Publications search

Slow inactivation has been described in multiple voltage-gated K+ channels and in great detail in the Drosophila Shaker channel. Structural studies have begun to facilitate a better understanding of the atomic details of this and other gating mechanisms. To date, the only voltage-gated potassium channels whose structure has been solved are KvAP (x-ray diffraction), the K(V)1.2-K(V)2.1 "paddle" chimera (x-ray diffraction and cryo-EM), K(V)1.2 (x-ray diffraction), and ether-a-go-go (cryo-EM); however, the structural details and mechanisms of slow inactivation in these channels are unknown or poorly characterized. Here, we present a detailed study of slow inactivation in the rat K(V)1.2 channel and show that it has some properties consistent with the C-type inactivation described in Shaker. We also study the effects of some mutations that are known to modulate C-type inactivation in Shaker and show that qualitative and quantitative differences exist in their functional effects, possibly underscoring subtle but important structural differences between the C-inactivated states in Shaker and K(V)1.2.

T-cell receptor (TCR)-based therapeutic cells and agents have emerged as a new class of effective cancer therapies. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on MHC receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high-throughput genetic platform (termed "PresentER") that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-and-off targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

Background. MK-8591 (4'-ethynyl-2-fluoro-2'-deoxyadenosine [EFdA]) is a novel reverse transcriptase-translocation inhibitor. Methods. We assessed MK-8591 as preexposure prophylaxis in the rhesus macaque model of intrarectal challenge with simian/human immunodeficiency virus (SHIV). In study 1, 8 rhesus macaques received 3.9 mg/kg of MK-8591 orally on day 0 and once weekly for the next 14 weeks. Eight controls were treated with vehicle. All rhesus macaques were challenged with SHIV109CP3 on day 6 and weekly for up to 12 challenges or until infection was confirmed. The dose of MK-8591 was reduced to 1.3 and 0.43 mg/kg/week in study 2 and further to 0.1 and 0.025 mg/kg/week in study 3. In studies 2 and 3, each dose was given up to 6 times once weekly, and animals were challenged 4 times once weekly with SHIV109CP3. Results. Control macaques were infected after a median of 1 challenge (range, 1-4 challenges). All treated animals in studies 1 and 2 were protected, consistent with a 41.5-fold lower risk of infection (P < .0001, by the log-rank test). In study 3, at a 0.1-mg/kg dose, 2 rhesus macaques became infected, consistent with a 7.2-fold lower risk of infection (P = .0003, by the log-rank test). The 0.025-mg/kg dose offered no protection. Conclusions. These data support MK-8591's potential as a preexposure prophylaxis agent.

The application of machine learning to longitudinal gene-expression profiles has demonstrated potential to decrease the assessment gap, between biochemical determination and clinical manifestation, of a patient's response to treatment. Although psoriasis is a proven testing ground for treatment-response prediction using transcriptomic data from clinically accessible skin biopsies, these biopsies are expensive, invasive, and challenging to obtain from certain body areas. Response prediction from blood biochemical measurements could be a cheaper, less invasive predictive platform. Longitudinal profiles for 92 inflammatory and 65 cardiovascular disease proteins were measured from the blood of psoriasis patients at baseline, and 4-weeks, following tofacitinib (janus kinase-signal transducer and activator of transcription-inhibitor) or etanercept (tumor necrosis factor-inhibitor) treatment, and predictive models were developed by applying machine-learning techniques such as bagging and ensembles. This data driven approach developed predictive models able to accurately predict the 12-week clinical endpoint for psoriasis following tofacitinib (area under the receiver operating characteristic curve [auROC] = 78%), or etanercept (auROC = 71%) treatment in a validation dataset, revealing a robust predictive protein signature including well-established psoriasis markers such as IL-17A and IL-17C, highlighting potential for biologically meaningful and clinically useful response predictions using blood protein data. Although most blood classifiers were outperformed by simple models trained using Psoriasis Area Severity Index scores, performance might be enhanced in future studies by measuring a wider variety of proteins.

A multi-person interview on the unrolling corona pandemic with Samuel Alizon, Akiko Iwasaki, Gerard Krause and Rino Rappuoli.

Interferon-induced transmembrane protein 3 (IFITM3) is a key interferon effector that broadly prevents infection by diverse viruses. However, the cellular factors that control IFITM3 homeostasis and antiviral activity have not been fully elucidated. Using site-specific photo-crosslinking and quantitative proteomic analysis, here we present the identification and functional characterization of VCP/p97 AAA-ATPase as a primary interaction partner of IFITM3. We show that IFITM3 ubiquitination at lysine 24 is crucial for VCP binding, trafficking, turnover, and engagement with incoming virus particles. Consistently, pharmacological inhibition of VCP/p97 ATPase activity leads to defective IFITM3 lysosomal sorting, turnover, and co-trafficking with virus particles. Our results showcase the utility of site-specific protein photo-crosslinking in mammalian cells and reveal VCP/p97 as a key cellular factor involved in IFITM3 trafficking and homeostasis.

Genetic polymorphisms of the L-type voltage-gated calcium channel (VGCC) are associated with psychiatric disorders including major depressive disorder. Alterations of S100A10 (p11) level are also implicated in the etiology of major depressive disorder. However, the existence of an endogenous regulator in the brain regulating p11, L-type VGCC, and depressive behavior has not been known. Here we report that Ahnak, whose function in the brain has been obscure, stabilizes p11 and Anxa2 proteins in the hippocampus and prefrontal cortex in the rodent brain. Protein levels of Ahnak, p11, and Anxa2 are highly and positively correlated in the brain. Together these data suggest the existence of an Ahnak/p11/Anxa2 protein complex. Ahnak is expressed in p11-positive as well as p11-negative neurons. Ahnak, through its N-terminal region, scaffolds the L-type pore-forming alpha 1 subunit and, through its C-terminal region, scaffolds the beta subunit of VGCC and the p11/Anxa2 complex. Cell surface expression of the alpha 1 subunits and L-type calcium current are significantly reduced in primary cultures of Ahnak knockout (KO) neurons compared to wild-type controls. A decrease in the L-type calcium influx is observed in both glutamatergic neurons and parvalbumin (PV) GABAergic interneurons of Ahnak KO mice. Constitutive Ahnak KO mice or forebrain glutamatergic neuron-selective Ahnak KO mice display a depression-like behavioral phenotype similar to that of constitutive p11 KO mice. In contrast, PV interneuron-selective Ahnak KO mice display an antidepressant-like behavioral phenotype. Our results demonstrate L-type VGCC as an effector of the Ahnak/p11/Anxa2 complex, revealing a novel molecular connection involved in the control of depressive behavior.

The original version of our manuscript, entitled, " The IL1RN mutation creating the most-upstream premature stop codon is hypomorphic because of a reinitiation of translation" unfortunately contained mistakes in Fig. 1a and d legends. The text should read as follows:

Type I interferonopathies are monogenic disorders characterized by enhanced type I interferon (IFN-I) cytokine activity. Inherited USP18 and ISG15 deficiencies underlie type I interferonopathies by preventing the regulation of late responses to IFN- I. Specifically, USP18, being stabilized by ISG15, sterically hinders JAK1 from binding to the IFNAR2 subunit of the IFN-I receptor. We report an infant who died of autoinflammation due to a homozygous missense mutation (R148Q) in STAT2. The variant is a gain of function (GOF) for induction of the late, but not early, response to IFN-I. Surprisingly, the mutation does not enhance the intrinsic activity of the STAT2-containing transcriptional complex responsible for IFN-I-stimulated gene induction. Rather, the STAT2 R148Q variant is a GOF because it fails to appropriately traffic USP18 to IFNAR2, thereby preventing USP18 from negatively regulating responses to IFN-I. Homozygosity for STAT2 R148Q represents a novel molecular and clinical phenocopy of inherited USP18 deficiency, which, together with inherited ISG15 deficiency, defines a group of type I interferonopathies characterized by an impaired regulation of late cellular responses to IFN-I.