The rockefeller university

Fanconi anemia (FA) is a rare genetic disease characterized by loss-of-function variants in any of the 22 previously identified genes (FANCA-FANCW) that encode proteins participating in the repair of DNA interstrand crosslinks (ICLs). Patient phenotypes are variable but may include developmental abnormalities, early-onset pancytopenia, and a predisposition to hematologic and solid tumors. Here, we describe 2 unrelated families with multiple pregnancy losses and offspring presenting with severe developmental and hematologic abnormalities leading to death in utero or in early life. Homozygous loss-of-function variants in FAAP100 were identified in affected children of both families. The FAAP100 protein associates with FANCB and FANCL, the E3 ubiquitin ligase responsible for the monoubiquitination of FANCD2 and FANCI, which is necessary for FA pathway function. Patient-derived cells exhibited phenotypes consistent with FA. Expression of the WT FAAP100 cDNA, but not the patient-derived variants, rescued the observed cellular phenotypes. This establishes FAAP100 deficiency as a cause of FA, with FAAP100 gaining an alias as FANCX. The extensive developmental malformations of individuals with FAAP100 loss-of-function variants are among the most severe across previously described FA phenotypes, indicating that the FA pathway is essential for human development.

Nanodiscs, small discoidal membrane patches stabilized by membrane-scaffold proteins (MSPs), are popular tools to stabilize integral membrane proteins (IMPs) for structural studies by cryogenic electron microscopy (cryo-EM). While nanodiscs provide a near-native membrane environment for the incorporated IMPs, they do not reproduce all characteristics of a native membrane. Also, IMPs must first be purified in detergent before they can be reconstituted into MSP-based nanodiscs, a problem that has been overcome by newer approaches, such as copolymer-based native nanodiscs and cell-derived vesicles. In this review, we argue that despite these advances, MSP-based nanodiscs remain a unique tool for the structural interrogation of IMPs.

Despite waning of virus-neutralizing antibodies, protection against severe SARS-CoV-2 in the majority of immune individuals remains high, but the underlying immune mechanisms are incompletely understood. Here, rhesus macaques with pre-existing immunity from Novavax WA-1 and/or P.1 vaccines and WA-1 or P.1 infection are immunized with a bivalent WA-1/Omicron BA.5 Novavax vaccine ten months after the last exposure. The boost vaccination primarily increases the frequency of cross-reactive spike (S)-specific antibodies and B cells instead of inducing de novo BA.5-specific responses. Reinfection with heterologous Omicron XBB.1.5 six months after the boost vaccination results in low levels of virus replication in the respiratory tract compared with virus-na & iuml;ve results from other studies. Whereas systemic S-specific immunity remains largely unchanged in all animals, the animals with complete protection from infection exhibit a stronger influx of S-specific IgG, monocytes, B cells and T cells into the bronchioalveolar space combined with expansion of CD69+CD103+ lung tissue-resident, S-specific CD8 T cells compared to actively infected animals. Our results underscore the importance of localized respiratory immune responses in mediating protection from Omicron reinfection and provide guidance for future vaccine development.

In the common cellular space, hundreds of binding reactions occur reliably and enable this remarkable compatibility have not yet been adequately elucidated. In order to delineate these principles, we consider the intracellular sensing of transition metals in bacteria-an integral part of cellular metal homeostasis. Protein cytosolic sensors typically interact with metals through three types of lateral chain residues, containing oxygen, nitrogen, or sulfur. The very existence of complete sets of mutually compatible sensors is a nontrivial problem solved by evolution, since each metal sensor has to bind to its cognate metal without being "mismetallated" by noncognate competitors. Here, based solely on theoretical considerations and limited information about binding transition-metal sensor sets are severely limited in their number by compatibility requirements, leaving only a handful of possible sensor compositions for each transition metal. Our theoretical results turn out to be broadly consistent with experimental data crucial role in the organization and functioning of intracellular processes.

The cGAS-STING pathway, well-known to elicit interferon (IFN) responses, is also a key inducer of autophagy upon virus infection or other stimuli. Whereas the mediators for cGAS-induced IFN responses are well characterized, much less is known about how cGAS elicits autophagy. Here, we report that TRIM23, a unique TRIM protein harboring both ubiquitin E3 ligase and GTPase activity, is crucial for cGAS-STING-dependent antiviral autophagy. Genetic ablation of TRIM23 impairs autophagic control of HSV-1 infection. HSV-1 infection or cGAS-STING stimulation induces TBK1-mediated TRIM23 phosphorylation at S39, which triggers TRIM23 autoubiquitination and GTPase activity and ultimately elicits autophagy. Fibroblasts from a patient with herpes simplex encephalitis heterozygous for a dominant-negative, kinase-inactivating TBK1 mutation fail to activate autophagy by TRIM23 and cGAS-STING. Our results thus identify the cGAS-STING-TBK1-TRIM23 axis as a key autophagy defense pathway and may stimulate new therapeutic interventions for viral or inflammatory diseases.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an enveloped, negative-sense RNA virus that is spread by ticks across Europe, Africa, and Asia and causes a lethal disease in humans (similar to 30%-40% case fatality). There are currently no approved vaccines or therapeutics. Antibody-based therapeutics targeting the CCHFV surface glycoproteins Gn and Gc, which are responsible for viral attachment and fusion during entry, are a promising therapeutic approach. We previously isolated three broadly neutralizing Gc-targeting human monoclonal antibodies (mAbs) and showed certain cocktails of these mAbs demonstrated synergistic virus neutralization. Furthermore, physical linkage of two of these mAbs into a dual variable domain (DVD) bispecific antibody (bsAb) DVD-121-801 resulted in improved neutralization and therapeutic protection against a lethal CCHFV challenge in mice. However, the molecular requirements for the activity of DVD-121-801, and why it is augmented over monospecific parental mAbs, remain the topic of investigation. Here, we generated a new panel of bsAb variants of DVD-121-801 to explore the spacing and avidity requirements and further optimize its protective efficacy against divergent CCHFV isolates. We evaluated these variants for neutralization, fusion inhibition, and protection with virus-like particles, authentic viruses, and in vivo challenge studies. We found that neutralization potency was relatively unaffected by spacing or identity of variable domains within the bsAb, but that one next-generation design employing longer and more flexible linkers between variable domains (DVD-121-801GS) had a greater breadth of therapeutic protection. Our efforts highlight the importance of antibody avidity and lead to an improved bsAb variant of DVD-121-801 for further therapeutic development. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus endemic to Europe, Africa, and Asia that causes severe disease in humans (30%-40% case fatality). There are currently no approved vaccines or therapeutics. In prior work, physical linkage of two Gc-specific monoclonal antibodies (mAbs) targeting distinct epitopes into a dual variable domain (DVD) bispecific antibody (bsAb), termed DVD-121-801, resulted in potent neutralization in vitro and therapeutic protection in vivo. Here, a panel of variants of this bsAb was developed and evaluated for neutralization potency, fusion inhibition, and therapeutic efficacy, and our work shows this panel to be effective against multiple isolates of CCHFV. Furthermore, incorporating longer, more flexible linkers between variable domains resulted in a lead candidate with improved activity and therapeutic potential compared to the parental bsAb. Utilizing this panel, we also explored the contribution of antibody avidity in antibody-mediated protection against CCHFV infection.

Gating in voltage-dependent ion channels is regulated by the transmembrane voltage. This form of regulation is enabled by voltage-sensing domains (VSDs) that respond to transmembrane voltage differences by changing their conformation and exerting force on the pore to open or close it. Here, we use cryogenic electron microscopy to study the neuronal K(v)2.1 channel in lipid vesicles with and without a voltage difference across the membrane. Hyperpolarizing voltage differences displace the positively charged S4 helix in the voltage sensor by one helical turn (similar to 5 angstrom). When this displacement occurs, the S4 helix changes its contact with the pore at two different interfaces. When these changes are observed in fewer than four voltage sensors, the pore remains open, but when they are observed in all four voltage sensors, the pore constricts. The constriction occurs because the S4 helix, as it displaces inward, squeezes the right-handed helical bundle of pore-lining S6 helices. A similar conformational change occurs upon hyperpolarization of the EAG1 channel but with two helical turns displaced instead of one. Therefore, while K(v)2.1 and EAG1 are from distinct architectural classes of voltage-dependent ion channels, called domain-swapped and non-domain-swapped, the way the voltage sensors gate their pores is very similar.

Changes in gene expression are a key driver of phenotypic evolution, leading to a persistent interest in the evolution of transcriptomes. Traditionally, gene expression is modeled as a continuous trait, leaving qualitative transitions largely unexplored. In this paper, we detail the development of new Bayesian inference techniques to study the evolutionary turnover of organ-specific transcriptomes, which we define as instances where orthologous genes gain or lose expression in a particular organ. To test these techniques, we analyze the transcriptomes of 2 male reproductive organs, testes and accessory glands, across 11 species of the Drosophila melanogaster species group. We first discretize gene expression states by estimating the probability that each gene is expressed in each organ and species. We then define a phylogenetic model of correlated transcriptome evolution in 2 or more organs and fit it to the expression state data. Inferences under this model imply that many genes have gained and lost expression in each organ, and that the 2 organs experienced accelerated transcriptome turnover on different branches of the Drosophila phylogeny.