kDa Subunit Research Articles

Med12 Mutations Promote Castration Resistant Prostate Cancer through Hyperactivated GLI3/SHH Signaling

Prostate cancer is the highest diagnosed and second most common cause of cancer death among US men. The onset and progression of prostate cancer is critically dependent upon androgens through their interaction with the androgen receptor (AR). Accordingly, it has long been established that androgen deprivation therapy leads to prostate cancer regression. Though androgen deprivation therapies are initially highly successful, most patients eventually suffer from recurrence of the disease in the form of castration resistant prostate cancer (CRPC). Currently there is no effective treatment for CRPC thereby highlighting the compelling need to address resistance mechanisms for patients that suffer from this lethal disease. Sonic hedgehog (SHH) signaling is a stromal‐epithelial interacting pathway that is critical for prostate development and cell growth. SHH signaling is induced by androgen deprivation thereby leading to re‐activation of AR target genes to promote prostate cancer cell growth in the absence of androgens. Furthermore, AR interacts with GLI proteins, downstream effectors in the SHH pathway. Therefore, GLI3‐dependent SHH signaling in androgen deprived cells could promote re‐activation of AR target genes and consequently lead to CRPC. It is currently unknown how GLI3‐dependent SHH signaling is induced in CRPC. In this study we show that stabilized GLI3 leads to the induction of GLI3‐dependent SHH signaling in prostate cancers that harbor mutations in MED12. MED12 is an Xq13‐encoded 230 kDa subunit of the RNA pol II transcriptional Mediator that has been found to be mutated in ~5% of prostate cancers. Through stable knockdown and luciferase assays, we show that MED12 mutations disrupt a Mediator‐imposed constraint on GLI3‐dependent SHH signaling. Furthermore, through stable knockdown and xenograft studies, we show that stable GLI3 is critically dependent for CRPC growth. The results of our study therefore indicate that prostate cancer patients with MED12 mutations relapse to CRPC after androgen deprivation therapy by hyperactivating the GLI3 dependent SHH signaling pathway. Overall, this study provides critical insight into possible treatment strategies for prostate cancer patients with MED12 mutations by using the GLI3/SHH signaling axis as a therapeutic target.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Site-specific observation of the conformational change of a protein with 15N-labeled Tyr residues using NMR

One of the reasons it is difficult to analyze protein structural dynamics at atomic resolution using NMR is the molecular size of the protein. The selective amino acid labeling method is one of the effective methods that can solve this problem. In this study, to determine the site-specific conformational change in 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD), which forms a dimer composed of two 26 kDa subunits, we expressed and purified 15N-Tyr labeled Ps3αHSD and its mutants, and analyzed the conformational change upon NADH binding. Using the Tyr substituted mutants, we first assigned the respective signals of four Tyr residues. In the titration experiments with NADH, the four Tyr signals changed uniquely; changes in chemical shift and signal broadening were observed. The NADH binding affinity, determined from the plots of the 1H and 15N chemical shift changes, was comparable to those reported previously. Together with the crystal structure information for Ps3αHSD in the NADH-free and -bound states, site-specific conformational changes including environmental changes could be deduced.

Cardiac proteomics reveals the potential mechanism of microtubule associated protein 4 phosphorylation-induced mitochondrial dysfunction

BackgroundOur previous work suggested that microtubule associated protein 4 (MAP4) phosphorylation led to mitochondrial dysfunction in MAP4 phosphorylation mutant mice with cardiomyopathy, but the detailed mechanism was still unknown. Thus, the aim of this study was to investigate the potential mechanism involved in mitochondrial dysfunction responsible for cardiomyopathy.MethodsThe present study was conducted to explore the potential mechanism underlying the mitochondrial dysfunction driven by MAP4 phosphorylation. Strain of mouse that mimicked constant MAP4 phosphorylation (S737 and S760) was generated. The isobaric tag for relative and absolute quantitation (iTRAQ) analysis was applied to the heart tissue. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) were all analyzed on the basis of differential expressed proteins (DEPs).ResultsAmong the 72 cardiac DEPs detected between the two genotypes of mice, 12 were upregulated and 60 were downregulated. GO analysis showed the biological process, molecular function, and cellular component of DEPs, and KEGG enrichment analysis linked DEPs to 96 different biochemical pathways. In addition, the PPI network was also extended on the basis of DEPs as the seed proteins. Three proteins, including mitochondrial ubiquitin ligase activator of NF-κB 1, reduced form of nicotinamide adenine dinucleotide (NADH)-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial and growth arrest, and DNA-damage-inducible proteins-interacting protein 1, which play an important role in the regulation of mitochondrial function, may correlate with MAP4 phosphorylation-induced mitochondrial dysfunction. Western blot was used to validate the expression of the three proteins, which was consistent with iTRAQ experiments.ConclusionsThese findings revealed that the DEPs caused by MAP4 phosphorylation in heart tissue using iTRAQ technique and may provide clues to uncover the potential mechanism of MAP4 phosphorylation-induced mitochondrial dysfunction.

Statistically optimized production of extracellular l-methionine γ-lyase by Streptomyces Sp. DMMMH60 and evaluation of purified enzyme in sub-culturing cell lines

The optimal conditions for the production of l-methionine γ-lyase (MGL) by a soil bacterium, Streptomyces DMMMH60, were established and their effects were compared using Plackett–Burman design (PBD). Accordingly, the optimal conditions of beet molasses, yeast extract, l-methionine, MgCl2, Tween 80, initial pH, aeration, temperature, inoculum size and incubation period for the maximum MGL production (60.7 U/mg) were 23.5 g/L, 0.79 g/L, 0.87 g/L, 0.85 g/L, 0.41%, 8.3, 50 ml, 33 °C, 5.2 ml, and 4 day, respectively. Initial pH was found to be the most significant factor for the production of MGL based on the calculated percentage of participation P (%) from an analysis of the variance (ANOVA). Additionally, MGL was purified from the supernatant of S. DMMMH60 using heat treatment, DEAE-cellulose and Sephadex G100, and the purified enzyme displaying 3.15-fold in comparison with the crude enzyme. Purified enzyme exhibiting a single band of 55 kDa subunits on the SDS-PAGE and the enzyme had maximum activity in pH 8 at 45 °C with ionic stability within pH range 6.5–8.5 and thermal stability below 60 °C. In-vitro, MGL showed a significant cytotoxic effect against cancer cell lines whereas those for normal cells was demonstrated a negligible toxicity.

Extraction and characterisation of analytical grade C-phycocyanin from Euhalothece sp.

C-Phycocyanin (C-PC) is a blue pigment that has great commercial value as a fluorophore in biomedical research, diagnostics therapeutics as well as sensitive fluorescent applications. However, the cost and application of C-PC is highly dependent on its purity ratio (A620nm/A280nm). In this study, freeze-thaw and precipitation are presented as relatively simple, efficient, low-cost C-PC extraction and purification methods from Euhalothece sp. Harvested biomass (0.03 g) was subjected to − 20 °C for 5 h, re-suspended in 50 mM, (pH 7) sodium phosphate buffer (containing 0.0025 mg mL−1 lysozyme), incubated at 37 °C thereafter thawed at 4 °C for 4 h. The optimised method yielded 75 mg g−1 of crude C-PC. A high purity ratio of 5 was achieved using activated charcoal and 30–60% (w/v) ammonium sulphate precipitation. SDS-PAGE of purified C-PC yielded two bands corresponding to α (17 kDa) and β (18 kDa) subunits. The purified C-PC was found to be highly stable at a pH range of 5–8 and a temperature of 45 °C. The addition of preservatives sodium azide and sodium citrate (at 4 °C) proved to be suitable for preservation of C-PC for up to 42 weeks. By following the optimised extraction and purification protocol, we were able to purify a high yield of stable analytical grade C-PC from Euhalothece sp.

Adaptive effect of sericin on hepatic mitochondrial conformation through its regulation of apoptosis, autophagy and energy maintenance: a proteomics approach

We recently demonstrated that in addition to its protective effect on pancreatic and adrenal biosynthesis, antioxidant properties of sericin decrease blood cholesterol levels and improve the liver mitochondrial architecture. However, little is known about the detailed mechanisms underlying these effects. Using proteomics and electron microscopy, we identified mitochondrial proteins that play important roles in the preservation of the mitochondrial ultrastructure and cholesterol-lowering properties of sericin. Our results showed that sericin maintains the mitochondrial architecture during conditions of high blood cholesterol by regulating apoptotic (NADH-ubiquinone oxidoreductase 75 kDa subunit) and autophagic (mitochondrial elongation factor Tu and prohibitin-2) proteins as well as energy maintenance proteins [haloacid dehalogenase-like hydrolase domain-containing protein 3, succinate dehydrogenase (ubiquinone) flavoprotein subunit, ATP synthase-α subunit precursor, enoyl-CoA hydratase domain-containing protein 3 and electron transfer flavoprotein subunit-α]. Sericin also exerts anti-oxidative properties via aconitate hydratase and Chain A, crystal structure of rat carnitine palmitoyltrasferase 2 proteins. Together, these activities may reduce hepatocytic triglyceride deposition, thereby decreasing steatosis, as demonstrated by the modulatory effects on ornithine aminotransferase, mitochondrial aspartate aminotransferase, acyl-CoA synthase, hydroxyacyl-CoA dehydrogenase and D-beta-hydroxybutyrate dehydrogenase. Sericin activity further balanced nitrogenous waste detoxification, characterised by carbamoyl-phosphate synthase (ammonia), aldehyde dehydrogenase and uricase, or folate biosynthesis via sarcosine dehydrogenase and dimethyl glycine dehydrogenase. These results suggest that sericin maintains the hepatic mitochondrial architecture through apoptotic, autophagic, energy maintenance and anti-oxidative mitochondrial proteins for alleviating hepatic steatosis and promoting liver function under conditions of hypercholesterolaemia.

Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato.

Shigella dysenteriae causing shigellosis is one of the diseases that threaten the health of human society in the developing countries. In Shigella, IpaD gene is one of the key pathogenic genes causing strong mucosal immune system reactions. Anthrax disease is caused by Bacillus anthracis. PA protective antigen is one of the subunits in anthrax toxin complex responsible for the transfer of other subunits into the cytosol of host cells. The 20 kDa subunit of PA (PA20) has the property of immunogenicity. CTxB or B subunit of Vibrio cholerae toxin (CT) is a non-toxic protein and has the function to transfer toxic subunit into cytosol of the host cells by binding to GM1 receptor. The aim of this study was to fuse PA20, ipaD and CTxB and transform tomato plants by this cassette in order to produce an oral vaccine against shigellosis, anthrax and cholera. CTxB was used for these two antigens as an immune adjuvant. IpaD and PA20 genes were cloned in pBI121 containing the CTxB gene and Extensin signal peptide. In order to evaluate the transient expression of Shigellosis, Anthrax and Cholera antigens, agro-infiltrated tomato tissues were inoculated with Agrobacterium tumefaciens containing the gene cassette. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. Expression of the antigens was examined by SDS-PAGE, dot blot and ELISA. Maturate green fruits demonstrated the highest expression of the recombinant proteins. The first phase of this study was carried out for cloning and expressing of CtxB, ipaD and PA20 antigens in tomato. In the next phase, we aim to analyze the immunogenicity of this vaccine candidate in laboratory animals.

Distinct substrate specificity and physicochemical characterization of native human hepatic thymidine phosphorylase.

Thymidine phosphorylase (TP; EC 2.4.2.4) is involved regulation of intra- or extracellular thymidine concentration, angiogenesis, cancer chemotherapy, radiotherapy, as well as tumor imaging. Although the liver is main site of pyrimidine metabolism and contains high levels of TP, nonetheless, purification and characterization of human hepatic TP has not been accomplished. We here report the purification and characterization of native human hepatic TP. The enzyme was purified to apparent homogeneity by a procedure shorter and more efficient than previously reported methods. Human hepatic TP has an apparent Kthymidine of 285 ± 55 μM. Like the enzyme from other tissues, it is highly specific to 2'-deoxyribosides. However, in contrast to TP from other normal tissues, the hepatic enzyme is active in the phosphorolysis of 5'-deoxy-5-fluorouridine, and the riboside 5-fluorouridine. Furthermore, native hepatic TP exists in different aggregates of 50 kDa subunits, with unknown aggregation factor(s) while TP from extra tissues exists as a homodimer. Isoelectric point was determined as 4.3. A total of 65 residues in the N-terminal were sequenced. The sequence of these 65 amino acids in hepatic TP has 100% sequence and location homology to the deduced amino acid sequence of the platelet derived-endothelial cell growth factor (PD-ECGF) cDNA. However, and contrary to PD-ECGF, the N-terminal of hepatic TP is blocked. The block was neither N-formyl nor pyrrolidone carboxylic acid moieties. The differences in substrate specificities, existence in multimers, and weak interaction with hydroxyapatite resin strongly suggest that hepatic TP is distinct from the enzyme in normal extrahepatic tissues. These results may have important clinical implications when TP is involved in activation or deactivation of chemotherapeutic agents in different tissues.

Clathrin-dependent endocytosis is associated with RNAi response in the western corn rootworm, Diabrotica virgifera virgifera LeConte.

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism.

Functional Characterization of a New Cold-Adapted β-Galactosidase from an Arctic Fjord Sediment Bacteria Enterobacter ludwigii MCC 3423

In the present study, a new cold-adapted β-galactosidase, BgalEL isolated from the fjord sediment bacteria, Enterobacter ludwigii MCC 3423, was purified and characterized. The phylogenetic analysis of partial sequence of bgalEL gene revealed 99% relatedness of the enzyme to Enterobacter cloacae β-galactosidase. BgalEL is a homotetramer with molecular weight 465 kDa composed of ~ 116.42 kDa subunits. The optimal pH range and temperature for maximum hydrolytic activity on ONPG were 7.0–8.0 and 45 °C respectively. BgalEL was stable at pH ranges 6.0–8.0. The Km and Vmax for BgalEL were recorded as 2.03 mM and 49.3 U mg−1 respectively. The product formation by BgalEL was modelled with various degradation kinetic models and zeroth was found as model was best-fitted model. The kinetic half-life period of ONPG was noted as 9.0 × 10−4. The presence of K+, Mg2+ and Mn2+ at 10 mM concentrations stimulated BgalEL activity by 31 ± 3.9, 34 ± 1.2 and 42 ± 1.9% respectively. It was interesting to see that BgalEL was least affected by the hydrolytic products, glucose and galactose. Noteworthy, effect of Ca2+ ions on BgalEL activity was negligible at concentrations of 10 mM. BgalEL hydrolyzed lactose in milk at refrigerated temperature and also displayed transglycosylation potential in the presence of substantial concentrations of lactose at 40 °C. Finally, this is the first report with such a detailed characterization of β-galactosidase enzyme from a gram-negative bacterium isolated from the Arctic region. In light of the above mentioned properties, we thus recommend the enzyme BgalEL for the large scale production lactose free milk for the safe consumption in lactose-intolerance and the prebiotic galactooligosaccharides as supplement for infant food formulas.

Mitochondrial complex I in the post-ischemic heart: reperfusion-mediated oxidative injury and protein cysteine sulfonation

A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of O2−. The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to injury. Herein we employed LC-MS/MS to identify protein sulfonation of complex I in mitochondria from the infarct region of rat hearts subjected to 30-min of coronary ligation and 24-h of reperfusion in vivo as well as the mitochondria of sham controls. Mitochondrial preparations from the I/R regions had enhanced sulfonation levels on the cysteine ligands of iron‑sulfur clusters, including N3 (C425), N1b (C92), N4 (C226), N2 (C158/C188), and N1a (C134/C139). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus sulfonation of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and an antioxidative function. Increased C134/C139 sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O2− generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C125, C187, C206, C238, C255, C286), 75 kDa (C367, C554, C564, C727), 49 kDa (C146, C326, C347), and PSST (C188). These results were consistent with the consensus indicating that 51 kDa and 75 kDa are two of major subunits hosting regulatory thiols, and their enhanced sulfonation by I/R predisposed the myocardium to further oxidant stress with impaired ubiquinone reduction. MS analysis further showed I/R-mediated enhanced sulfonation at the supernumerary subunits of 42 kDa (C67, C112, C183, C253), 15 kDa (C43), and 13 kDa (C79). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C43 of the 15 kDa subunit forms an intramolecular disulfide bond with C56, which was reported to stabilize complex I structure. C79 of the 13 kDa subunit is involved in Zn2+-binding, which was reported functionally important for complex I assembly. C79 sulfonation by I/R was found to impair Zn2+-binding. No significant enhancement of protein sulfonation was observed in mitochondrial complex I from the rat heart subjected to 30-min ischemia alone in vivo despite a decreased state 3 respiration, suggesting that the physiologic conditions of hyperoxygenation during reperfusion mediated an increase in complex I sulfonation and oxidative injury. In conclusion, sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increasing •O2− production, and mediating redox dysfunction of complex I.

Production of a monoclonal antibody against a mannose-binding protein of Acanthamoeba culbertsoni and its localization

Amoebae from the genus Acanthamoeba are facultative pathogens of humans and other animals. In humans they most frequently infect the eye causing a sight threatening infection known as Acanthamoeba keratitis (AK), and also cause an often fatal encephalitis (GAE). A mannose-binding protein (MBP) has been identified as being important for Acanthamoeba infection especially in AK. This lectin has previously been characterized from Acanthamoeba castellanii as consisting of multiple 130 kDa subunits. MBP expression correlates with pathogenic potential and is expressed in a number of Acanthamoeba species. Here we report the purification of a similar lectin from Acanthamoeba culbertsoni and the production of a monoclonal antibody to it. The A. culbertsoni MBP was isolated by affinity chromatography using α-D-mannose agarose and has an apparent molecular weight of 83 kDa. The monoclonal antibody is an IgM that is useful in both western blots and immunofluorescence. We expect that this antibody will be useful in the study of the pathology of A. culbertsoni and in its identification in clinical samples.

Nitrate Assimilation Limits Nitrogen Use Efficiency (NUE) in Maize (Zea mays L.)

Grain yield in maize responds to N fertility in a linear-plateau fashion with nitrogen use efficiency (NUE) higher under lower N fertilities and less as grain yield plateaus. Field experiments were used to identify plant parameters relative for improved NUE in maize and then experiments were performed under controlled conditions to elucidate metabolism controlling these parameters. Field experiments showed reproductive parameters, including R1 ear-weight, predictive of N response under both high and low NUE conditions. R1 ear-weight could be changed by varying nitrate concentrations early during reproductive development but from V12 onward R1 ear-weight could be changed little by increasing or decreasing nitrate fertility. Ammonia, on the other hand, could rescue R1 ear-weight as late as V15 suggesting nitrate assimilation (NA) limits ear development response to N fertility since bypassing NA can rescue R1 ear-weight. Nitrate reductase activity (NRA (in vitro)) increases linearly with nitrate fertility but in vivo nitrate reductase activity (NRA (in vivo)) follows organic N accumulation, peaking at sufficient levels of nitrate fertility. The bulk of the increase in total plant N at high levels of nitrate fertility is due to increased plant nitrate concentration. Increasing NADH levels by selective co-suppression of ubiquinone oxidoreductase 51 kDa subunit (Complex I) was associated with improved grain yield by increasing ear size, as judged by increased kernel number plant−1 (KNP), and increased NRA (in vivo) without a change in NRA (in vitro). These results support NUE is limited in maize by NA but not by nitrate uptake or NRA (in vitro).

Accurate Sequence Analysis of a Monoclonal Antibody by Top-Down and Middle-Down Orbitrap Mass Spectrometry Applying Multiple Ion Activation Techniques.

Targeted top-down (TD) and middle-down (MD) mass spectrometry (MS) offer reduced sample manipulation during protein analysis, limiting the risk of introducing artifactual modifications to better capture sequence information on the proteoforms present. This provides some advantages when characterizing biotherapeutic molecules such as monoclonal antibodies, particularly for the class of biosimilars. Here, we describe the results obtained analyzing a monoclonal IgG1, either in its ∼150 kDa intact form or after highly specific digestions yielding ∼25 and ∼50 kDa subunits, using an Orbitrap mass spectrometer on a liquid chromatography (LC) time scale with fragmentation from ion-photon, ion-ion, and ion-neutral interactions. Ultraviolet photodissociation (UVPD) used a new 213 nm solid-state laser. Alternatively, we applied high-capacity electron-transfer dissociation (ETD HD), alone or in combination with higher energy collisional dissociation (EThcD). Notably, we verify the degree of complementarity of these ion activation methods, with the combination of 213 nm UVPD and ETD HD producing a new record sequence coverage of ∼40% for TD MS experiments. The addition of EThcD for the >25 kDa products from MD strategies generated up to 90% of complete sequence information in six LC runs. Importantly, we determined an optimal signal-to-noise threshold for fragment ion deconvolution to suppress false positives yet maximize sequence coverage and implemented a systematic validation of this process using the new software TDValidator. This rigorous data analysis should elevate confidence for assignment of dense MS2 spectra and represents a purposeful step toward the application of TD and MD MS for deep sequencing of monoclonal antibodies.

Ubiquitination of ABCE1 by NOT4 in Response to Mitochondrial Damage Links Co-translational Quality Control to PINK1-Directed Mitophagy

Translation of mRNAs is tightly regulated and constantly surveyed for errors. Aberrant translation can trigger co-translational protein and RNA quality control processes, impairments of which cause neurodegeneration by still poorly understood mechanism(s). Here we show that quality control of translation of mitochondrial outer membrane (MOM)-localized mRNA intersects with the turnover of damaged mitochondria, both orchestrated by the mitochondrial kinase PINK1. Mitochondrial damage causes stalled translation of complex-I 30 kDa subunit (C-I30) mRNA on MOM, triggering the recruitment of co-translational quality control factors Pelo, ABCE1, and NOT4 to the ribosome/mRNA-ribonucleoprotein complex. Damage-induced ubiquitination of ABCE1 by NOT4 generates poly-ubiquitin signals that attract autophagy receptors to MOM to initiate mitophagy. In the Drosophila PINK1 model, these factors act synergistically to restore mitophagy and neuromuscular tissue integrity. Thus ribosome-associated co-translational quality control generates an early signal to trigger mitophagy. Our results have broad therapeutic implications for the understanding and treatment of neurodegenerative diseases.

Effect of tRNA on the Maturation of HIV-1 Reverse Transcriptase

The mature HIV-1 reverse transcriptase is a heterodimer that comprises 66 kDa (p66) and 51 kDa (p51) subunits. The latter is formed by HIV-1 protease-catalyzed removal of a C-terminal ribonuclease H domain from a p66 subunit. This proteolytic processing is a critical step in virus maturation and essential for viral infectivity. Here, we report that tRNA significantly enhances in vitro processing even at a substoichiometric tRNA:p66/p66 ratio. Other double-stranded RNAs have considerably less pronounced effect. Our data support a model where interaction of p66/p66 with tRNA introduces conformational asymmetry in the two subunits, permitting specific proteolytic processing of one p66 to provide the mature RT p66/p51 heterodimer.

Structural characterization and antioxidant potential of phycocyanin from the cyanobacterium Geitlerinema sp. H8DM

We are reporting characterization of phycocyanin (PC), a prime light harvesting pigment, composed of nearly the same molecular weight of alpha- (17.5 kDa) and beta- (18.1 kDa) subunits in Geitlerinema sp. H8DM. Phycocyanin was purified using ammonium sulfate and anionic exchange chromatography. The fluorescence emission spectrum of PC was 637 nm when excited over 589 nm, which denoted integrity and functionality of protein structure. PC was depicted as a single subunit of ~18 kDa based on SDS-PAGE. However, similarity between both subunits was proven by two spots on gel using two dimensional gel electrophoresis. MALDI ToF/ToF showed structure similarities with alpha and beta subunits of PC. Urea induced Gibbs free energy denoted that folding and structural stability of this pigment is similar to phycocyanin of other species. Biophysical properties of peptide sequence were analyzed for several parameters and 3D model for its correlation as antioxidant. Furthermore, verification of in-silico data of PC was done by in vitro anti-oxidant assays. Hence, due to high availability, stability and antioxidant properties it can be used for auxiliary applications as colorant and therapeutic agent against oxidative stress.

Purification of R-phycoerythrin from Gracilaria lemaneiformis by centrifugal precipitation chromatography

Centrifugal precipitation chromatography (CpC) is a powerful chromatographic technique invented in the year 2000 but so far very little applied. The method combines dialysis, counter-current and salting out processes. The separation rotor consists of two identical spiral channels separated by a dialysis membrane (6–8 K MW cut-off) in which the upper channel is eluted with an ammonium sulfate gradient and the lower channel with water, and the mixtures are separated according to their solubility in ammonium sulfate as a chromatographic technique. In the present study, the method was successfully applied for separation and purification of R-phycoerythrin (R-PE), a protein widely used as a fluorescent probe, from the red alga Gracilaria lemaneiformis. The separation was performed with the elution of ammonium sulfate from 50% to 0% in 21.5 h at a flow rate of 0.5 ml/min, while the lower channel was eluted with water at a flow rate of 0.05 ml/min after sample charge, and the column was rotated at 200 rpm. After a single run, the absorbance ratio A565/A280 (a criterion for the purity of R-PE) was increased from 0.5 of the crude to 6.5. The purified R-PE exhibited a typical “three peaks” spectrum with absorbance maximum at 497, 538 and 565 nm. The Native-PAGE showed one single protein band and 20 kDa (subunits α and β) and 30 kDa (subunit γ) can be observed in SDS-PAGE analysis which were consistent with the (αβ)6γ subunit composition of R-PE. The results indicated that CpC is an efficient method to obtain protein with the high purity from a complex source.

A role for focal adhesion kinase in facilitating the contractile responses of murine gastric fundus smooth muscles.

Activation of focal adhesion kinase (FAK) by integrin signalling facilitates smooth muscle contraction by transmitting the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here we report that electrical field stimulation (EFS) of cholinergic motor neurons activates FAK in gastric fundus smooth muscles, and that FAK activation by EFS is atropine-sensitive but nicardipine-insensitive. PDBu and calyculin A contracted gastric fundus muscles Ca2+ -independently and also activated FAK. Inhibition of FAK activation inhibits the contractile responses evoked by EFS, and inhibits CPI-17 phosphorylation at T38. This study indicates that mechanical force or tension is sufficient to activate FAK, and that FAK appears to be involved in the activation of the protein kinase C-CPI-17 Ca2+ sensitization pathway in gastric fundus smooth muscles. These results reveal a novel role for FAK in gastric fundus smooth muscle contraction by facilitating CPI-17 phosphorylation. Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. C-kinase potentiated protein phosphatase-1 inhibitor of 17kDa (CPI-17) and myosin phosphatase targeting subunit of myosin light-chain phosphatase (MYPT1) are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including focal adhesion kinase (FAK), to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of Nω -nitro-l-arginine methyl ester and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF-431396 inhibited the contractions and the increase in pY397. PF-431396 also inhibited the electrical field stimulation-induced increase in CPI-17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca2+ influx was unaffected by PF-431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. Phorbol 12,13-dibutyrate or calyculin A contracted gastric fundus smooth muscle strips Ca2+ independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction and reveal a novel role of FAK in the regulation of CPI-17 phosphorylation.

Global collective motions in the mammalian and bacterial respiratory complex I

The respiratory complex I is an enzyme responsible for the conversion of chemical energy into an electrochemical proton motive force across the membrane. Despite extensive studies, the mechanism by which the activity of this enormous, ca. 1 MDa, redox-coupled proton pump is regulated still remains unclear. Recent structural studies (Zhu et al., Nature 2016; Fiedorczuk et al., Nature 2016) resolved complex I in different conformations connected to the active-to-deactive (A/D) transition that regulate complex I activity in several species. Based on anisotropic network models (ANM) and principal component analysis (PCA), we identify here transitions between experimentally resolved structures of the mammalian complex I as low-frequency collective motions of the enzyme, highlighting similarities and differences between the bacterial and mammalian enzymes. Despite the reduced complexity of the smaller bacterial enzyme, our results suggest that the global dynamics of complex I is overall conserved. We further probe how the supernumerary subunits could be involved in the modulation of the A/D-transition, and show that in particular the 42 kDa and B13 subunits affect the global motions of the mammalian enzyme.