Viruses employ a variety of molecular strategies to carve out an existence in their host and to thwart host defenses. Crystal structures of viral proteins and of the host proteins deployed as ...molecular weapons contribute enormously to our understanding of viral pathogenesis and our efforts to combat viral infection. Mosquito‐borne flaviviruses, including the dengue, Zika, yellow fever, Japanese encephalitis and West Nile viruses, cause serious human diseases in much of the world. A growing number of diverse functions has been discovered for the enigmatic virulence factor known as NS1: it is an essential cofactor in viral genome replication, a mediator of the host immune response, and a trigger of host vascular leakage. NS1 lacks precedent in the structure and sequence databases, so an accurate 3D structure was key to understanding the basis for its various functions. A high‐throughput survey of expression conditions in baculovirus‐infected insect cells was essential to identifying conditions for production of recombinant NS1 in its natural glycosylated, disulfide‐linked form. We solved a crystal structure of West Nile virus NS1 from the anomalous scattering of the native sulfur atoms using an 18‐crystal data set 1,2. The structure revealed a previously unknown protein fold with a fundamentally dimeric architecture, and led to assigned functions for two of the three NS1 structural domains. Subsequent structures of NS1 from dengue virus and Zika viruses established the basis for its membrane association in cells and its lipid encapsidation when secreted 3. Recently discovered functions of this remarkable virulence factor emerged in follow‐up studies that built upon the crystal structures. Prime among these is the discovery that the NS1 protein, in a mouse model of dengue disease and in absence of virus, can induce the vascular leak that is a hallmark of severe dengue disease 4,5. Clues to the molecular mechanism of NS1‐induced endothelial dysfunction emerged from a study of the protection afforded by a monoclonal antibody and the structure of the Fab fragment bound to the NS1 epitope 6.
1 Akey DL et al. & Smith JL (2014) Science 343, 881‐885.
2 Akey DL et al. & Smith JL (2014) Acta Crystallogr D70, 2719‐2729.
3 Brown WC et al. & Smith JL (2016) Nature Struct Molec Biol 23, 856‐867.
4 Beatty PR et al. & Harris E (2015) Science Trans Med 7, 304ra141.
5 Modhiran N et al. & Young PR (2015) Science Trans Med 7, 304ra142.
6 Biering SB, Akey DL et al. Smith JL & Harris E (2021) Science 371, 194‐200.
This report presents the conclusions of the X-ray Validation Task Force of the worldwide Protein Data Bank (PDB). The PDB has expanded massively since current criteria for validation of deposited ...structures were adopted, allowing a much more sophisticated understanding of all the components of macromolecular crystals. The size of the PDB creates new opportunities to validate structures by comparison with the existing database, and the now-mandatory deposition of structure factors creates new opportunities to validate the underlying diffraction data. These developments highlighted the need for a new assessment of validation criteria. The Task Force recommends that a small set of validation data be presented in an easily understood format, relative to both the full PDB and the applicable resolution class, with greater detail available to interested users. Most importantly, we recommend that referees and editors judging the quality of structural experiments have access to a concise summary of well-established quality indicators.
► Validation criteria used by the PDB for X-ray crystal structures have been reassessed ► Key scores should be presented prominently in an easily understood format ► A concise validation report should be available to referees of papers on crystal structures
Micro-crystallography comes of age Smith, Janet L; Fischetti, Robert F; Yamamoto, Masaki
Current opinion in structural biology,
10/2012, Letnik:
22, Številka:
5
Journal Article
Recenzirano
Odprti dostop
► X-ray micro-beams permit structure determination from small or imperfect crystals. ► Robust micro-beams require source, optics and sample support to be extremely stable. ► Four different approaches ...to making a 1–20-μm beam have been implemented. ► A growing demand has led to ∼20 beamlines for micro-crystallography worldwide. ► Micro-beams enable data collection from LCP-grown crystals and crystals in situ.
The latest revolution in macromolecular crystallography was incited by the development of dedicated, user friendly, micro-crystallography beam lines. Brilliant X-ray beams of diameter 20μm or less, now available at most synchrotron sources, enable structure determination from samples that previously were inaccessible. Relative to traditional crystallography, crystals with one or more small dimensions have diffraction patterns with vastly improved signal-to-noise when recorded with an appropriately matched beam size. Structures can be solved from isolated, well diffracting regions within inhomogeneous samples. This review summarizes the technological requirements and approaches to producing micro-beams and how they continue to change the practice of crystallography.
DnaA, the replication initiation protein in bacteria, is an AAA+ ATPase that binds and hydrolyzes ATP and exists in a heterogeneous population of ATP-DnaA and ADP-DnaA. DnaA binds cooperatively to ...the origin of replication and several other chromosomal regions, and functions as a transcription factor at some of these regions. We determined the binding properties of Bacillus subtilis DnaA to genomic DNA in vitro at single nucleotide resolution using in vitro DNA affinity purification and deep sequencing (IDAP-Seq). We used these data to identify 269 binding regions, refine the consensus sequence of the DnaA binding site, and compare the relative affinity of binding regions for ATP-DnaA and ADP-DnaA. Most sites had a slightly higher affinity for ATP-DnaA than ADP-DnaA, but a few had a strong preference for binding ATP-DnaA. Of the 269 sites, only the eight strongest binding ones have been observed to bind DnaA in vivo, suggesting that other cellular factors or the amount of available DnaA in vivo restricts DnaA binding to these additional sites. Conversely, we found several chromosomal regions that were bound by DnaA in vivo but not in vitro, and that the nucleoid-associated protein Rok was required for binding in vivo. Our in vitro characterization of the inherent ability of DnaA to bind the genome at single nucleotide resolution provides a backdrop for interpreting data on in vivo binding and regulation of DnaA, and is an approach that should be adaptable to many other DNA binding proteins.
Primary vitreoretinal lymphoma (PVRL), also known as primary intraocular lymphoma, is a rare malignancy typically classified as a diffuse large B‐cell lymphoma and most frequently develops in elderly ...populations. PVRL commonly masquerades as posterior uveitis and has a unique tropism for the retina and central nervous system (CNS). Over 15% of primary CNS lymphoma patients develop intraocular lymphoma, usually occurring in the retina and/or vitreous. Conversely, 65%–90% of PVRL patients develop CNS lymphoma. Consequently, PVRL is often fatal because of ultimate CNS association. Current PVRL animal models are limited and require further development. Typical clinical findings include vitreous cellular infiltration (lymphoma and inflammatory cells) and subretinal tumor infiltration as determined using dilated fundoscopy, fluorescent angiography, and optical coherent tomography. Currently, PVRL is most often diagnosed using both histology to identify lymphoma cells in the vitreous or retina and immunohistochemistry to indicate monoclonality. Additional adjuncts in diagnosing PVRL exist, including elevation of interleukin‐10 levels in ocular fluids and detection of IgH or T‐cell receptor gene rearrangements in malignant cells. The optimal therapy for PVRL is not defined and requires the combined effort of oncologists and ophthalmologists. PVRL is sensitive to radiation therapy and exhibits high responsiveness to intravitreal methotrexate or rituximab. Although systemic chemotherapy alone can result in high response rates in patients with PVRL, there is a high relapse rate. Because of the disease rarity, international, multicenter, collaborative efforts are required to better understand the biology and pathogenesis of PVRL as well as to define both diagnostic markers and optimal therapies.
摘要
原发性玻璃体视网膜淋巴瘤（PVRL），又称原发性眼内淋巴瘤，是一种罕见的恶性肿瘤，通常被归为弥漫性大B细胞淋巴瘤，老年人多见。PVRL仅发生于视网膜和中枢神经系统，常被误认为后色素层炎。超过15%的原发性中枢神经系统淋巴瘤患者发生眼内淋巴瘤，通常发生在视网膜和/或玻璃体。相反，65%~90%的PVRL患者发生中枢神经系统淋巴瘤。由于PVRL最终与中枢神经系统相关联，因此往往是致命的。目前的PVRL动物模型有局限性，需进一步开发。典型临床表现为在散瞳眼底镜检查、荧光血管造影和 光学相干断层扫描 下可见玻璃体细胞浸润（淋巴瘤和炎症细胞）和视网膜下肿瘤浸润。目前，PVRL最常用的诊断方法是通过组织学确定玻璃体或视网膜的淋巴瘤细胞、以及免疫组化提示单克隆性。其他PVRL辅助诊断方法包括眼内液白介素‐10增高、恶性细胞中检测出IgH 或T细胞受体基因重排。PVRL的最佳治疗方案尚未确定，需要肿瘤科医生和眼科医生通力合作。PVRL对放疗敏感，并对玻璃体内注射甲氨蝶呤或利妥昔单抗具有高反应性。虽然PVRL采用全身化疗缓解率较高，但复发率也较高。由于本病罕见，因此需要国际性多中心的协作努力，才能更透彻地了解PVRL的生物学特性和发病机制，确定其诊断标记物和最佳疗法。
A symposium on primary vitreoretinal lymphoma held at the Fifth Annual, National Cancer Institute–sponsored International Primary Central Nervous System Lymphoma Collaborative Group conference, a multidisciplinary meeting, is summarized herein. Tumor biology, nomenclature, epidemiology and prognosis, biology and pathogenesis, animal models, clinical manifestations, diagnosis, therapeutics, and future investigations are reviewed.
Covering: up to the end of 2018
Polyketides are a valuable source of bioactive and clinically important molecules. The biosynthesis of these chemically complex molecules has led to the discovery of ...equally complex polyketide synthase (PKS) pathways. Crystallography has yielded snapshots of individual catalytic domains, di-domains, and multi-domains from a variety of PKS megasynthases, and cryo-EM studies have provided initial views of a PKS module in a series of defined biochemical states. Here, we review the structural and biochemical results that shed light on the protein-protein interactions critical to catalysis by PKS systems with an embedded acyltransferase. Interactions include those that occur both within and between PKS modules, as well as with accessory enzymes.
Protein-protein interactions of
cis
-AT polyketide synthases are dominated by the travels of the ACP domain to the active site entrance of each catalytic domain.
The HIV Tat protein competes with the 7SK:HEXIM interaction to hijack pTEFb from 7SK snRNP and recruit it to the TAR motif on stalled viral transcripts. Here we solve structures of 7SK stemloop-1 and ...TAR in complex with Tat's RNA binding domain (RBD) to gain insights into this process. We find that 7SK is peppered with arginine sandwich motifs (ASM)-three classical and one with a pseudo configuration. Despite having similar RBDs, the presence of an additional arginine, R52, confers Tat the ability to remodel the pseudo configuration, required for HEXIM binding, into a classical sandwich, thus displacing HEXIM. Tat also uses R52 to remodel the TAR bulge into an ASM whose structure is identical to that of the remodeled ASM in 7SK. Together, our structures reveal a dual structural mimicry wherein viral Tat and TAR have co-opted structural motifs present in cellular HEXIM and 7SK for productive transcription of its genome.
Severe prenatal undernutrition is usually associated with low birth weights in offspring and disorders including hypertension, obesity, and diabetes. Whether alterations in maternal nutrition ...insufficient to impair birth weight or prenatal growth impact the cardiovascular, stress, or metabolic systems is unknown. In addition, little is known about the effects of maternal dietary restriction on development of the reproductive system in mammals. Here, we use the bovine model, which has a gestational length and birth rate similar to humans, to show that offspring from nutritionally restricted dams (during the first trimester) were born with identical birth weights and had similar postnatal growth rates (to 95 wk of age), puberty, glucose metabolism, and responses to stress compared to offspring from control mothers. However, an increase in maternal testosterone concentrations was detected during dietary restriction, and these dams had offspring with a diminished ovarian reserve (as assessed by a reduction in antral follicle count, reduced concentrations of anti-Müllerian hormone, and increased follicle-stimulating hormone concentrations), enlarged aorta, and increased arterial blood pressure compared with controls. Our study links transient maternal undernutrition and enhanced maternal androgen production with a diminished ovarian reserve as well as potential suboptimal fertility, enlarged aortic trunk size, and enhanced blood pressure independent of alterations in birth weight, postnatal growth, or stress response and glucose tolerance. The implications are that relatively mild transient reductions in maternal nutrition during the first trimester of pregnancy (even those that do not affect gross development) should be avoided to ensure healthy development of reproductive and cardiovascular systems in offspring.
Individuals participating in biobanks and other large research projects are increasingly asked to provide broad consent for open-ended research use and widespread sharing of their biosamples and ...data. We assessed willingness to participate in a biobank using different consent and data sharing models, hypothesizing that willingness would be higher under more restrictive scenarios. Perceived benefits, concerns, and information needs were also assessed. In this experimental survey, individuals from 11 US healthcare systems in the Electronic Medical Records and Genomics (eMERGE) Network were randomly allocated to one of three hypothetical scenarios: tiered consent and controlled data sharing; broad consent and controlled data sharing; or broad consent and open data sharing. Of 82,328 eligible individuals, exactly 13,000 (15.8%) completed the survey. Overall, 66% (95% CI: 63%–69%) of population-weighted respondents stated they would be willing to participate in a biobank; willingness and attitudes did not differ between respondents in the three scenarios. Willingness to participate was associated with self-identified white race, higher educational attainment, lower religiosity, perceiving more research benefits, fewer concerns, and fewer information needs. Most (86%, CI: 84%–87%) participants would want to know what would happen if a researcher misused their health information; fewer (51%, CI: 47%–55%) would worry about their privacy. The concern that the use of broad consent and open data sharing could adversely affect participant recruitment is not supported by these findings. Addressing potential participants’ concerns and information needs and building trust and relationships with communities may increase acceptance of broad consent and wide data sharing in biobank research.
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