The rockefeller university

There is substantial evidence for extensive nonvesicular sterol transport in cells. For example, lipid transfer by the steroidogenic acute regulator-related proteins (StarD) containing a StarT domain has been shown to involve several pathways of nonvesicular trafficking. Among the soluble StarT domain-containing proteins, StarD4 is expressed in most tissues and has been shown to be an effective sterol transfer protein. However, it was unclear whether the lipid composition of donor or acceptor membranes played a role in modulating StarD4-mediated transport. Here, we used fluorescence-based assays to demonstrate a phosphatidylinositol phosphate (PIP)-selective mechanism by which StarD4 can preferentially extract sterol from liposome membranes containing certain PIPs (especially, PI(4,5)P2 and to a lesser degree PI(3,5)P2). Monophosphorylated PIPs and other anionic lipids had a smaller effect on sterol transport. This enhancement of transport was less effective when the same PIPs were present in the acceptor membranes. Furthermore, using molecular dynamics (MD) simulations, we mapped the key interaction sites of StarD4 with PIP-containing membranes and identified residues that are important for this interaction and for accelerated sterol transport activity. We show that StarD4 recognizes membrane-specific PIPs through specific interaction with the geometry of the PIP headgroup as well as the surrounding membrane environment. Finally, we also observed that StarD4 can deform membranes upon longer incubations. Taken together, these results suggest a mechanism by which PIPs modulate cholesterol transfer activity via StarD4.

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and mutations in myosin-15 cause hereditary deafness. In addition to transporting actin regulatory machinery to stereocilia tips, myosin-15 directly nucleates actin filament ("F-actin") assembly, which is disrupted by a progressive hearing loss mutation (p.D1647G, "Jordan"). Here, we present cryo-electron microscopy structures of myosin-15 bound to F-actin, providing a framework for interpreting the impacts of deafness mutations on motor activity and actin nucleation. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin's D-loop, which mediates inter-subunit contacts, while the Jordan mutant locks the D-loop in a single conformation. Adenosine diphosphate-bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin's structural plasticity in a myosin nucleotide state-dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

The Omicron variant of SARS-CoV-2 infected many vaccinated and convalescent individuals(1-)(3). Despite the reduced protection from infection, individuals who received three doses of an mRNA vaccine were highly protected from more serious consequences of infection(4). Here we examine the memory B cell repertoire in a longitudinal cohort of individuals receiving three mRNA vaccine doses(5,6). We find that the third dose is accompanied by an increase in, and evolution of, receptor-binding domain (RBD)-specific memory B cells. The increase is due to expansion of memory B cell clones that were present after the second dose as well as the emergence of new clones. The antibodies encoded by these cells showed significantly increased potency and breadth when compared with antibodies obtained after the second dose. Notably, the increase in potency was especially evident among newly developing clones of memory cells, which differed from persisting clones in targeting more conserved regions of the RBD. Overall, more than 50% of the analysed neutralizing antibodies in the memory compartment after the third mRNA vaccine dose neutralized the Omicron variant. Thus, individuals receiving three doses of an mRNA vaccine have a diverse memory B cell repertoire that can respond rapidly and produce antibodies capable of clearing even diversified variants such as Omicron. These data help to explain why a third dose of a vaccine that was not specifically designed to protect against variants is effective against variant-induced serious disease.

Background Autosomal recessive (AR) complete IRF8 deficiency is a rare severe inborn error of immunity underlying an absence of blood myeloid mononuclear cells, intracerebral calcifications, and multiple infections. Only three unrelated patients have been reported. Materials and Methods We studied an Argentinian child with multiple infectious diseases and severe pulmonary alveolar proteinosis (PAP). We performed whole-exome sequencing (WES) and characterized his condition by genetic, immunological, and clinical means. Results The patient was born and lived in Argentina. He had a history of viral pulmonary diseases, disseminated disease due to bacillus Calmette-Guerin (BCG), PAP, and cerebral calcifications. He died at the age of 10 months from refractory PAP. WES identified two compound heterozygous variants in IRF8: c.55del and p.R111*. In an overexpression system, the p.R111* cDNA was loss-of-expression, whereas the c.55del cDNA yielded a protein with a slightly lower molecular weight than the wild-type protein. The mutagenesis of methionine residues downstream from c.55del revealed a re-initiation of translation. However, both variants were loss-of-function in a luciferase assay, suggesting that the patient had AR complete IRF8 deficiency. The patient had no blood monocytes or dendritic cells, associated with neutrophilia, and normal counts of NK and other lymphoid cell subsets. Conclusion We describe the fourth patient with AR complete IRF8 deficiency. This diagnosis should be considered in children with PAP, which is probably due to the defective development or function of alveolar macrophages.

Cyclin-dependent kinases (CDKs) lie at the heart of eukaryotic cell cycle control, with different cyclin-CDK complexes initiating DNA replication (S-CDKs) and mitosis (M-CDKs)(1,2). However, the principles on which cyclin-CDK complexes organize the temporal order of cell cycle events are contentious(3). One model proposes that S-CDKs and M-CDKs are functionally specialized, with substantially different substrate specificities to execute different cell cycle events4-6. A second model proposes that S-CDKs and M-CDKs are redundant with each other, with both acting as sources of overall CDK activity(7,8). In this model, increasing CDK activity, rather than CDK substrate specificity, orders cell cycle events(9,10). Here we reconcile these two views of core cell cycle control. Using phosphoproteomic assays of in vivo CDK activity in fission yeast, we find that S-CDK and M-CDK substrate specificities are remarkably similar, showing that S-CDKs and M-CDKs are not completely specialized for S phase and mitosis alone. Normally, S-CDK cannot drive mitosis but can do so when protein phosphatase 1 is removed from the centrosome. Thus, increasing S-CDK activity in vivo is sufficient to overcome substrate specificity differences between S-CDK and M-CDK, and allows S-CDK to carry out M-CDK function. Therefore, we unite the two opposing views of cell cycle control, showing that the core cell cycle engine is largely based on a quantitative increase in CDK activity through the cell cycle, combined with minor and surmountable qualitative differences in catalytic specialization of S-CDKs and M-CDKs.

The hepatitis B virus (HBV) is one of the smallest but most highly infectious human pathogens. With a DNA genome of only 3.2 kb and only four genes, HBV successfully completes its life cycle by using intricate processes to hijack the host machinery. HBV infects non-dividing liver cells in which dNTPs are limited. As a DNA virus, HBV requires dNTPs for its replication. HBV induces the ATR-mediated cellular DNA damage response pathway to overcome this constraint. This pathway upregulates R2 (RRM2) expression in generating an active RNR holoenzyme catalyzing de novo dNTP synthesis. Previously we reported that ERE, a small RNA fragment within the HBx ORF, is sufficient to induce R2 upregulation. Interestingly, there is high sequence similarity between ERE and a region within the R2 5 ' UTR that we named R2-box. Here, we established a mutant cell line in the R2-box region of the R2 gene using CRISPR-Cas9 technology to investigate the R2 regulation by ERE. This cell line expresses a much lower R2 level than the parental cell line. Interestingly, the HBV infection and life cycle were severely impaired. These cells became permissive to HBV infection upon ectopically R2 expression. These results validate the requirement of the R2 gene expression for HBV replication. Remarkably, the R2-box mutated cells became ERE refractory, suggesting that the homology region between ERE and R2 gene is critical for ERE-mediated R2 upregulation. Thus, along with the induction of the ATR pathway of the DNA damage response, ERE might also directly target the R2 gene via the R2-box.

Alzheimer's disease (AD) is a fatal cognitive disorder with proteinaceous brain deposits, neuroinflammation, cerebrovascular dysfunction, and extensive neuronal loss over time. AD is a multifactorial disease, and lifestyle factors, including diet, are likely associated with the development of AD pathology. Since obesity and diabetes are recognized as risk factors for AD, it might be predicted that a high-fat diet (HFD) would worsen AD pathology. However, modeling HFD-induced obesity in AD animal models has yielded inconclusive results. Some studies report a deleterious effect of HFD on A beta accumulation, neuroinflammation, and cognitive function, while others report that HFD worsens memory without affecting AD brain pathology. Moreover, several studies report no major effect of HFD on AD-related phenotypes in mice, while other studies show that HFD might, in fact, be protective. The lack of a clear association between dietary fat consumption and AD-related pathology and cognitive function in AD mouse models might be explained by experimental variations, including AD mouse model, sex and age of the animals, composition of the HFD, and timeline of HFD consumption. In this review, we summarize recent studies that aimed at elucidating the effect of HFD-induced obesity on AD-related pathology in mice and provide an overview of the factors that may have contributed to the results reported in these studies. Based on the heterogeneity of these animal model studies and given that the human population itself is quite disparate, it is likely that people will benefit most from individualized nutritional plans based on their medical history and clinical profiles.

Translation is a tightly regulated process that ensures optimal protein quality and enables adaptation to energy/nutrient availability. The alpha-kinase eukaryotic elongation factor 2 kinase (eEF-2K), a key regulator of translation, specifically phosphorylates the guanosine triphosphatase eEF-2, thereby reducing its affinity for the ribosome and suppressing the elongation phase of protein synthesis. eEF-2K activation requires calmodulin binding and autophosphorylation at the primary stimulatory site, T348. Biochemical studies predict a calmodulin-mediated activation mechanism for eEF-2K distinct from other calmodulin-dependent kinases. Here, we resolve the atomic details of this mechanism through a 2.3-angstrom crystal structure of the heterodimeric complex of calmodulin and the functional core of eEF-2K (eEF-2K(TR)). This structure, which represents the activated T348-phosphorylated state of eEF-2K(TR), highlights an intimate association of the kinase with the calmodulin C-lobe, creating an "activation spine" that connects its amino-terminal calmodulin-targeting motif to its active site through a conserved regulatory element.

Microbiome data have several specific characteristics (sparsity and compositionality) that introduce challenges in data analysis. The integration of prior information regarding the data structure, such as phylogenetic structure and repeated-measure study designs, into analysis, is an effective approach for revealing robust patterns in microbiome data. Past methods have addressed some but not all of these challenges and features: for example, robust principal-component analysis (RPCA) addresses sparsity and compositionality; compositional tensor factorization (CIF) addresses sparsity, compositionality, and repeated measure study designs; and UniFrac incorporates phylogenetic information. Here we introduce a strategy of incorporating phylogenetic information into RPCA and CTF. The resulting methods, phylo-RPCA, and phylo-CTF, provide substantial improvements over state-of-the-art methods in terms of discriminatory power of underlying clustering ranging from the mode of delivery to adult human lifestyle. We demonstrate quantitatively that the addition of phylogenetic information improves effect size and classification accuracy in both data-driven simulated data and real microbiome data. IMPORTANCE Microbiome data analysis can be difficult because of particular data features, some unavoidable and some due to technical limitations of DNA sequencing instruments. The first step in many analyses that ultimately reveals patterns of similarities and differences among sets of samples (e.g., separating samples from sick and healthy people or samples from seawater versus soil) is calculating the difference between each pair of samples. We introduce two new methods to calculate these differences that combine features of past methods, specifically being able to take into account the principles that most types of microbes are not in most samples (sparsity), that abundances are relative rather than absolute (compositionality), and that all microbes have a shared evolutionary history (phylogeny). We show using simulated and real data that our new methods provide improved classification accuracy of ordinal sample clusters and increased effect size between sample groups on beta-diversity distances.