CATALYSIS AND INHIBITION OF MYCOBACTERIUM TUBERCULOSIS METHIONINE AMINOPEPTIDASE

Tuberculosis, caused by Mycobacterium tuberculosis, is the leading cause of death due to infectious disease. Now, the prevalence of multidrug-resistant and extensively drug-resistant TB, and the emergence of co-infection of TB and HIV have highlighted the need for new antibiotics with novel mechanisms of action. Methionine aminopeptidase (MetAP) is a ubiquitous enzyme found in both prokaryotic and eukaryotic cells and carries out an important cotranslational modification of newly synthesized proteins. The MetAPs can be divided into type I and type II based on the existence of an insert in the catalytic domain. Prokaryotic cells have only one type of MetAP, either type I or type II; encoded by a single gene. MetAP is essential for cell viability, which is demonstrated by gene deletion experiment in E.coli and Salmonella typhimurium. Therefore, MetAP is a promising target for developing novel drugs against bacterial infection, including TB-causing drug resistance bacteria. Two genes, mapA and mapB , were found in Mycobacterium tuberculosis H37Rv. They encode two type I MetAP enzymes, MtMetAP Ia and MtMetAP Ic, respectively. Both MtMetAP proteins were over-expressed and purified in homogeneity as apoenzyme. Biochemical characterization using a fluorogenic substrate (Met-AMC) was carried out with both MtMetAP Ia and MtMetAP Ic. Both MtMetAPs can be activated by divalent metals, including Ni(II), Co(II), Mn(II) and Fe(II). Ni(II) is the best activator for both MtMetAPs, followed by Co(II) . Mn(II) and Fe(II) are the least efficient to activate MtMetAP Ia and MtMetAP Ic, respectively. Metal titration assays were used to determine the metal binding affinity to each MtMetAP. In both MtMetAP Ia and MtMetAP Ic, Co(II) and Fe(II) are the tightest binding metals, as indicated by their smallest Kd values. Mn(II) gives the weakest binding in MtMetAP Ia and Ni(II) shows a weakest binding to MtMetAP Ic. Growth complementation experiments were employed to evaluate the cellular function of MtMetAP in the E. coli that had an amber mutation in the chromosomal EcMetAP gene, and a pBAD plasmid, which encoded a suppressor tRNA to suppress the lethal effect of the amber mutation. The existence of glucose or arabinose in the culture medium could suppress or express the tRNA respectively, therefore result the death or survival of the E. coli, respectively. The plasmid-expressed MtMetAP Ic in the amber mutant rescued the E coli from death and supported cell growth. A set of inhibitors with selectivity for different metalated MetAPs were tested on both MtMetAPs. For MtMetAP Ib, all tested compounds retained their inhibitory activities and metal selectivity. However, in MtMetAP Ia, the Co(II)-, Mn(II)- and Fe(II)-selective inhibitors did not show inhibition. Only Fe(II)-selective inhibitors retained their inhibition, whereas they lost their metal selectivity. An amino acid sequence alignment suggested some differences in the active sites between MtMetAP 1a and MtMetAP Ic. A homology model of MtMetAP Ia based on MtMetAP Ic structure was generated. A similar active site is observed in this virtual structure of MtMetAP Ia. Given the size of the tested compound library, the failure to find an inhibitor specific for MtMetAP Ia may be due to the limited number of compounds in the library. Screening of a compound library consisting of a larger number of molecules with more structural diversity will possibly identify inhibitors for MtMetAP Ia. The inhibitors of MtMetAP Ic were further tested for their inhibition on cellular growth. The fact that only the Fe(II)-form selective inhibitors inhibited the cellular MtMetAP Ic activity and inhibited the MtMetAP Ic-complemented cell growth, suggested that Fe(II) was the native metal used by MtMetAP1c in an E. coli cellular environment. X-ray structures of MtMetAP Ic in complex with three metalloform-selective inhibitors were analyzed. The results demonstrated different binding modes and different interactions with metal ions and active site residues for these inhibitors. The MtMetAP1c inhibitors with metalloform selectivity are potential leads for antitubercular drugs. Understanding the catalytic mechanism and inhibition of the mycobacterial MetAP is an essential step towards discovering and developing effective MetAP inhibitors as therapeutics. The compounds with potent inhibition and high metal selectivity toward MtMetAP may be therapeutically useful for improved TB treatment

Role of Phosphatidylinositol-3-Kinase Pathway in Head and Neck Squamous Cell Carcinoma

Activation of the phosphatidylinositol-3-kinase (PI3K) pathway is one of the most frequently observed molecular alterations in many human malignancies, including head and neck squamous cell carcinoma (HNSCC). A growing body of evidence demonstrates the prime importance of the PI3K pathway at each stage of tumorigenesis, that is, tumor initiation, progression, recurrence, and metastasis. Expectedly, targeting the PI3K pathway yields some promising results in both preclinical studies and clinical trials for certain cancer patients. However, there are still many questions that need to be answered, given the complexity of this pathway and the existence of its multiple feedback loops and interactions with other signaling pathways. In this paper, we will summarize recent advances in the understanding of the PI3K pathway role in human malignancies, with an emphasis on HNSCC, and discuss the clinical applications and future direction of this field

Doc2b serves as a scaffolding platform for concurrent binding of multiple Munc18 isoforms in pancreatic islet β-cells

Biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells involves soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) protein-regulated exocytosis. SNARE complex assembly further requires the regulatory proteins Munc18c, Munc18-1 and Doc2b. Munc18-1 and Munc18c are required for first- and second-phase GSIS respectively. These distinct Munc18-1 and Munc18c roles are related to their transient high-affinity binding with their cognate target (t-)SNAREs, Syntaxin 1A and Syntaxin 4 respectively. Doc2b is essential for both phases of GSIS, yet the molecular basis for this remains unresolved. Because Doc2b binds to Munc18-1 and Munc18c via its distinct C2A and C2B domains respectively, we hypothesized that Doc2b may provide a plasma membrane-localized scaffold/platform for transient docking of these Munc18 isoforms during GSIS. Towards this, macromolecular complexes composed of Munc18c, Doc2b and Munc18-1 were detected in β-cells. In vitro interaction assays indicated that Doc2b is required to bridge the interaction between Munc18c and Munc18-1 in the macromolecular complex; Munc18c and Munc18-1 failed to associate in the absence of Doc2b. Competition-based GST-Doc2b interaction assays revealed that Doc2b could simultaneously bind both Munc18-1 and Munc18c. Hence these data support a working model wherein Doc2b functions as a docking platform/scaffold for transient interactions with the multiple Munc18 isoforms operative in insulin release, promoting SNARE assembly

Cytoplasmic p21 induced by p65 prevents doxorubicin-induced cell death in pancreatic carcinoma cell line

<p>Abstract</p> <p>Background</p> <p>Studies have shown the existence of p21 induction in a p53-dependent and -independent pathway. Our previous study indicates that DOX-induced p65 is able to bind the p21 promoter to activate its transactivation in the cells.</p> <p>Methods</p> <p>Over-expression and knock-down experiments were performed in Human Pancreatic Carcinoma (PANC1) cells. Cell cycle and cell death related proteins were assessed by Western Blotting. Cytotoxicity assay was checked by CCK-8 kit. Cell growth was analyzed by flow cytometers.</p> <p>Results</p> <p>Here we showed that over-expression of p65 decreased the cytotoxic effect of DOX on PANC1 cells, correlating with increased induction of cytoplasmic p21. We observed that pro-caspase-3 physically associated with cytoplasmic p21, which may be contribution to prevent p21 translocation into the nucleus. Our data also suggested that no clear elevation of nuclear p21 by p65 provides a survival advantage by progression cell cycle after treatment of DOX. Likewise, down-regulation of p65 expression enhanced the cytotoxic effect of DOX, due to a significant decrease of mRNA levels of anti-apoptotic genes, such as the cellular inhibitor of apoptosis-1 (c-IAP1), and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2), leading to efficient induction of caspase-3 cleavage in the cells. More, we present evidence that over-expression of p53 or p53/p65 in the PANC1 cells were more sensitive to DOX treatment, correlated with activation of caspase-3 and clear elevation of nuclear p21 level. Our previous data suggested that expression of p21 increases Gefitinib-induced cell death by blocking the cell cycle at the G1 and G2 phases. The present findings here reinforced this idea by showing p21's ability of potentiality of DOX-induced cell death correlated with its inhibition of cell cycle progression after over-expression of p53 or p53/p65.</p> <p>Conclusion</p> <p>Our data suggested p65 could increase p53-mediated cell death in response to DOX in PANC1 cells. Thus, it is worth noting that in p53 null or defective tumors, targeting in down-regulation of p65 may well be useful, leading to the potentiality of chemotherapeutic drugs.</p

Two-dimensional Kagome-in-Honeycomb materials (MN4_4)3_3C32_{32} (M=Pt or Mn)

We propose two novel two-dimensional (2D) topological materials, (PtN$_4$)$_3$C$_{32}$ and (MnN$_4$)$_3$C$_{32}$, with a special geometry that we named as kagome-in-honeycomb (KIH) lattice structure, to illustrate the coexistence of the paradigmatic states of kagome physics, Dirac fermions and flat bands, that are difficult to be simultaneously observed in three-dimensional realistic systems. In such system, MN$_4$(M=Pt or Mn) moieties are embedded in honeycomb graphene sheet according to kagome lattice structure, thereby resulting in a KIH lattice. Using the first-principles calculations, we have systemically studied the structural, electronic, and topological properties of these two materials. In the absence of spin-orbit coupling (SOC), they both exhibit the coexistence of Dirac/quadratic-crossing cone and flat band near the Fermi level. When SOC is included, a sizable topological gap is opened at the Dirac/quadratic-crossing nodal point. For nonmagnetic (PtN$_4$)$_3$C$_{32}$, the system is converted into a $\mathbb{Z}_2$ topological quantum spin Hall insulator defined on a curved Fermi level, while for ferromagnetic (MnN$_4$)$_3$C$_{32}$, the material is changed from a half-semi-metal to a quantum anomalous Hall insulator with nonzero Chern number and nontrivial chiral edge states. Our findings not only predict a new family of 2D quantum materials, but also provide an experimentally feasible platform to explore the emergent kagome physics, topological quantum Hall physics, strongly correlated phenomena, and theirs fascinating applications.Comment: 6 figures. arXiv admin note: text overlap with arXiv:2207.0370

Filamin B Regulates Chondrocyte Proliferation and Differentiation through Cdk1 Signaling

Humans who harbor loss of function mutations in the actin-associated filamin B (FLNB) gene develop spondylocarpotarsal syndrome (SCT), a disorder characterized by dwarfism (delayed bone formation) and premature fusion of the vertebral, carpal and tarsal bones (premature differentiation). To better understand the cellular and molecular mechanisms governing these seemingly divergent processes, we generated and characterized FlnB knockdown ATDC5 cell lines. We found that FlnB knockdown led to reduced proliferation and enhanced differentiation in chondrocytes. Within the shortened growth plate of postnatal FlnB−/− mice long bone, we observed a similarly progressive decline in the number of rapidly proliferating chondrocytes and premature differentiation characterized by an enlarged prehypertrophic zone, a widened Col2a1+/Col10a1+ overlapping region, but relatively reduced hypertrophic zone length. The reduced chondrocyte proliferation and premature differentiation were, in part, attributable to enhanced G2/M phase progression, where fewer FlnB deficient ATDC5 chondrocytes resided in the G2/M phase of the cell cycle. FlnB loss reduced Cdk1 phosphorylation (an inhibitor of G2/M phase progression) and Cdk1 inhibition in chondrocytes mimicked the null FlnB, premature differentiation phenotype, through a β1-integrin receptor- Pi3k/Akt (a key regulator of chondrocyte differentiation) mediated pathway. In this context, the early prehypertrophic differentiation provides an explanation for the premature differentiation seen in this disorder, whereas the progressive decline in proliferating chondrocytes would ultimately lead to reduced chondrocyte production and shortened bone length. These findings begin to define a role for filamin proteins in directing both cell proliferation and differentiation through indirect regulation of cell cycle associated proteins

Faktor-Faktor Yang Berhubungan Dengan Pemanfaatan Penolong Persalinan Di Desa Moyongkota Baru Kecamatan Modayag Barat

Latar belakang : Persalinan merupakan hal yang sangat kompleks karena disatusisi terjadi kebahagiaan menjelang kelahiran anak tetapi di sisilain terjadi resiko-resiko yang mungkin mengancam keselamatan ibu dan bayi. Di desa Moyongkota Baru Kecamatan Modayag Barat sebagian besar ibu bersalin memanfaatkan dukun sebagai penolong persalinannya dibandingkan dengan pemanfaatan penolong persalinan oleh tenaga kesehatan.Tujuan : Penelitian ini bertujuan untuk mengetahui faktor - faktor yang berhubungan dengan pemanfaatan penolong persalinan pada ibu bersalin di desa Moyongkota Baru Kecamatan Modayag Barat.Metode : Penelitian ini menggunakan desain penelitian observasional analitik dengan rancangan penelitian cross sectional study. Populasi dalam penelitian ini yaitu seluruh ibu yang bersalin pada bulan September – Oktober 2013 di Desa Moyongkota Baru Kecamatan Modayag Barat. Sampel yang digunakan adalah Quota sampling yaitu sampel dikumpulkan sampai mencapai jumlah yang diinginkan, jumlah sampel yang diinginkan adalah 50 responden.Hasil Penelitian : Berdasarkan hasil uji chi square diketahui bahwa faktor pengetahuan (ρ=0,006) dan dukungan suami (ρ=0,001) berhubungan signifikan terhadap pemanfaatan penolong persalinan, sedang kanfaktor status ekonomi tidak berhubungan signifikan dengan pemanfaatan penolong persalinan dengan nilai ρ=0,206.Kesimpulan : 58% ibu bersalin di desa Moyongkota Baru Kecamatan Modayag Barat Kabupaten Bolaang Mongondow Timur memanfaatkan penolong persalinan oleh dukun/paraji dibandingkan ibu bersalin yang memanfaatkan penolong persalinan oleh bidan (14%) dan penolong persalinan olehdokter (28%)

Two-dimensional anisotropic Dirac materials PtN4C2 and Pt2N8C6 with quantum spin and valley Hall effects

We propose two novel two-dimensional topological Dirac materials, planar PtN4C2 and Pt2N8C6, which exhibit graphene-like electronic structures with linearly dispersive Dirac-cone states exactly at the Fermi level. Moreover, the Dirac cone is anisotropic, resulting in anisotropic Fermi velocities and making it possible to realize orientation-dependent quantum devices. Using the first-principles electronic structure calculations, we have systemically studied the structural, electronic, and topological properties. We find that spin-orbit coupling opens a sizable topological band gap so that the materials can be classified as quantum spin Hall insulators as well as quantum valley Hall insulators. Helical edge states that reside in the insulating band gap connecting the bulk conduction and valence bands are observed. Our work not only expands the Dirac cone material family, but also provides a new avenue to searching for more two-dimensional topological quantum spin and valley Hall insulators.Comment: 6 pages, 4 figure