Catalytic Turnover Triggers Exchange of Subunits of the Magnesium Chelatase AAA+ Motor Unit

The ATP-dependent insertion of Mg²⁺ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg²⁺ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity.     

Wiskott-Aldrich Syndrome Protein (WASp) Controls the Delivery of Platelet Transforming Growth Factor-β1

Platelets are immunologically competent cells containing cytokines such as TGF-β1 that regulate cell-mediated immunity. However, the mechanisms underlying cytokine secretion from platelets are undefined. The Wiskott-Aldrich syndrome protein (WASp) regulates actin polymerization in nucleated hematopoietic cells but has other role(s) in platelets. WASp-null (WASp^−/−) platelets stimulated with a PAR-4 receptor agonist had increased TGF-β1 release compared with WT platelets; inhibiting WASp function with wiskostatin augmented TRAP-induced TGF-β1 release in human platelets. TGF-β1 release is dissociated from α-granule secretion (P-selectin up-regulation) and occurs more gradually, with ∼10–15% released after 30–60 min. Blockade of Src family kinase-mediated WASp Tyr-291/Tyr-293 phosphorylation increased TGF-β1 release, with no additive effect in WASp^−/− platelets, signifying that phosphorylation is critical for WASp-limited TGF-β1 secretion. Inhibiting F-actin assembly with cytochalasin D enhanced secretion in WT platelets and further increased TGF-β1 release in WASp^−/− platelets, indicating that WASp and actin assembly independently regulate TGF-β1 release. A permeabilized platelet model was used to test the role of upstream small GTPases in TGF-β1 release. N17Cdc42, but not Rac1 mutants, increased TGF-β1 secretion and abrogated WASp phosphorylation. We conclude that WASp function restricts TGF-β1 secretion in a Cdc42- and Src family kinase-dependent manner and independently of actin assembly.     

The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding to Metabolic Intermediates

Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.     

Rotavirus Viroplasm Proteins Interact with the Cellular SUMOylation System: Implications for Viroplasm-Like Structure Formation

Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasm-like structures.     

Egg predation within the nests of social wasps: a new genus and species of Phlaeothripidae,and evolutionary consequences of Thysanoptera invasive behaviour

The insects known as thrips are commonly thought of as flower‐living and pestiferous organisms, but we report here a novel interaction between a phlaeothripine thrips species, Mirothrips arbiter gen. et sp. nov. and three species of social paper wasps in Brazil. This thrips species breeds inside the wasp colonies, and larval and adult thrips feed on wasp eggs, which become severely damaged. Infested nests can contain up to 300 M. arbiter gen. et sp. nov. individuals. The closest relatives of M. arbiter are two presumably predaceous species: Mirothrips bicolor sp. nov. , which inhabits abandoned Cecidomyiidae galls, and Mirothrips analis comb. nov. , described from individuals collected in the silken bags of the caterpillars of Psychidae moths. The behaviour exhibited by M. arbiter represents one of the most evolutionarily advanced lifestyles known among Thysanoptera, and we predict that other polistine species serve as hosts for this thrips in Brazil. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109 , 332–341.     

Immunologic evaluation and validation of methods using synthetic peptides derived from Mycobacterium tuberculosis for the diagnosis of tuberculosis infection

The goal of this study was to demonstrate the usefulness of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of pulmonary tuberculosis (PTB) and extrapulmonary TB (EPTB). This assay used 20 amino acid-long, non-overlapped synthetic peptides that spanned the complete Mycobacterium tuberculosis ESAT-6 and Ag85A sequences. The validation cohort consisted of 1,102 individuals who were grouped into the following five diagnostic groups: 455 patients with PTB, 60 patients with EPTB, 40 individuals with non-EPTB, 33 individuals with leprosy and 514 healthy controls. For the PTB group, two ESAT-6 peptides (12033 and 12034) had the highest sensitivity levels of 96.9% and 96.2%, respectively, and an Ag85A-peptide (29878) was the most specific (97.4%) in the PTB groups. For the EPTB group, two Ag85A peptides (11005 and 11006) were observed to have a sensitivity of 98.3% and an Ag85A-peptide (29878) was also the most specific (96.4%). When combinations of peptides were used, such as 12033 and 12034 or 11005 and 11006, 99.5% and 100% sensitivities in the PTB and EPTB groups were observed, respectively. In conclusion, for a cohort that consists entirely of individuals from Venezuela, a multi-antigen immunoassay using highly sensitive ESAT-6 and Ag85A peptides alone and in combination could be used to more rapidly diagnose PTB and EPTB infection.     

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.     

Characterisation of pks15/1 in clinical isolates of Mycobacterium tuberculosis from Mexico

Tuberculosis (TB) is an infectocontagious respiratory disease caused by members of the Mycobacterium tuberculosis complex. A 7 base pair (bp) deletion in the locus polyketide synthase (pks)15/1 is described as polymorphic among members of the M. tuberculosis complex, enabling the identification of Euro-American, Indo-Oceanic and Asian lineages. The aim of this study was to characterise this locus in TB isolates from Mexico. One hundred twenty clinical isolates were recovered from the states of Veracruz and Estado de Mexico. We determined the nucleotide sequence of a ± 400 bp fragment of the locus pks15/1, while genotypic characterisation was performed by spoligotyping. One hundred and fifty isolates contained the 7 bp deletion, while five had the wild type locus. Lineages X (22%), LAM (18%) and T (17%) were the most frequent; only three (2%) of the isolates were identified as Beijing and two (1%) EAI-Manila. The wild type pks15/1 locus was observed in all Asian lineage isolates tested. Our results confirm the utility of locus pks15/1 as a molecular marker for identifying Asian lineages of the M. tuberculosis complex. This marker could be of great value in the epidemiological surveillance of TB, especially in countries like Mexico, where the prevalence of such lineages is unknown.