Commentary: The carboxyl-terminal Crk SH3 domain: Regulatory strategies and new perspectives

Since their discovery as cellular counterparts of viral oncogenes more than two decades ago, enormous progress has been made in unraveling the complex regulatory pathways of signal transduction initiated by the Crk family of proteins. New structural and biochemical studies have uncovered novel insights into both negative and positive regulation of Crk mediated by its atypical carboxyl-terminal SH3 domain (SH3C). Moreover, SH3C is tyrosine phosphorylated by receptor tyrosine kinases and non-receptor tyrosine kinases, thereby permitting assemblages of other SH2/PTB domain containing proteins. Such non-canonical signaling by the Crk SH3C reveals new regulatory strategies for adaptor proteins.     

Molecular hybridization of bioactives: Synthesis and antitubercular evaluation of novel dibenzofuran embodied homoisoflavonoids via Baylis–Hillman reaction

A novel series of natural product like dibenzofuran embodied homoisoflavonoids [(E)-3-(dibenzo[b,d]furan-2-ylmethylene)chroman-4-ones] designed by molecular hybridization were synthesized in very good yields via a sequence of reactions involving base catalyzed Baylis–Hillmann (BH) reaction of 2-dibenzofuran carboxaldehyde and methyl acrylate; bromination of BH adduct; condensation of resulted allylic bromide with substituted phenols or 2-dibenzofuranol followed by cyclization. Among the all 11 new compounds screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (MTB), (E)-3-(dibenzo[b,d]furan-2-ylmethylene)-6-fluorochroman-4-one (7f) and (E)-3-(dibenzo[b,d] furan-2-ylmethylene)-6-fluorochroman-4-one (7g) were found to be active with MIC 12.5 μg/mL.     

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galβ1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.     

Evidence of mTOR Activation by an AKT-Independent Mechanism Provides Support for the Combined Treatment of PTEN-Deficient Prostate Tumors with mTOR and AKT Inhibitors

Activation of the phosphoinositide 3-kinase pathway is commonly observed in human prostate cancer. Loss of function of phosphatase and tensin homolog (PTEN) is associated with the activation of AKT and mammalian target of rapamycin (mTOR) in many cancer cell lines as well as in other model systems. However, activation of mTOR is also dependent of kinases other than AKT. Here, we show that activation of mTOR is not dependent on AKT in a prostate-specific PTEN-deficient mouse model of prostate cancer. Pathway bifurcation of AKT and mTOR was noted in both mouse and human prostate tumors. We demonstrated for the first time that cotargeting mTOR and AKT with ridaforolimus/MK-8669 and M1K-2206, respectively, delivers additive antitumor effects in vivo when compared to single agents. Our preclinical data suggest that the combination of AKT and mTOR inhibitors might be more effective in treating prostate cancer patients than current treatment regimens or either treatment alone.     

Conjugation of Proteins by Installing BIO-Orthogonally Reactive Groups at Their N-Termini

N-terminal site-specific modification of a protein has many advantages over methods targeting internal positions, but it is not easy to install reactive groups onto a protein in an N-terminal specific manner. We here report a strategy to incorporate amino acid analogues specifically in the N-terminus of a protein in vivo and demonstrate it by preparing green fluorescent protein (GFP) having bio-orthogonally reactive groups at its N-terminus. In the first step, GFP was engineered to be a foldable, internal methionine-free sequence via the semi-rational mutagenesis of five internal methionine residues and the introduction of mutations for GFP folding enhancement. In the second step, the N-terminus of the engineered protein was modified in vivo with bio-orthogonally functional groups by reassigning functional methionine surrogates such as L-homopropargylglycine and L-azidohomoalanine into the first methionine codon of the engineered internal methionine-free GFP. The N-terminal specific incorporation of unnatural amino acids was confirmed by ESI-MS analysis and the incorporation did not affect significantly the specific activity, refolding rate and folding robustness of the protein. The two proteins which have alkyne or azide groups at their N-termini were conjugated each other by bio-orthogonal Cu(I)-catalyzed click chemistry. The strategy used in this study is expected to facilitate bio-conjugation applications of proteins such as N-terminal specific glycosylation, labeling of fluorescent dyes, and immobilization on solid surfaces.     

nNOS mediated mitochondrial injury in LPS stimulated oligodendrocytes

Products of inflammation and the activation of nitric oxide synthase have been proposed as a mechanism of oligodendrocyte injury in CNS inflammation. There are currently three well described and known isoforms of NOS. Of these, neuronal NOS (nNOS) was initially discovered in neurons, endothelial NOS (eNOS) in vascular endothelium, while the inducible form of NOS (iNOS) is known to be activated in oligodendrocytes, astrocytes and microglia. We examined the activation of nNOS and the down stream effects of NO production in oligodendrocyte precursor cells (OPC) and MO3.13 cell line following culture with LPS. Our studies show that both MO3.13 cells and OPC are susceptible to the cellular injury resulting from LPS mediated activation and NO production. Activation of the TLR4 receptor with LPS led to decrease in cell viability that was associated with loss of mitochondrial membrane potential and impaired enzymatic activity of complex I and complex IV protein of the respiratory chain. 7-NI, a known inhibitor of nNOS was able to rescue of cells from LPS mediated mitochondrial damage. Loss of mitochondrial function was associated with translocation of cytochrome C and apoptosis inducing factor to the cytosol, setting the stage for apoptosis. Phosphorylation of PI3K and Akt was required for optimal activation of NOS. These studies provide a biochemical basis for nNOS mediated oligodendrocyte injury and suggest similar mechanisms may play a role in diseases characterized by oligodendrocyte loss and demyelination.     

Structural and functional consequences of the replacement of proximal residues Cys(172) and Cys(192) in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys(172) has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys(172) and Cys(192) by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared with the wild-type enzyme. The specificity factor of C192S Rubisco was not altered. The V(c) (V(max) for carboxylation) was similar to that of wild-type Rubisco in the case of the C172S enzyme, but approx. 30% lower for the C192S Rubisco. In contrast, the K(m) for CO(2) and O(2) was similar for C192S and wild-type enzymes, but distinctly higher (approximately double) for the C172S enzyme. C172S Rubisco showed a critical denaturation temperature approx. 2 degrees C lower than wild-type Rubisco and a distinctly higher denaturation rate at 55 degrees C, whereas C192S Rubisco was only slightly more sensitive to temperature denaturation than the wild-type enzyme. X-ray crystal structures reveal that the C172S mutation causes a shift of the main-chain backbone atoms of beta-strand 1 of the alpha/beta-barrel affecting a number of amino acid side chains. This may cause the exceptional catalytic features of C172S. In contrast, the C192S mutation does not produce similar structural perturbations.     

Fate of membrane-bound reactants and products during the activation of human prothrombin by prothrombinase

Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg(320) yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg(271), yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg(271). We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg(271), accumulates on the surface because of a approximately 20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.     

Effect of thyroid hormone on the distribution and activity of Na, K-ATPase in ventricular myocardium

Employing detergent-free sucrose-density gradient fractionation method we isolated cholesterol-rich lighter membrane fractions containing ∼10% of protein, ∼30% of cholesterol in membranes of ventricular myocardium. Cholesterol-rich lighter membrane fractions contain >70% of Na, K-ATPase and caveolins 1 and 3 and <10% of β-actin. Treatment of hypothyroid rats with T₃ increased the relative abundance of both α1 and β1 Na, K-ATPase subunits in total membranes by 4- to 5-fold (with no change in caveolin-3), and resulted in 1.9-fold increase in enzyme activity. T₃-induced Na, K-ATPase subunits were preferentially distributed to the lighter fractions (#s 4, 5 and 6); and increased abundance of α1 and β1 were 34-70% and 43-68%, respectively. We conclude that the activity of Na, K-ATPase is not uniform in cardiac membranes, and while a significant amount of Na, K-ATPase is present in cardiac cholesterol-rich membrane fractions, the intrinsic activity is significantly less than the enzyme present in relatively cholesterol-poor membranes.