Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain

Background: IPS-1/MAVS/VISA/Cardif is an adaptor protein that plays a crucial role in the induction of interferons in response to viral infection. In the initial stage of the intracellular antiviral response two RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-association gene 5 (MDA5), are independently able to bind viral RNA in the cytoplasm. The 62 kDa protein IPS-1/MAVS/VISA/Cardif contains an N-terminal caspase activation and recruitment (CARD) domain that associates with the CARD regions of RIG-I and MDA5, ultimately leading to the induction of type I interferons. As a first step towards understanding the molecular basis of this important adaptor protein we have undertaken structural studies of the IPS-1 MAVS/VISA/Cardif CARD region. Results: The crystal structure of human IPS-1/MAVS/VISA/Cardif CARD has been determined to 2.1 angstrom resolution. The protein was expressed and crystallized as a maltose-binding protein (MBP) fusion protein. The MBP and IPS-1 components each form a distinct domain within the structure. IPS-1/MAVS/VISA/Cardif CARD adopts a characteristic six-helix bundle with a Greek-key topology and, in common with a number of other known CARD structures, contains two major polar surfaces on opposite sides of the molecule. One face has a surface-exposed, disordered tryptophan residue that may explain the poor solubility of untagged expression constructs. Conclusion: The IPS-1/MAVS/VISA/Cardif CARD domain adopts the classic CARD fold with an asymmetric surface charge distribution that is typical of CARD domains involved in homotypic protein-protein interactions. The location of the two polar areas on IPS-1/MAVS/VISA/Cardif CARD suggest possible types of associations that this domain makes with the two CARD domains of MDA5 or RIG-I. The N-terminal CARD domains of RIG-I and MDA5 share greatest sequence similarity with IPS-1/MAVS/VISA/Cardif CARD and this has allowed modelling of their structures. These models show a very different charge profile for the equivalent surfaces compared to IPS-1/MAVS/VISA/Cardif CARD.Publisher PDFPeer reviewe

Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain

<p>Abstract</p> <p>Background</p> <p>IPS-1/MAVS/VISA/Cardif is an adaptor protein that plays a crucial role in the induction of interferons in response to viral infection. In the initial stage of the intracellular antiviral response two RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-association gene 5 (MDA5), are independently able to bind viral RNA in the cytoplasm. The 62 kDa protein IPS-1/MAVS/VISA/Cardif contains an N-terminal caspase activation and recruitment (CARD) domain that associates with the CARD regions of RIG-I and MDA5, ultimately leading to the induction of type I interferons. As a first step towards understanding the molecular basis of this important adaptor protein we have undertaken structural studies of the IPS-1 MAVS/VISA/Cardif CARD region.</p> <p>Results</p> <p>The crystal structure of human IPS-1/MAVS/VISA/Cardif CARD has been determined to 2.1Å resolution. The protein was expressed and crystallized as a maltose-binding protein (MBP) fusion protein. The MBP and IPS-1 components each form a distinct domain within the structure. IPS-1/MAVS/VISA/Cardif CARD adopts a characteristic six-helix bundle with a Greek-key topology and, in common with a number of other known CARD structures, contains two major polar surfaces on opposite sides of the molecule. One face has a surface-exposed, disordered tryptophan residue that may explain the poor solubility of untagged expression constructs.</p> <p>Conclusion</p> <p>The IPS-1/MAVS/VISA/Cardif CARD domain adopts the classic CARD fold with an asymmetric surface charge distribution that is typical of CARD domains involved in homotypic protein-protein interactions. The location of the two polar areas on IPS-1/MAVS/VISA/Cardif CARD suggest possible types of associations that this domain makes with the two CARD domains of MDA5 or RIG-I. The N-terminal CARD domains of RIG-I and MDA5 share greatest sequence similarity with IPS-1/MAVS/VISA/Cardif CARD and this has allowed modelling of their structures. These models show a very different charge profile for the equivalent surfaces compared to IPS-1/MAVS/VISA/Cardif CARD.</p

Red Clover Variety Trials through 1982

Care should be taken by growers to obtain red clover seed of known origin, variety, germination, and purity. Whenever possible, purchase of certified seed of adapted varieties is strongly advised. Varieties such as Altaswede, Norlac, and Ottawa from Canada; Arlington and Lakeland from Wisconsin; Pennscott from Pennsylvania; Chesapeake from Maryland; and Tensas from Louisiana are not as well suited for use in Kentucky as are Kenland, Kenstar, and a few other varieties developed for the general area of Kentucky

Structure of the catalytic domain of Streptococcus pneumoniae sialidase NanA

The structure of a catalytically active subdomain of the NanA sialidase from S. pneumoniae is reported to a resolution of 2.5 Å. The complex with the inhibitor Neu5Ac2en identifies the key catalytic residues and provides a platform for structure-based development of specific inhibitors

Alfalfa, White Clover, and Red Clover Variety Trials

This note is a brief summary of results obtained in 1981 from one white clover, five alfalfa, and three red clover trials. Yields in the seeding year, and from the first full year of production thereafter, are not the best estimate of the full potential of most forage legumes. The third season stand and yield are a good measure of persistency with red clover and white clover. At times it may be the fourth or fifth season with alfalfa before stands begin to thin. To supply information on the best varieties available there is a continuous monitoring of new varieties and a few experimental strains about ready for release. Results of tests of experimental strains are found in Forage Variety Progress Reports along with descriptions of most varieties named in this report

Streptococcus pneumoniae NanC. Structural insights into the specificity and mechanism of a sialidase that produces a sialidase inhibitor

This work was supported by the Biotechnology and Biological Sciences Research Council (UK) and the Medical Research Council (UK).Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2-3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2-3- and α2-6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.Publisher PDFPeer reviewe

Promiscuity in the part-phosphorylative Entner–Doudoroff pathway of the archaeon Sulfolobus solfataricus

AbstractThe hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a ‘promiscuous’ non-phosphorylative variant of the Entner–Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner–Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of d-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose

Obligate Heterodimerization of the Archaeal Alba2 Protein with Alba1 Provides a Mechanism for Control of DNA Packaging

SummaryOrganisms growing at elevated temperatures face a particular challenge to maintain the integrity of their genetic material. All thermophilic and hyperthermophilic archaea encode one or more copies of the Alba (Sac10b) gene. Alba is an abundant, dimeric, highly basic protein that binds cooperatively and at high density to DNA. Sulfolobus solfataricus encodes a second copy of the Alba gene, and the Alba2 protein is expressed at ∼5% of the level of Alba1. We demonstrate by NMR, ITC, and crystallography that Alba2 exists exclusively as a heterodimer with Alba1 at physiological concentrations and that heterodimerization exerts a clear effect upon the DNA packaging, as observed by EM, potentially by changing the interface between adjacent Alba dimers in DNA complexes. A functional role for Alba2 in modulation of higher order chromatin structure and DNA condensation is suggested

PRIMA-1Met suppresses colorectal cancer independent of p53 by targeting MEK

This work was supported by Grant No. 81201779 (Hua Xiong) from the National Natural Science Youth Foundation; Grant No. 81502118 (Yanmei Zou) from the National Natural Science Youth Foundation; Grant No. 2014CFB250 (Yanmei Zou) from the Natural Science Foundation of Hubei Province; Grant No. 81372434 (Huihua Xiong) from the National Natural Science Foundation.PRIMA-1Met is the methylated PRIMA-1 (p53 reactivation and induction of massive apoptosis) and could restore tumor suppressor function of mutant p53 and induce p53 dependent apoptosis in cancer cells harboring mutant p53. However, p53 independent activity of PRIMA-1Met remains elusive. Here we reported that PRIMA-1Met attenuated colorectal cancer cell growth irrespective of p53 status. Kinase profiling revealed that mitogen-activated or extracellular signal-related protein kinase (MEK) might be a potential target of PRIMA-1Met. Pull-down binding and ATP competitive assay showed that PRIMA-1Met directly bound MEK in vitro and in cells. Furthermore, the direct binding sites of PRIMA-1Met were explored by using a computational docking model. Treatment of colorectal cancer cells with PRIMA-1Met inhibited p53-independent phosphorylation of MEK, which in turn impaired anchorage-independent cell growth in vitro. Moreover, PRIMA-1Met suppressed colorectal cancer growth in xenograft mouse model by inhibiting MEK1 activity. Taken together, our findings demonstrate a novel p53-independent activity of PRIMA-1Met to inhibit MEK and suppress colorectal cancer growth.Publisher PDFPeer reviewe