Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase
Byung-Kuk Yoo, Isabelle Lamarre, Jean-Louis Martin, Fabrice Rappaport, and Michel Negrerie
Soluble guanylate cyclase is the mammalian endogenous nitric oxide (NO) receptor that controls numerous signaling physiological processes. Time-resolved spectroscopy allowed us to probe the dynamics of the heme coordination after NO release and binding. After photodissociation of NO, all heme transitions are identified within the time range of 1 ps to 0.2 s, notably the bond breaking and reformation between the heme iron and proximal His, which are major events for the activation/deactivation processes. It is thus possible to demonstrate that the structural allosteric transition occurs in the time range 1–50 μs, which remarkably matches the time range observed for hemoglobin, the prototypic protein for allostery. These findings (pp. E1697–E1704) relate not only to NO signaling but also to general allostery in heme proteins.
Altered cofactor regulation with disease-associated p97/VCP mutations
Xiaoyi Zhang, Lin Gui, Xiaoyan Zhang, Stacie L. Bulfer, Valentina Sanghez, Daniel E. Wong, YouJin Lee, Lynn Lehmann, James Siho Lee, Pei-Yin Shih, Henry J. Lin, Michelina Iacovino, Conrad C. Weihl, Michelle R. Arkin, Yanzhuang Wang, and Tsui-Fen Chou
Age-associated degenerative diseases have similar pathogenic mechanisms related to defects in protein homeostasis. p97/VCP (valosin-containing protein) is essential for coordinating protein degradation and is mutated in a multisystem degenerative disease that affects the central nervous system, muscle, and bone. p97/VCP is an enzyme in the AAA ATPases (ATPases associated with diverse cellular activities) family, which takes apart ATP and uses this energy to perform pivotal functions. We found (pp. E1705–E1714) that p97/VCP cofactors control its enzymatic activity. p97/VCP disease mutants behave abnormally due to lack of appropriate control by these cofactors. Correcting the function of the disease-associated proteins may be a desirable approach to developing safe treatment for fatal degenerative diseases. The next steps are to screen and characterize large panels of compounds to identify potential drugs that may correct the malfunction.
Single-molecule analysis of RAG-mediated V(D)J DNA cleavage
Geoffrey A. Lovely, Robert C. Brewster, David G. Schatz, David Baltimore, and Rob Phillips
V(D)J recombination is essential for assembling immunoglobulin and T-cell receptor genes. The lymphoid-specific RAG recombinase proteins (RAG1 and RAG2) perform DNA cleavage at recombination signal sequences (RSSs) during V(D)J recombination. Although RAG-mediated DNA cleavage has been analyzed in bulk, it has not at the single-molecule level. We used singly tethered DNA particle motion to examine the process in real time (pp. E1715–E1723). We have found that RAG–RSS binding is reversible, with measurable dwell times before release. We also directly observed a reversible synaptic complex before RAG-mediated hairpin formation and measured its dwell time. These biochemical findings are compatible with a model for V(D)J recombination in vivo, in which the RAG recombinase often samples multiple partner RSSs before initiating the V(D)J recombination reaction.
Oncogenesis driven by the Ras/Raf pathway requires the SUMO E2 ligase Ubc9
Bing Yu, Stephen Swatkoski, Alesia Holly, Liam C. Lee, Valentin Giroux, Chih-Shia Lee, Dennis Hsu, Jordan L. Smith, Garmen Yuen, Junqiu Yue, David K. Ann, R. Mark Simpson, Chad J. Creighton, William D. Figg, Marjan Gucek, and Ji Luo
Currently there are no targeted therapies for KRAS mutant cancer. Our study (pp. E1724–E1733) uncovers a critical role of the small ubiquitin-like modifier (SUMO) E2 ligase Ubc9 in sustaining the transformation growth of KRAS mutant colorectal cancer cells, thus establishing a functional link between the SUMO pathway and the KRAS oncogene. SUMO ligases are poorly explored drug targets; our work suggests that targeting the SUMO pathway, and Ubc9 in particular, could be potentially useful for the treatment of KRAS mutant colorectal cancers.
Emergence of hematopoietic stem and progenitor cells involves a Chd1-dependent increase in total nascent transcription
Fong Ming Koh, Carlos O. Lizama, Priscilla Wong, John S. Hawkins, Ann C. Zovein, and Miguel Ramalho-Santos
Adult hematopoietic stem and progenitor cells (HSPCs) develop from a small number of specialized endothelial cells in the embryo. Very little is known about how this process, known as the endothelial-to-hematopoietic transition, is regulated. In this paper (pp. E1734–E1743), we used mouse genetic knockout models to establish Chd1 as the first chromatin remodeler, to our knowledge, shown to regulate this transition. Chd1 is not required in the endothelium prior to the transition, nor in the blood system after the transition. We found that the emergence of HSPCs involves an increase in total nascent transcription that is dependent on Chd1. These results reveal a new paradigm of regulation of a developmental transition by modulation of transcriptional output that may be relevant in other stem/progenitor cell contexts.
T-cell receptor α enhancer is inactivated in αβ T lymphocytes
Beatriz del Blanco, Úrsula Angulo, Michael S. Krangel, and Cristina Hernández-Munain
The Tcra enhancer (Eα) is essential during thymocyte development for Tcra germline transcription, Vα-to-Jα rearrangement, and the generation of αβ T-lymphocytes. It has been assumed that Eα displays constitutive activity to drive the expression of rearranged Tcra genes in mature T cells. We show (pp. E1744–E1753) that Eα is dramatically inhibited in peripheral T-lymphocytes and is not reactivated in the context of antigen-mediated stimulation or T helper differentiation. We conclude that the major function of Eα is to coordinate the formation of a chromatin hub that drives germline transcription and primary Tcra rearrangements in DP thymocytes. Our results also imply the existence of an Eα-independent molecular mechanism that directs transcription of the rearranged Tcra locus in αβ T cells.
TCR contact residue hydrophobicity is a hallmark of immunogenic CD8+ T cell epitopes
Diego Chowell, Sri Krishna, Pablo D. Becker, Clément Cocita, Jack Shu, Xuefang Tan, Philip D. Greenberg, Linda S. Klavinskis, Joseph N. Blattman, and Karen S. Anderson
The design of effective T-cell vaccines against pathogens and tumor antigens is challenged by the highly inefficient identification of the subset of peptides from a given antigen that effectively stimulate an immune response. Here (pp. E1754–E1762) we report that the relative hydrophobicity of T-cell receptor contact residues is markedly enriched in immunogenic major histocompatibility complex class I epitopes in both human and murine MHCs, and in both self and pathogen-derived immunogenic epitopes. Incorporating hydrophobicity into T-cell epitope prediction models increases the efficiency of epitope identification, which will manifest in the time and cost of T-cell vaccine development. Amino acid hydrophobicity may represent a biochemical basis by which T cells discriminate immunogenic epitopes within the background of self peptides.
An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis
Jordan B. Jastrab, Tong Wang, J. Patrick Murphy, Lin Bai, Kuan Hu, Remco Merkx, Jessica Huang, Champak Chatterjee, Huib Ovaa, Steven P. Gygi, Huilin Li, and K. Heran Darwin
The proteasome of Mycobacterium tuberculosis is required to cause lethal infections and is thus a potential drug target. Bacterial proteasomes degrade proteins modified by pupylation, but evidence suggests that the M. tuberculosis proteasome possesses additional functions. In this work (pp. E1763–E1772), we describe a degradation pathway in M. tuberculosis controlled by a previously unidentified proteasomal cofactor, Rv3780. Rv3780 enhanced the ATP-independent proteasomal degradation of peptides and proteins and was required to maintain levels of a unique set of putative proteasome substrates. Importantly, an Rv3780 mutant was attenuated for growth in mice. To our knowledge, these studies show for the first time that an ATP-independent proteasomal-degradation pathway plays a role in the physiology of an important human pathogen.
cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection
Megan H. Orzalli, Nicole M. Broekema, Benjamin A. Diner, Dustin C. Hancks, Nels C. Elde, Ileana M. Cristea, and David M. Knipe
Interferon γ-inducible protein 16 (IFI16) and cGMP-AMP synthase (cGAS) have both been proposed to directly detect herpesviral DNA in herpes simplex virus (HSV)-infected cells and initiate interferon regulatory factor-3 signaling, but it has been unclear how two DNA sensors could both be required. Both are required in human fibroblasts for detection of HSV and transfected DNAs. We found evidence that IFI16 plays a direct role in HSV DNA sensing, whereas cGAS produces low amounts of cGAMP and promotes the stability of IFI16. Our results (pp. E1773–E1781) demonstrate a new function for cGAS in the maintenance of normal levels of IFI16 and provide an explanation for the multiple proposed DNA sensors.
RNA virus replication depends on enrichment of phosphatidylethanolamine at replication sites in subcellular membranes
Kai Xu and Peter D. Nagy
Positive-strand RNA viruses are major pathogens of plants, animals, and humans. These viruses subvert intracellular membranes for virus replication, and lipids are critical due to interaction with viral and coopted host proteins. To dissect the roles of various lipids in Tomato bushy stunt virus (TBSV) replication, we have developed artificial vesicle-based replication assay. Vesicles consisting of a major phospholipid, namely phosphatidylethanolamine (PE), can support TBSV replication by assembling viral replicase complexes and performing a complete replication cycle (pp. E1782–E1791). Monitoring PE distribution reveals that PE is enriched at the sites of TBSV replication in plant and yeast cells. Increasing PE level in cells leads to enhanced replication of TBSV and other viruses, suggesting that abundant PE in subcellular membranes has proviral function.
Structure of the paramyxovirus parainfluenza virus 5 nucleoprotein–RNA complex
Maher Alayyoubi, George P. Leser, Christopher A. Kors, and Robert A. Lamb
Paramyxoviruses, the cause of many important human and animal diseases, constitute a large family of enveloped negative-stranded RNA viruses including parainfluenza virus 5 (PIV5). The virion RNA is associated with ∼2,600 protomers of N-protein in the form of a helical ribonucleoprotein (RNP) (nucleocapsid). The RNP serves as the template for the viral polymerase in vivo. When expressed, N forms a 13 member N-ring that resembles the building block of the RNP. We have determined the atomic structure of the N-ring from PIV5 with 78 bound RNA residues (pp. E1792–E1799). Precisely, six nucleotides of RNA are associated with each N protomer. Modeling PIV5-N in an “open conformation” in the ring structure indicates how transcription/replication could occur with minimal changes to the nucleocapsid structure.
Cross-talk between PKA-Cβ and p65 mediates synergistic induction of PDE4B by roflumilast and NTHi
Seiko Susuki-Miyata, Masanori Miyata, Byung-Cheol Lee, Haidong Xu, Hirofumi Kai, Chen Yan, and Jian-Dong Li
Chronic obstructive pulmonary disease (COPD) is the fourth-leading cause of death worldwide. Roflumilast has been approved for COPD. However, its efficacy has been hampered by the development of tolerance, and the underlying mechanism remains unknown but may be attributed to phosphodiesterase (PDE) 4B up-regulation. Here (pp. E1800–E1809) we found that PDE4B2 is synergistically up-regulated by inflammatory stimuli (e.g. bacteria) and cAMP-elevating agents (e.g. roflumilast) via cross-talk between protein kinase A catalytic subunit β and NF-κB p65 in a cAMP-dependent manner. Up-regulated PDE4B2 contributes to the induction of certain key chemokines in both enzymatic activity-dependent and activity-independent manners. Our findings may explain, at least in part, the development of tolerance and help develop new therapeutics to improve efficacy of roflumilast.