The store-operated calcium (Ca
) channel Orai governs Ca
influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca
stored in ...the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of
Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca
selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, we propose a sequence for store-operated activation.
To investigate the cyanylated cysteine vibrational probe group’s ability to report on binding-induced changes along a protein–protein interface, the probe group was incorporated at several sites in a ...peptide of the calmodulin (CaM)-binding domain of skeletal muscle myosin light chain kinase. Isothermal titration calorimetry was used to determine the binding thermodynamics between calmodulin and each peptide. For all probe positions, the binding affinity was nearly identical to that of the unlabeled peptide. The CN stretching infrared band was collected for each peptide free in solution and bound to calmodulin. Binding-induced shifts in the IR spectral frequencies were correlated with estimated solvent accessibility based on molecular dynamics simulations. This work generally suggests (1) that site-specific incorporation of this vibrational probe group does not cause major perturbations to its local structural environment and (2) that this small probe group might be used quite broadly to map dynamic protein-binding interfaces. However, site-specific perturbations due to artificial labeling groups can be somewhat unpredictable and should be evaluated on a site-by-site basis through complementary measurements. A fully quantitative, simulation-based interpretation of the rich probe IR spectra is still needed but appears to be possible given recent advances in simulation techniques.
The capacity of respiratory viruses to undergo evolution within the respiratory tract raises the possibility of evolution under the selective pressure of the host environment or drug treatment. ...Long-term infections in immunocompromised hosts are potential drivers of viral evolution and development of infectious variants. We showed that intrahost evolution in chronic human parainfluenza virus 3 (HPIV3) infection in immunocompromised individuals elicited mutations that favored viral entry and persistence, suggesting that similar processes may operate across enveloped respiratory viruses. We profiled longitudinal HPIV3 infections from 2 immunocompromised individuals that persisted for 278 and 98 days. Mutations accrued in the HPIV3 attachment protein hemagglutinin-neuraminidase (HN), including the first in vivo mutation in HN's receptor binding site responsible for activating the viral fusion process. Fixation of this mutation was associated with exposure to a drug that cleaves host-cell sialic acid moieties. Longitudinal adaptation of HN was associated with features that promote viral entry and persistence in cells, including greater avidity for sialic acid and more active fusion activity in vitro, but not with antibody escape. Long-term infection thus led to mutations promoting viral persistence, suggesting that host-directed therapeutics may support the evolution of viruses that alter their biophysical characteristics to persist in the face of these agents in vivo.
Abstract
Background
Biomarkers to predict the severity of lung damage due to COVID-19 are urgently needed to inform management and treatment decisions. Our objective was to investigate the predictive ...value of host proteins for worsening respiratory failure in one of the by COVID-19 most affected and diverse patient populations in the US.
Methods
We performed a prospective single-center cross-sectional study of 34 adult patients admitted to Montefiore Medical Center in the Bronx, New York, for respiratory symptoms due to PCR-confirmed COVID-19. Exclusion criteria were age < 21, history of prior SARS-CoV-2 infection, and/or underlying severe chronic lung diseases requiring home O2 and/or high dose steroids. We stratified and compared patients by whether they developed worsening respiratory failure, necessitating transfer to the intensive care unit (ICU) during their hospital stay. Using a custom Luminex Assay, we measured hospital admission serum concentrations of 8 host proteins, representing respiratory-associated epithelial (RAGE, SP-D, CC16), endothelial (Ang-2, vWF), and immune pathways (S100A12, ICAM-1, VCAM-1).
Results
Except for race and WHO COVID-19 scores, demographics, co-morbidities, symptoms, and symptom duration were not statistically significantly different between patients requiring transfer to the ICU (n=15) and non-ICU patients (n=19). Higher log-transformed levels for 5/8 proteins (S100A12, ICAM-1, Ang-2, RAGE, SP-D) showed significant or marginally significant increased cause-specific hazard for ICU transfer (n=15). Estimated cumulative incidence functions further showed a significantly or near significantly increased risk for ICU transfer for patients with above the median values of S100A12 or ICAM-1 (p=0.013), Ang-2 (p=0.056) or RAGE (p=0.077), respectively (Figure 1). Host proteins predicting need for ICU transfer did not correlate strongly with other clinical laboratory markers for COVID-19 severity (CRP, LDH, D-Dimer, Fibrinogen, Ferritin).
Figure 1. Patients with above median levels of host protein markers S100A12, ICAM-1, Ang-2, and RAGE have a significantly or near significantly increased risk for severe respiratory failure requiring transfer to the ICU.
Comparison of estimated cumulative incidence at 7 days post admission for host protein markers above and below median levels for (A) S10012 (median 96,675 pg/ml); (B) ICAM-1 (median (1,192,277 pg/ml); (C) Ang-2 (median 3463 pg/ml); (D) RAGE (median 6356 pg/ml); and (E) SP-D (median 11,832 pg/ml).
Conclusion
These results suggest that host proteins have additional predictive value for the severity of COVID-19-associated lung damage at time of presentation to the hospital.
Disclosures
Inessa Gendlina, Nothing to disclose
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