34 research outputs found
Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin
One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΞΞG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution
Effective Prevention of Arterial Thrombosis with Albumin-Thrombin Inhibitor Conjugates
Thromboprophylaxis
is indicated in patients at an elevated risk
of developing thrombotic disorders, typically using direct oral anticoagulants
or low-molecular-weight heparins. We postulated that transient thromboprophylaxis
(daysβweeks) could be provided by a single dose of an anticoagulant
engineered for prolonged pharmacokinetics. In the present work, d-phenylalanyl-l-prolyl-l-arginine chloromethyl
ketone (PPACK) was used as a model anticoagulant to test the hypothesis
that conjugation of thrombin inhibitors to the surface of albumin
would provide durable protection against thrombotic insults. Covalent
conjugates were formed between albumin and PPACK using click chemistry,
and they were tested in vitro using a thrombin activity
assay and a clot formation assay. Thromboprophylactic efficacy was
tested in mouse models of arterial thrombosis, both chemically induced
(FeCl3) and following ischemia-reperfusion (transient middle
cerebral artery occlusion; tMCAO). Albumin-PPACK conjugates were shown
to have nanomolar potency in both in vitro assays,
and following intravenous injection had prolonged circulation. Conjugates
did not impact hemostasis (tail clipping) or systemic coagulation
parameters in normal mice. Intravenous injection of conjugates prior
to FeCl3-induced thrombosis provided significant protection
against occlusion of the middle cerebral and common carotid arteries,
and injection immediately following ischemia-reperfusion reduced stroke
volume measured 3 days after injury by βΌ40% in the tMCAO model.
The data presented here provide support for the use of albumin-linked
anticoagulants as an injectable, long-circulating, safe thromboprophylactic
agent. In particular, albumin-PPACK provides significant protection
against thrombosis induced by multiple mechanisms, without adversely
affecting hemostasis
Quantitative differences in xanthine dehydrogenase activity in wild-type strains ofDrosophila melanogaster
Gene regulation in Drosophila: Independent expression of closely linked, related structural loci
Supramolecular arrangement of protein in nanoparticle structures predicts nanoparticle tropism for neutrophils in acute lung inflammation
Myerson JW, Patel PN, Rubey KM, et al. Supramolecular arrangement of protein in nanoparticle structures predicts nanoparticle tropism for neutrophils in acute lung inflammation. Nature Nanotechnology. 2022;17(1):86-97.This study shows that the supramolecular arrangement of proteins in nanoparticle structures predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of nanoparticles with agglutinated protein (NAPs), defined by arrangement of protein in or on the nanoparticles via hydrophobic interactions, crosslinking and electrostatic interactions. Nanoparticles with symmetric protein arrangement (for example, viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We demonstrate diagnostic imaging of ALI with NAPs; show NAP tropism for inflamed human donor lungs; and show that NAPs can remediate pulmonary oedema in ALI. This work demonstrates that structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI