of GSK 3beta occurs on its K(292) residue, and this modification promotes its nuclear localization in COS-1. Additionally, our data showed that the GSK 3beta SUMO mutant (K292R) decreased its kinase activity and protein stability, affecting cell death. Therefore, our observations at first time suggested that SUMOylation on the K(292) residue of GSK 3beta might be a GSK 3beta regulation mechanism for its kinase activation, subcellular localization, protein stability, and cell apoptosis.
The role of herpes simplex virus-1 thymidine kinase alanine 168 in substrate specificity.
Candice L W, Django S, Margaret E B
Open Biochem J · 2008 · PMID 18949076 · Full text
Herpes simplex virus type 1 (HSV) thymidine kinase (TK) has been widely used in suicide gene therapy for the treatment of cancer due to its broad substrate specificity and the inability of the endogenous human TK to phos...
Herpes simplex virus type 1 (HSV) thymidine kinase (TK) has been widely used in suicide gene therapy for the treatment of cancer due to its broad substrate specificity and the inability of the endogenous human TK to phosphorylate guanosine analogs such as ganciclovir (GCV). The basis of suicide gene therapy is the introduction of a gene that encodes a prodrug-activating enzyme into tumor cells. After administration, the prodrug is selectively converted to a toxic drug by the suicide gene product thereby bringing about the eradication of the cancer cells. A major drawback to this therapy is the low activity the enzyme displays towards the prodrugs, requiring high prodrug doses that result in adverse side effects. Earlier studies revealed two HSV TK variants (SR39 and mutant 30) derived by random mutagenesis with enhanced activities towards GCV in vitro and in vivo. While these mutants contain multiple amino acid substitutions, molecular modeling suggests that substitutions at alanine 168 (A168) may be responsible for the observed increase in prodrug sensitivity. To evaluate this, site-directed mutagenesis was used to individually substitute A168 with phenylalanine or tyrosine to reflect the mutations found in SR39 and mutant 30, respectively. Additionally, kinetic parameters and the ability of these mutants to sensitize tumor cells to GCV in comparison to wild-type thymidine kinase were determined.
Signal transduction of fertilization in frog eggs and anti-apoptotic mechanism in human cancer cells: common and specific functions of membrane microdomains.
Sato K
Open Biochem J · 2008 · PMID 18949075 · Full text
Membrane microdomains or lipid/membrane rafts are distinct areas on the plasma membranes, where a specific subset of lipids (e.g. cholesterol, sphingolipids) and proteins (e.g. glycosylphosphatidylinositol-anchored prote...
Membrane microdomains or lipid/membrane rafts are distinct areas on the plasma membranes, where a specific subset of lipids (e.g. cholesterol, sphingolipids) and proteins (e.g. glycosylphosphatidylinositol-anchored proteins, growth factor receptor/kinases) are getting together and functioning for several aspects of cellular functions. Our recent investigation has revealed that fertilization of African clawed frog, Xenopus laevis, requires cholesterol-dependent nature of egg membrane microdomains. Moreover, fertilization of Xenopus eggs involves proteolytic cleavage of the extracellular part and subsequent phosphorylation of a cytoplasmic tyrosine residue of uroplakin III, an egg membrane microdomain-associated protein. Protease activity toward uroplakin III seems to be derived from fertilizing sperm, while phosphorylation of uroplakin III seems to be catalyzed by the egg tyrosine kinase Src, whose activation is required for cytoplasmic rearrangement of fertilized eggs; so-called 'egg activation'. Therefore, it is assumed that uroplakin III serves an integral part of signal transduction in fertilization of Xenopus. Our more recent study on human cancer cells has revealed that a similar but distinct scheme of signal transduction operates in anti-apoptotic growth of cells. Namely, in human bladder carcinoma cells, cooperation of uroplakin III and Src, both of which localize to the membrane microdomains, allows cells to escape from apoptotic cell death and proliferate under culture conditions deprived of serum. In this review, I briefly introduce about biology of fertilization and cancer, and then present and discuss our experimental data on general importance and specific features of membrane microdomains in Xenopus fertilization and anti-apoptosis in human bladder carcinoma cells.
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