Introduction: Modern Technologies for In-Cell Biochemistry [Protein Structure and Folding]

December 16th, 2015 by Lutsenko, S.

The last decade has seen enormous progress in the exploration and understanding of the behavior of molecules in their natural cellular environments at increasingly high spatial and temporal resolution. Advances in microscopy, development of new fluorescent reagents, and genetic editing have enabled quantitative analysis of protein interactions, intracellular trafficking, metabolic changes, and signaling. Modern biochemistry now faces new and exciting challenges. Can traditionally "in vitro" experiments, e.g. analysis of protein folding and conformational transitions, be done in cells? Can the structure and behavior of endogenous and/or non-tagged recombinant proteins be analyzed and altered within the cell or in cellular compartments? How can molecules and their action be studied mechanistically in tissues and organs? Is personalized cellular biochemistry a reality? This Thematic Series summarizes recent studies that illustrate some first steps towards successfully answering these modern biochemical questions. The first Minireview focuses on utilization of three-dimensional primary enteroids and organoids for mechanistic studies of intestinal biology with molecular resolution. The second Minireview describes application of single chain antibodies (nanobodies) for monitoring and regulating protein dynamics in vitro and in cells. The third Minireview highlights advances in using NMR spectroscopy for analysis of protein folding and assembly in cells.

Pseudomonas aeruginosa EftM is a Thermoregulated Methyltransferase [Microbiology]

December 16th, 2015 by

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-Tu (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25°C), but not at higher temperatures (37°C). We have previously identified the gene, eftM (for EF-Tu modifying enzyme), responsible for this modification and shown its activity to be associated with increase adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-L-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50, in the predicted SAM-binding domain lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, pre-incubation of EftM at 37°C abolished methyltransferase activity, while this activity was retained when EftM was pre-incubated at 25°C. Irreversible protein unfolding at 37°C was observed and we propose is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.

Rapid activation of bone morphogenic protein 9 by receptor-mediated displacement of pro-domains [Protein Structure and Folding]

December 16th, 2015 by

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β (TGFβ) family. In case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9-pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin (ENG), or to mature BMP9 domain targeting antibodies, leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. While mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9-pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation, and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Mutational constraints on local unfolding inhibit the rheological adaptation of von Willebrand factor [Molecular Bases of Disease]

December 16th, 2015 by

Unusually large von Willebrand factor (ULVWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease (VWD), two rare mutations (G1324A and G1324S) within the platelet GPIbα binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limited proteolysis. Collagen binding is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The enhanced stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to H1322 across the β2-β3 hairpin in the GPIbα binding interface which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIbα binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.

C-terminal Domain of Leucyl-tRNA Synthetase from Pathogenic Candida albicans Recognizes Both tRNASer and tRNALeu [RNA]

December 16th, 2015 by Ji, Q.-Q., Fang, Z.-P., Ye, Q., Ruan, Z.-R., Zhou, X.-L., Wang, E.-D.

Leucyl-tRNA synthetase (LeuRS) is a multi-domain enzyme that catalyzes Leu-tRNALeu formation and is classified into bacterial and archaeal/eukaryotic types, with significant diversity in the C-terminal domain (CTD). CTDs of both bacterial and archaeal LeuRSs have been reported to recognize tRNALeu through different modes of interaction. In the human pathogen, Candida albicans, the cytoplasmic LeuRS (CaLeuRS) is distinguished by its capacity to recognize a uniquely evolved chimeric tRNASer [CatRNASer(CAG)] in addition to its cognate CatRNALeu, leading to CUG codon reassignment. Our previous study showed that eukaryotic but not archaeal LeuRSs recognize this peculiar tRNASer, suggesting the significance of their highly divergent CTDs in tRNASer recognition. The results of this study provided the first evidence of the indispensable function of eukaryotic LeuRS's CTD in recognizing non-cognate CatRNASer and cognate CatRNALeu. Three lysine residues were identified as involved in mediating enzyme-tRNA interaction in leucylation process: mutation of all three sites totally ablated the leucylation activity. The importance of the three lysine residues was further verified by gel mobility shift assays and complementation of a yeast leuS gene knockout strain.

Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy [Glycobiology and Extracellular Matrices]

December 16th, 2015 by Kunze, G., Kohling, S., Vogel, A., Rademann, J., Huster, D.

The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10-GAG interaction is unclear. In particular, experimental evidence about IL-10-GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Whereas no co-crystal structure of the IL-10-GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts (PCSs) of IL-10 and its heparin ligand as induced by a protein-attached lanthanide spin label provided structural restraints for the protein-ligand complex. Using these restraints, PCS-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines R102, R104, R106, R107 and lysines K117 and K119. This study represents the first experimental characterization of the IL-10-GAG complex structure and provides the starting point for revealing the biological significance of IL-10's interaction with GAGs.

Mitochondrial sulfide quinone oxidoreductase prevents activation of the unfolded protein response in hydrogen sulfide [Protein Synthesis and Degradation]

December 16th, 2015 by Horsman, J. W., Miller, D. L.

Hydrogen sulfide (H2S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H2S improves survival of ischemia/reperfusion. We have previously shown that exposure to H2S increases the lifespan and thermotolerance in C. elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H2S metabolism. SQRD-1 is generally considered important to detoxify H2S. Here, we show that SQRD-1 is also required to maintain protein translation in H2S. In sqrd-1 mutant animals, exposure to H2S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H2S or in hif-1(ia04) mutants that die when exposed to low H2S. We demonstrate that both gcn‑2 and pek‑1 kinases are involved in the H2S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H2S, but not in wild-type animals. We speculate that SQRD-1 activity in H2S may coordinate proteostasis responses in multiple cellular compartments.
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Inhibition of G-protein-coupled receptor kinase 2 prevents the dysfunctional cardiac substrate metabolism in fatty acid synthase-transgenic mice [Signal Transduction]

December 15th, 2015 by Abd Alla, J., Graemer, M., Fu, X., Quitterer, U.

Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN-transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN-transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN-transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK (extracellular signal-regulated kinase) axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine-273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart.
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A Novel Serpin Regulatory Mechanism: SERPINB9 is Reversibly Inhibited By Vicinal Disulfide Bond Formation in the Reactive Center Loop. [Immunology]

December 15th, 2015 by

The intracellular protease inhibitor, SERPINB9 (Sb9), is a regulator of the cytotoxic lymphocyte protease, granzyme B (GzmB). Although primarily involved in the destruction of compromised cells, recent evidence suggests that GzmB is also involved in lysosome-mediated death of the cytotoxic lymphocyte itself. Sb9 protects the cell from GzmB released from lysosomes into the cytosol. Here we show that reactive oxygen species (ROS) generated within cytotoxic lymphocytes by receptor stimulation are required for lyososomal permeabilization, and release of GzmB into the cytosol. Importantly, ROS also inactivate Sb9 by oxidizing a highly conserved cysteine pair (P1-P1′ in rodents; P1′-P2′ in other mammals) in the reactive center loop to form a vicinal disulfide bond. Replacement of the P4-P3′ reactive center loop residues of the prototype serpin, SERPINA1, with the P4-P5′ residues of Sb9 containing the cysteine pair is sufficient to convert SERPINA1 into a ROS-sensitive GzmB inhibitor. Conversion of the cysteine pair to serines in either human or mouse Sb9 results in a functional serpin that inhibits GzmB and resists ROS inactivation. We conclude that ROS sensitivity of Sb9 allows the threshold for GzmB-mediated suicide to be lowered, as part of a conserved post-translational homeostatic mechanism regulating lymphocyte numbers or activity. It follows for example that antioxidants may improve NK cell viability in adoptive immunotherapy applications by stabilizing Sb9.
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Involvement of receptor activator of nuclear factor kappa-B ligand-induced incomplete cytokinesis in polyploidization of osteoclasts [Developmental Biology]

December 15th, 2015 by

Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation.
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