Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.     

High-performance liquid chromatographic assay for the determination of sulfadoxine and N-acetyl sulfadoxine in plasma from patients infected with sensitive and resistant Plasmodium falciparum malaria

A reversed-phase high-performance liquid chromatographic method using a mobile phase of acetonitrile-methanol-trifluoroacetic acid-water (16.1:7.2:0.1:76.6, v/v/v/v) at a flow rate of 1.0 ml min(-1) on a LiChrospher RP-18 column with UV (254 nm) detection has been developed for the separation of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma. No interferences due to endogenous compounds or common antimalarial drugs were noticed. The limit of detection for sulfadoxine and N-acetyl sulfadoxine was 0.01 microg ml(-1) with a signal-to-noise ratio of 5:1 while the limit of quantification was 2.5 microg ml(-1). Intra-day mean relative standard deviations (RSD's) for sulfadoxine and N-acetyl sulfadoxine were 2.6 and 2.8%, respectively, while mean inter-day RSD's for sulfadoxine and N-acetyl sulfadoxine were 2.4 and 2.8%, respectively. Extraction recoveries averaged 90.6% for sulfadoxine and 86.9% for N-acetyl sulfadoxine. The method was applied for the assay of sulfadoxine and its metabolite N-acetyl sulfadoxine in plasma from Plasmodium falciparum malaria patients. Mean plasma sulfadoxine concentrations on day 2 (51 h) from samples collected from sensitive and resistant P. falciparum patients treated with three tablets of Fansidar were 62.8 and 60.5 microg ml(-1), respectively. Mean ratio of N-acetyl sulfadoxine to sulfadoxine was 9.1% for responders and 13.9% for non-responders which revealed that higher amounts of the metabolite N-acetyl sulfadoxine were present in non-responders. The method described should find an application in the therapeutic monitoring of malaria patients.     

Computer aided screening and evaluation of herbal therapeutics against MRSA infections

Methicillin resistant Staphylococcus aureus (MRSA), a pathogenic bacterium that causes life threatening outbreaks such as community-onset and nosocomial infections has emerged as 'superbug'. The organism developed resistance to all classes of antibiotics including the best known Vancomycin (VRSA). Hence, there is a need to develop new therapeutic agents. This study mainly evaluates the potential use of botanicals against MRSA infections. Computer aided design is an initial platform to screen novel inhibitors and the data finds applications in drug development. The drug-likeness and efficiency of various herbal compounds were screened by ADMET and docking studies. The virulent factor of most of the MRSA associated infections are Penicillin Binding Protein 2A (PBP2A) and Panton-Valentine Leukocidin (PVL). Hence, native structures of these proteins (PDB: 1VQQ and 1T5R) were used as the drug targets. The docking studies revealed that the active component of Aloe vera, β-sitosterol (3S, 8S, 9S, 10R, 13R, 14S, 17R) -17- [(2R, 5R)-5-ethyl-6-methylheptan-2-yl] -10, 13-dimethyl 2, 3, 4, 7, 8, 9, 11, 12, 14, 15, 16, 17- dodecahydro-1H-cyclopenta [a] phenanthren-3-ol) showed best binding energies of -7.40 kcal/mol and -6.34 kcal/mol for PBP2A and PVL toxin, respectively. Similarly, Meliantriol (1S-1-[ (2R, 3R, 5R)-5-hydroxy-3-[(3S, 5R, 9R, 10R, 13S, 14S, 17S)-3-hydroxy 4, 4, 10, 13, 14-pentamethyl-2, 3, 5, 6, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta[a] phenanthren-17-yl] oxolan-2-yl] -2- methylpropane-1, 2 diol), active compound in Azadirachta indica (Neem) showed the binding energies of -6.02 kcal/mol for PBP2A and -8.94 for PVL toxin. Similar studies were conducted with selected herbal compound based on pharmacokinetic properties. All in silico data tested in vitro concluded that herbal extracts of Aloe-vera, Neem, Guava (Psidium guajava), Pomegranate (Punica granatum) and tea (Camellia sinensis) can be used as therapeutics against MRSA infections.     

Polymeric black tea polyphenols modulate the localization and activity of 12-O-tetradecanoylphorbol-13-acetate-mediated kinases in mouse skin: Mechanisms of their anti-tumor-promoting action

Polymeric black tea polyphenols (PBPs) have been shown to possess anti-tumor-promoting effects in two-stage skin carcinogenesis. However, their mechanisms of action are not fully elucidated. In this study, mechanisms of PBP-mediated antipromoting effects were investigated in a mouse model employing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Compared to controls, a single topical application of TPA to mouse skin increased the translocation of protein kinase C (PKC) from cytosol to membrane. Pretreatment with PBPs 1-3 decreased TPA-induced translocation of PKC isozymes (α, β, η, γ, ε) from cytosol to membrane, whereas PBPs 4 and 5 were less effective. The levels of PKCs δ and ζ in cytosol/membrane were similar in all the treatment groups. Complementary confocal microscopic evaluation showed a decrease in TPA-induced PKCα fluorescence in PBP-3-pretreated membranes, whereas pretreatment with PBP-5 did not show a similar decrease. Based on the experiments with specific enzyme inhibitors and phosphospecific antibodies, both PBP-3 and PBP-5 were observed to decrease TPA-induced level and/or activity of phosphatidylinositol 3-kinase (PI3K) and AKT1 (pS473). An additional ability of PBP-3 to inhibit site-specific phosphorylation of PKCα at all three positions responsible for its activation [PKCα (pT497), PKC PAN (βII pS660), PKCα/βII (pT638/641)] and AKT1 at the Thr308 position, along with a decrease in TPA-induced PDK1 protein level, correlated with the inhibition of translocation of PKC, which may impart relatively stronger chemoprotective activity to PBP-3 than to PBP-5. Altogether, PBP-mediated decrease in TPA-induced PKC phosphorylation correlated well with decreased TPA-induced NF-κB phosphorylation and downstream target proteins associated with proliferation, apoptosis, and inflammation in mouse skin. Results suggest that the antipromoting effects of PBPs are due to modulation of TPA-induced PI3K-mediated signal transduction.     

An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain

Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 CATD is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation.     

QuickNGS elevates Next-Generation Sequencing data analysis to a new level of automation

Background

Next-Generation Sequencing (NGS) has emerged as a widely used tool in molecular biology. While time and cost for the sequencing itself are decreasing, the analysis of the massive amounts of data remains challenging. Since multiple algorithmic approaches for the basic data analysis have been developed, there is now an increasing need to efficiently use these tools to obtain results in reasonable time.

Results

We have developed QuickNGS, a new workflow system for laboratories with the need to analyze data from multiple NGS projects at a time. QuickNGS takes advantage of parallel computing resources, a comprehensive back-end database, and a careful selection of previously published algorithmic approaches to build fully automated data analysis workflows. We demonstrate the efficiency of our new software by a comprehensive analysis of 10 RNA-Seq samples which we can finish in only a few minutes of hands-on time. The approach we have taken is suitable to process even much larger numbers of samples and multiple projects at a time.

Conclusion

Our approach considerably reduces the barriers that still limit the usability of the powerful NGS technology and finally decreases the time to be spent before proceeding to further downstream analysis and interpretation of the data.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1695-x) contains supplementary material, which is available to authorized users.     

Endomembrane Trafficking Protein SEC24A Regulates Cell Size Patterning in Arabidopsis

Size is a critical property of a cell, but how it is determined is still not well understood. The sepal epidermis of Arabidopsis (Arabidopsis thaliana) contains cells with a diversity of sizes ranging from giant cells to small cells. Giant cells have undergone endoreduplication, a specialized cell cycle in which cells replicate their DNA but fail to divide, becoming polyploid and enlarged. Through forward genetics, we have identified a new mutant with ectopic giant cells covering the sepal epidermis. Surprisingly, the mutated gene, SEC24A, encodes a coat protein complex II vesicle coat subunit involved in endoplasmic reticulum-to-Golgi trafficking in the early secretory pathway. We show that the ectopic giant cells of sec24a-2 are highly endoreduplicated and that their formation requires the activity of giant cell pathway genes LOSS OF GIANT CELLS FROM ORGANS, DEFECTIVE KERNEL1, and Arabidopsis CRINKLY4. In contrast to other trafficking mutants, cytokinesis appears to occur normally in sec24a-2. Our study reveals an unexpected yet specific role of SEC24A in endoreduplication and cell size patterning in the Arabidopsis sepal.Size is a fundamental characteristic of a cell, but how cell size is determined is still not well understood in most living organisms (Marshall et al., 2012). Cells of different types typically have characteristic sizes, indicating that size is carefully regulated to fit cell functions during differentiation. At the simplest level, cell size is determined by growth and division. Although many factors regulating these two processes have been studied, how they are comprehensively regulated to achieve specific size outcomes remains unclear.The sepal of Arabidopsis (Arabidopsis thaliana) is an excellent model to study the regulation of cell size because it exhibits a characteristic pattern of giant cells interspersed in between small cells. The giant cells are large cells that span about one-fifth the length of the sepal (approximately 360 μm), while the smallest cells only reach to about 10 μm (Roeder et al., 2010). Previously, we have shown that variability in cell division times is sufficient to produce the cell size pattern (Roeder et al., 2010). The giant cells stop dividing and enter endoreduplication, a specialized cell cycle in which the cell replicates its DNA but skips mitosis to continue growing (Edgar and Orr-Weaver, 2001; Sugimoto-Shirasu and Roberts, 2003; Inzé and De Veylder, 2006; Breuer et al., 2010). Alongside the giant cells, the smaller cells continue dividing mitotically. Giant cells and small cells are different cell types, as they can be distinguished by the expression pattern of two independent enhancers. Furthermore, mutant screens have shown that genes involved in epidermal specification and cell cycle regulation are crucial for sepal cell size patterning. DEFECTIVE KERNEL1 (DEK1), Arabidopsis thaliana MERISTEM LAYER1 (ATML1), Arabidopsis CRINKLY4 (ACR4), and HOMEODOMAIN GLABROUS11 first establish the identity of giant cells, and then the cyclin dependent kinase inhibitor LOSS OF GIANT CELLS FROM ORGANS (LGO) influences the probability with which cells enter endoreduplication. Endoreduplication can further suppress the identity of small cells through an unknown mechanism (Roeder et al., 2010, 2012). The number of giant cells influences the curvature of the sepal, which is important for protecting the flower (Roeder et al., 2012). Therefore, cell size patterning ensures the protective role of sepals at the physiological level.The secretory pathway in eukaryotes is crucial for cells to maintain membrane homeostasis and protein localization. Proteins destined for the cell surface are first translated on the rough endoplasmic reticulum (ER) and then incorporated into coat protein complex II (COPII) vesicles that bud from ER membranes on the way to the Golgi apparatus. COPII machinery is highly conserved in eukaryotes, and each COPII component acts sequentially on the surface of the ER (Bickford et al., 2004; Marti et al., 2010; Zanetti et al., 2012). Vesicle coat assembly is initiated by SEC12, an ER membrane-anchored guanine nucleotide exchange factor (Barlowe and Schekman, 1993). SEC12 exchanges GDP with GTP on the small GTPase Secretion-associated RAS-related protein1 (SAR1), which increases the membrane affinity of SAR1. The ER membrane-bound SAR1 subsequently brings the SEC23/SEC24 subunits to form the prebudding complex, and eventually SEC13/SEC31 are recruited to increase rigidity of the COPII vesicle coat (Nakano and Muramatsu, 1989; Barlowe et al., 1994; Shaywitz et al., 1997; Aridor et al., 1998; Kuehn et al., 1998; Stagg et al., 2006; Copic et al., 2012). For COPII vesicles to fuse with the target membrane, superfamily N-ethylmaleimide-sensitive factor adaptor protein receptors (SNAREs) must be incorporated by SEC24 (Mossessova et al., 2003; Lipka et al., 2007; Mancias and Goldberg, 2008). In addition to its role in SNARE packaging, SEC24 also binds and loads secretory cargo proteins (Miller et al., 2003). Both the cargo and SNARE specificities are determined by the correspondence between the SEC24 isoform and the various ER export signals of cargoes and SNAREs (Barlowe., 2003; Miller et al., 2003; Mossessova et al., 2003; Mancias and Goldberg, 2008). The Arabidopsis genome encodes four SEC24 isoforms, SEC24A to SEC24D; how they differentially regulate trafficking is unknown (Bassham et al., 2008). Likewise, SEC24-cargo/SNARE interactions remain elusive in plants.Secretion defects in plants often lead to cell division defects due to the unique mechanisms of plants cytokinesis (Sylvester, 2000; Jürgens, 2005). In many eukaryotes other than plants, cytokinesis is accomplished by contraction of the cleavage furrow at the division plane. By contrast, cytokinesis in plants requires de novo secretion of vesicles to the division plane, after formation of the phragmoplast as the scaffold for delivery. Homotypic vesicle fusion sets up the early cell plate, which then expands laterally by fusing with other arriving vesicles (Balasubramanian et al., 2004; Jürgens, 2005; Reichardt et al., 2007). Hence, disruption of secretion in plants can often result in cytokinesis defects. For instance, a mutation in the SNARE KNOLLE leads to enlarged embryo cells with multiple nuclei (Lukowitz et al., 1996).Another common phenotype observed in secretion-deficient plants is abnormal auxin responses. The phytohormone auxin acts as a prominent signal in Arabidopsis development, and auxin influx/efflux carriers are essential in directing auxin transport and creating local maxima in an auxin gradient (Reinhardt et al., 2003; Heisler et al., 2005; Jönsson et al., 2006; Smith et al., 2006; Vanneste and Friml, 2009). To maintain appropriate auxin gradients, the subcellular localization of auxin carriers must be delicately regulated. Thus, auxin responses are highly sensitive to trafficking perturbations in plants (Geldner et al., 2003; Grunewald and Friml, 2010).Here, we have identified a new mutant with ectopic giant cells. Through positional cloning, we determined that the mutation occurs in the SEC24A gene, which encodes the cargo-binding subunit of the COPII vesicle complex. In addition to altered cell size, this unique sec24a-2 allele shows pleiotropic defects, including dwarfism, which have not been reported previously for other SEC24A alleles (Faso et al., 2009; Nakano et al., 2009; Conger et al., 2011). Although the mutant is developmentally aberrant, both cytokinesis and auxin response appear normal in sec24a-2, unlike other transport mutants. Instead, we find SEC24A regulates cell size specifically via the giant cell development pathway. Thus, our data reveal an unexpected role of SEC24A in endoreduplication and cell size patterning in the Arabidopsis sepal.     

Coreceptor phenotype of natural human immunodeficiency virus with nef deleted evolves in vivo,leading to increased virulence

The Sydney Blood Bank Cohort is a group of patients with slowly progressive infection by a human immunodeficiency virus strain containing spontaneous deletions within the nef long terminal repeat region. In 1999, 18 years after the initial infection, one of the members (D36) developed AIDS. In this work, we used an ex vivo human lymphoid cell culture system to analyze two viral isolates obtained from this patient, one prior to the onset of AIDS in 1995 and one after disease progression in 1999. Both D36 isolates were less potent in depleting CD4(+) T cells than a reference dualtropic, nef-bearing viral isolate. However, the 1999 isolate was measurably more cytotoxic to CD4(+) T cells than the 1995 isolate. Interestingly, although both isolates were nearly equally potent in depleting CCR5(+) CD4(+) T cells, the cytotoxic effect of the 1999 isolate toward CCR5(-) CD4(+) T cells was significantly higher. Furthermore, GHOST cell infection assays and blocking experiments with the CXCR4 inhibitor AMD3100 showed that the later D36 1999 isolate could infect both CCR5(+) and CCR5(-) CXCR4(+) cells efficiently, while infection by the 1995 isolate was nearly completely restricted to CCR5(+) cells. Sequence analysis of the V1/V2 and V3 regions of the viral envelope protein gp120 revealed that the more efficient CXCR4 usage of the later isolate might be caused by an additional potential N-glycosylation site in the V1/V2 loop. In conclusion, these data show that an in vivo evolution of the tropism of this nef-deleted strain toward an X4 phenotype was associated with a higher cytopathic potential and progression to AIDS.     

Prognostic Stratification of GBMs Using Combinatorial Assessment of IDH1 Mutation,MGMT Promoter Methylation,and TERT Mutation Status: Experience from a Tertiary Care Center in India

This study aims to establish the best and simplified panel of molecular markers for prognostic stratification of glioblastomas (GBMs). One hundred fourteen cases of GBMs were studied for IDH1, TP53, and TERT mutation by Sanger sequencing; EGFR and PDGFRA amplification by fluorescence in situ hybridization; NF1expression by quantitative real time polymerase chain reaction (qRT-PCR); and MGMT promoter methylation by methylation-specific PCR. IDH1 mutant cases had significantly longer progression-free survival (PFS) and overall survival (OS) as compared to IDH1 wild-type cases. Combinatorial assessment of MGMT and TERT emerged as independent prognostic markers, especially in the IDH1 wild-type GBMs. Thus, within the IDH1 wild-type group, cases with only MGMT methylation (group 1) had the best outcome (median PFS: 83.3 weeks; OS: not reached), whereas GBMs with only TERT mutation (group 3) had the worst outcome (PFS: 19.7 weeks; OS: 32.8 weeks). Cases with both or none of these alterations (group 2) had intermediate prognosis (PFS: 47.6 weeks; OS: 89.2 weeks). Majority of the IDH1 mutant GBMs belonged to group 1 (75%), whereas only 18.7% and 6.2% showed group 2 and 3 signatures, respectively. Interestingly, none of the other genetic alterations were significantly associated with survival in IDH1 mutant or wild-type GBMs.Based on above findings, we recommend assessment of three markers, viz., IDH1, MGMT, and TERT, for GBM prognostication in routine practice. We show for the first time that IDH1 wild-type GBMs which constitute majority of the GBMs can be effectively stratified into three distinct prognostic subgroups based on MGMT and TERT status, irrespective of other genetic alterations.