Three-Dimensional Architecture of the Human BRCA1-A Histone Deubiquitinase Core Complex.

BRCA1 is a tumor suppressor found to be mutated in hereditary breast and ovarian cancer and plays key roles in the maintenance of genomic stability by homologous recombination repair. It is recruited to damaged chromatin as a component of the BRCA1-A deubiquitinase, which cleaves K63-linked ubiquitin chains attached to histone H2A and H2AX. BRCA1-A contributes to checkpoint regulation, repair pathway choice, and HR repair efficiency through molecular mechanisms that remain largely obscure. The structure of an active core complex comprising two Abraxas/BRCC36/BRCC45/MERIT40 tetramers determined by negative-stain electron microscopy (EM) reveals a distorted V-shape architecture in which a dimer of Abraxas/BRCC36 heterodimers sits at the base, with BRCC45/Merit40 pairs occupying each arm. The location and ubiquitin-binding activity of BRCC45 suggest that it may provide accessory interactions with nucleosome-linked ubiquitin chains that contribute to their efficient processing. Our data also suggest how ataxia telangiectasia mutated (ATM)-dependent BRCA1 dimerization may stabilize self-association of the entire BRCA1-A complex.

Crystal structure of vaccinia virus mRNA capping enzyme provides insights into the mechanism and evolution of the capping apparatus.

Vaccinia virus capping enzyme is a heterodimer of D1 (844 aa) and D12 (287 aa) polypeptides that executes all three steps in m(7)GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1⋅D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB-fold domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1⋅D12 structure complements and rationalizes four decades of biochemical studies of this enzyme, which was the first capping enzyme to be purified and characterized, and provides new insights into the origins of the capping systems of other large DNA viruses.

Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyl-transferase.

The vaccinia virus mRNA capping enzyme is a multifunctional heterodimeric protein associated with the viral polymerase that both catalyses the three steps of mRNA capping and regulates gene transcription. The structure of a subcomplex comprising the C-terminal N7-methyl-transferase (MT) domain of the large D1 subunit, the stimulatory D12 subunit and bound S-adenosyl-homocysteine (AdoHcy) has been determined at 2.7 A resolution and reveals several novel features of the poxvirus capping enzyme. The structure shows for the first time the critical role played by the proteolytically sensitive N-terminus of the MT domain in binding the methyl donor and in catalysis. In addition, the poxvirus enzyme has a completely unique mode of binding of the adenosine moiety of AdoHcy, a feature that could be exploited for design of specific anti-poxviral compounds. The structure of the poxvirus-specific D12 subunit suggests that it was originally an RNA cap 2'O-MT that has evolved to a catalytically inactive form that has been retained for D1 stabilisation and MT activity enhancement through an allosteric mechanism.

Crystal structure of the catalytic domain of DESC1, a new member of the type II transmembrane serine proteinase family.

DESC1 was identified using gene-expression analysis between squamous cell carcinoma of the head and neck and normal tissue. It belongs to the type II transmembrane multidomain serine proteinases (TTSPs), an expanding family of serine proteinases, whose members are differentially expressed in several tissues. The biological role of these proteins is currently under investigation, although in some cases their participation in specific functions has been reported. This is the case for enteropeptidase, hepsin, matriptase and corin. Some members, including DESC1, are associated with cell differentiation and have been described as tumor markers. TTSPs belong to the type II transmembrane proteins that display, in addition to a C-terminal trypsin-like serine proteinase domain, a differing set of stem domains, a transmembrane segment and a short N-terminal cytoplasmic region. Based on sequence analysis, the TTSP family is subdivided into four subfamilies: hepsin/transmembrane proteinase, serine (TMPRSS); matriptase; corin; and the human airway trypsin (HAT)/HAT-like/DESC subfamily. Members of the hepsin and matriptase subfamilies are known structurally and here we present the crystal structure of DESC1 as a first member of the HAT/HAT-like/DESC subfamily in complex with benzamidine. The proteinase domain of DESC1 exhibits a trypsin-like serine proteinase fold with a thrombin-like S1 pocket, a urokinase-type plasminogen activator-type S2 pocket, to accept small residues, and an open hydrophobic S3/S4 cavity to accept large hydrophobic residues. The deduced substrate specificity for DESC1 differs markedly from that of other structurally known TTSPs. Based on surface analysis, we propose a rigid domain association for the N-terminal SEA domain with the back site of the proteinase domain.

Crystal structures of the tricorn interacting factor F3 from Thermoplasma acidophilum, a zinc aminopeptidase in three different conformations.

The tricorn interacting factor F3 is an 89 kDa zinc aminopeptidase from the archaeon Thermoplasma acidophilum. Together with the tricorn interacting factors F1 and F2, F3 degrades the tricorn protease products and thus completes the proteasomal degradation pathway by generating free amino acids. Here, we present the crystal structures of F3 in three different conformations at 2.3 A resolution. The zinc aminopeptidase is composed of four domains: an N-terminal saddle-like beta-structure domain; a thermolysin-like catalytic domain; a small barrel-like beta-structure domain; and an alpha-helical C-terminal domain, the latter forming a deep cavity at the active site. Three crystal forms provide snapshots of the molecular dynamics of F3 where the C-terminal domain can adapt to form an open, an intermediate and a nearly closed cavity, respectively. With the conserved Zn(2+)-binding motifs HEXXH and NEXFA as well as the N-terminal substrate-anchoring glutamate residues, F3 together with the leukotriene A4 hydrolase, represents a novel gluzincin subfamily of aminoproteases. We discuss the functional implications of these structures with respect to the underlying catalytic mechanism, substrate recognition and processing, and possible component interactions.

[Seasonal variation of intestinal protozoa infections in outpatients of the north section of Santiago, Chile. 1995-1996]. / Variación estacional de las infecciones por protozoos intestinales en pacientes ambulatorios del sector norte de Santiago, Chile, 1995-1996.

Formalin preserved fecal samples from 6,058 and 5,863 outpatients were examined for intestinal parasites during 1995 and 1996 respectively. Prevalence rates of infections by intestinal protozoa in both years were similar. By age group (0-9, 10-19 and > 20 years old) Blastocystis hominis was observed in 18.6-19.3, 37.0-31.1 and 25.3-25.4% in 1995-1996 respectively. Prevalence of Giardia intestinalis infections decreased from 16.6-17.4% in the 0-9 year-old children group to 4.1-4.5% in patients over 20 years. Overall percentages of infection by Entamoeba histolytica varied between 4.2 and 10.9. Rates of infections by G. intestinalis, E. histolytica, and Entamoeba coli observed during rainy-cold months (april-september) of the year versus drywarmy period (october-march) were the same. On the contrary, more cases of B. hominis infection 25.8% versus 18.2% (this difference being statistically significant, p > 0.001) were observed during rainy-cold months of the year.

[Human infection by intestinal protozoa and helminths in Calbuco County, X Region, Chile, 1997]. / Infección humana por protozoos y helmintos intestinales en la Comuna de Calbuco, X Región de Chile, 1997.

By the performance of parasitological examination of one fecal sample per individual, a total of 256 persons from a rural county in the X Region (41 degrees 50 minutes South lat., 73 degrees 05 minutes West long.) were studied. The general rates of infection by intestinal parasite and/or commensal protozoa and helminths found were: Giardia intestinalis 14.1%, Entamoeba histolytica 11.7%, Blastocystis hominis 36.0%, Entamoeba coli 9.8%, Endolimax nana 16.4%, Iodamoeba buetschlii 1.2%, Chilomastix mesnili 0.8%, Ascaris lumbricoides 13.7% and Trichuris trichiura 9.8%. The prevalence rates of intestinal infection led us to conclude that environmental conditions favorable for its transmission remain and show that intestinal parasitoses are still a public health problem in this region, affecting mostly children.