Structural Studies of a Four-MBT Repeat Protein MBTD1

The Polycomb group (PcG) of proteins is a family of important developmental regulators. The respective members function as large protein complexes involved in establishment and maintenance of transcriptional repression of developmental control genes. MBTD1, Malignant Brain Tumor domain-containing protein 1, is one such PcG protein. MBTD1 contains four MBT repeats.We have determined the crystal structure of MBTD1 (residues 130-566aa covering the 4 MBT repeats) at 2.5 A resolution by X-ray crystallography. The crystal structure of MBTD1 reveals its similarity to another four-MBT-repeat protein L3MBTL2, which binds lower methylated lysine histones. Fluorescence polarization experiments confirmed that MBTD1 preferentially binds mono- and di-methyllysine histone peptides, like L3MBTL1 and L3MBTL2. All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a "cavity insertion recognition mode" to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts [1]. Nevertheless, our mutagenesis data based on L3MBTL1 suggested that the histone peptides could not bind to MBT repeats in any orientation.The four MBT repeats in MBTD1 exhibits an asymmetric rhomboid architecture. Like other MBT repeat proteins characterized so far, MBTD1 binds mono- or dimethylated lysine histones through one of its four MBT repeats utilizing a semi-aromatic cage.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Rare Variant Analysis of Human and Rodent Obesity Genes in Individuals with Severe Childhood Obesity

A. Palotie on työryhmän UK10K Consortium jäsen.Obesity is a genetically heterogeneous disorder. Using targeted and whole-exome sequencing, we studied 32 human and 87 rodent obesity genes in 2,548 severely obese children and 1,117 controls. We identified 52 variants contributing to obesity in 2% of cases including multiple novel variants in GNAS, which were sometimes found with accelerated growth rather than short stature as described previously. Nominally significant associations were found for rare functional variants in BBS1, BBS9, GNAS, MKKS, CLOCK and ANGPTL6. The p.S284X variant in ANGPTL6 drives the association signal (rs201622589, MAF similar to 0.1%, odds ratio = 10.13, p-value = 0.042) and results in complete loss of secretion in cells. Further analysis including additional case-control studies and population controls (N = 260,642) did not support association of this variant with obesity (odds ratio = 2.34, p-value = 2.59 x 10(-3)), highlighting the challenges of testing rare variant associations and the need for very large sample sizes. Further validation in cohorts with severe obesity and engineering the variants in model organisms will be needed to explore whether human variants in ANGPTL6 and other genes that lead to obesity when deleted in mice, do contribute to obesity. Such studies may yield druggable targets for weight loss therapies.Peer reviewe

Altered ureteric branching morphogenesis and nephron endowment in offspring of diabetic and insulin-treated pregnancy

<div><p>There is strong evidence from human and animal models that exposure to maternal hyperglycemia during <i>in utero</i> development can detrimentally affect fetal kidney development. Notwithstanding this knowledge, the precise effects of diabetic pregnancy on the key processes of kidney development are unclear due to a paucity of studies and limitations in previously used methodologies. The purpose of the present study was to elucidate the effects of hyperglycemia on ureteric branching morphogenesis and nephrogenesis using unbiased techniques. Diabetes was induced in pregnant C57Bl/6J mice using multiple doses of streptozotocin (STZ) on embryonic days (E) 6.5-8.5. Branching morphogenesis was quantified <i>ex vivo</i> using Optical Projection Tomography, and nephrons were counted using unbiased stereology. Maternal hyperglycemia was recognised from E12.5. At E14.5, offspring of diabetic mice demonstrated fetal growth restriction and a marked deficit in ureteric tip number (control 283.7±23.3 vs. STZ 153.2±24.6, mean±SEM, <i>p</i>&lt;0.01) and ureteric tree length (control 33.1±2.6 mm vs. STZ 17.6±2.7 mm, <i>p</i> = 0.001) vs. controls. At E18.5, fetal growth restriction was still present in offspring of STZ dams and a deficit in nephron endowment was observed (control 1246.2±64.9 vs. STZ 822.4±74.0, <i>p&lt;</i>0.001). Kidney malformations in the form of duplex ureter and hydroureter were a common observation (26%) in embryos of diabetic pregnancy compared with controls (0%). Maternal insulin treatment from E13.5 normalised maternal glycaemia but did not normalise fetal weight nor prevent the nephron deficit. The detrimental effect of hyperglycemia on ureteric branching morphogenesis and, in turn, nephron endowment in the growth-restricted fetus highlights the importance of glycemic control in early gestation and during the initial stages of renal development.</p> </div

Heterokaryon Incompatibility Is Suppressed Following Conidial Anastomosis Tube Fusion in a Fungal Plant Pathogen

It has been hypothesized that horizontal gene/chromosome transfer and parasexual recombination following hyphal fusion between different strains may contribute to the emergence of wide genetic variability in plant pathogenic and other fungi. However, the significance of vegetative (heterokaryon) incompatibility responses, which commonly result in cell death, in preventing these processes is not known. In this study, we have assessed this issue following different types of hyphal fusion during colony initiation and in the mature colony. We used vegetatively compatible and incompatible strains of the common bean pathogen Colletotrichum lindemuthianum in which nuclei were labelled with either a green or red fluorescent protein in order to microscopically monitor the fates of nuclei and heterokaryotic cells following hyphal fusion. As opposed to fusion of hyphae in mature colonies that resulted in cell death within 3 h, fusions by conidial anastomosis tubes (CAT) between two incompatible strains during colony initiation did not induce the vegetative incompatibility response. Instead, fused conidia and germlings survived and formed heterokaryotic colonies that in turn produced uninucleate conidia that germinated to form colonies with phenotypic features different to those of either parental strain. Our results demonstrate that the vegetative incompatibility response is suppressed during colony initiation in C. lindemuthianum. Thus, CAT fusion may allow asexual fungi to increase their genetic diversity, and to acquire new pathogenic traits

Human vaccination against RH5 induces neutralizing antimalarial antibodies that inhibit RH5 invasion complex interactions.

The development of a highly effective vaccine remains a key strategic goal to aid the control and eventual eradication of Plasmodium falciparum malaria. In recent years, the reticulocyte-binding protein homolog 5 (RH5) has emerged as the most promising blood-stage P. falciparum candidate antigen to date, capable of conferring protection against stringent challenge in Aotus monkeys. We report on the first clinical trial to our knowledge to assess the RH5 antigen - a dose-escalation phase Ia study in 24 healthy, malaria-naive adult volunteers. We utilized established viral vectors, the replication-deficient chimpanzee adenovirus serotype 63 (ChAd63), and the attenuated orthopoxvirus modified vaccinia virus Ankara (MVA), encoding RH5 from the 3D7 clone of P. falciparum. Vaccines were administered i.m. in a heterologous prime-boost regimen using an 8-week interval and were well tolerated. Vaccine-induced anti-RH5 serum antibodies exhibited cross-strain functional growth inhibition activity (GIA) in vitro, targeted linear and conformational epitopes within RH5, and inhibited key interactions within the RH5 invasion complex. This is the first time to our knowledge that substantial RH5-specific responses have been induced by immunization in humans, with levels greatly exceeding the serum antibody responses observed in African adults following years of natural malaria exposure. These data support the progression of RH5-based vaccines to human efficacy testing

Structural Basis for Specific Binding of Human MPP8 Chromodomain to Histone H3 Methylated at Lysine 9

. MPP8 binding to methylated H3K9 is suggested to recruit the H3K9 methyltransferases GLP and ESET, and DNA methyltransferase 3A to the promoter of the E-cadherin gene, mediating the E-cadherin gene silencing and promote tumor cell motility and invasion. MPP8 contains a chromodomain in its N-terminus, which is used to bind the methylated H3K9. HP1, a chromodomain containing protein that binds to methylated H3K9 as well. The structure also reveals that the human MPP8 chromodomain forms homodimer, which is mediated via an unexpected domain swapping interaction through two β strands from the two protomer subunits.Our findings reveal the molecular mechanism of selective binding of human MPP8 chromodomain to methylated histone H3K9. The observation of human MPP8 chromodomain in both solution and crystal lattice may provide clues to study MPP8-mediated gene regulation furthermore

Comparative Structural Analysis of Human DEAD-Box RNA Helicases

DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members

Biomarkers of Dietary Omega-6 Fatty Acids and Incident Cardiovascular Disease and Mortality: An Individual-Level Pooled Analysis of 30 Cohort Studies

BACKGROUND: Global dietary recommendations for and cardiovascular effects of linoleic acid, the major dietary omega-6 fatty acid, and its major metabolite, arachidonic acid, remain controversial. To address this uncertainty and inform international recommendations, we evaluated how in vivo circulating and tissue levels of linoleic acid (LA) and arachidonic acid (AA) relate to incident cardiovascular disease (CVD) across multiple international studies. METHODS: We performed harmonized, de novo, individual-level analyses in a global consortium of 30 prospective observational studies from 13 countries. Multivariable-adjusted associations of circulating and adipose tissue LA and AA biomarkers with incident total CVD and subtypes (coronary heart disease, ischemic stroke, cardiovascular mortality) were investigated according to a prespecified analytic plan. Levels of LA and AA, measured as the percentage of total fatty acids, were evaluated linearly according to their interquintile range (ie, the range between the midpoint of the first and fifth quintiles), and categorically by quintiles. Study-specific results were pooled using inverse-variance–weighted meta-analysis. Heterogeneity was explored by age, sex, race, diabetes mellitus, statin use, aspirin use, omega-3 levels, and fatty acid desaturase 1 genotype (when available). RESULTS: In 30 prospective studies with medians of follow-up ranging 2.5 to 31.9 years, 15 198 incident cardiovascular events occurred among 68 659 participants. Higher levels of LA were significantly associated with lower risks of total CVD, cardiovascular mortality, and ischemic stroke, with hazard ratios per interquintile range of 0.93 (95% CI, 0.88–0.99), 0.78 (0.70–0.85), and 0.88 (0.79–0.98), respectively, and nonsignificantly with lower coronary heart disease risk (0.94; 0.88–1.00). Relationships were similar for LA evaluated across quintiles. AA levels were not associated with higher risk of cardiovascular outcomes; in a comparison of extreme quintiles, higher levels were associated with lower risk of total CVD (0.92; 0.86–0.99). No consistent heterogeneity by population subgroups was identified in the observed relationships. CONCLUSIONS: In pooled global analyses, higher in vivo circulating and tissue levels of LA and possibly AA were associated with lower risk of major cardiovascular events. These results support a favorable role for LA in CVD prevention

Analyses of human vaccine-specific circulating and bone marrow-resident B cell populations reveal benefit of delayed vaccine booster dosing with blood-stage malaria antigens

We have previously reported primary endpoints of a clinical trial testing two vaccine platforms for the delivery of Plasmodium vivax malaria DBPRII: viral vectors (ChAd63, MVA), and protein/adjuvant (PvDBPII with 50µg Matrix-M™ adjuvant). Delayed boosting was necessitated due to trial halts during the pandemic and provides an opportunity to investigate the impact of dosing regimens. Here, using flow cytometry – including agnostic definition of B cell populations with the clustering tool CITRUS – we report enhanced induction of DBPRII-specific plasma cell and memory B cell responses in protein/adjuvant versus viral vector vaccinees. Within protein/adjuvant groups, delayed boosting further improved B cell immunogenicity compared to a monthly boosting regimen. Consistent with this, delayed boosting also drove more durable anti-DBPRII serum IgG. In an independent vaccine clinical trial with the P. falciparum malaria RH5.1 protein/adjuvant (50µg Matrix-M™) vaccine candidate, we similarly observed enhanced circulating B cell responses in vaccinees receiving a delayed final booster. Notably, a higher frequency of vaccine-specific (putatively long-lived) plasma cells was detected in the bone marrow of these delayed boosting vaccinees by ELISPOT and correlated strongly with serum IgG. Finally, following controlled human malaria infection with P. vivax parasites in the DBPRII trial, in vivo growth inhibition was observed to correlate with DBPRII-specific B cell and serum IgG responses. In contrast, the CD4+ and CD8+ T cell responses were impacted by vaccine platform but not dosing regimen and did not correlate with in vivo growth inhibition in a challenge model. Taken together, our DBPRII and RH5 data suggest an opportunity for protein/adjuvant dosing regimen optimisation in the context of rational vaccine development against pathogens where protection is antibody-mediated