The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: interactions at ferrous heme and cysteine thiols

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols

Peripheral Nervous System Genes Expressed in Central Neurons Induce Growth on Inhibitory Substrates

Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS’s enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons

Experimental Estimation of the Effects of All Amino-Acid Mutations to HIV’s Envelope Protein on Viral Replication in Cell Culture

<div><p>HIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV’s most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV’s replication in cell culture. We compare our experimental measurements of each site’s preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. Our data enable us to quantify the inherent mutational tolerance of each site in Env. We show that the epitopes of broadly neutralizing antibodies have a significantly reduced inherent capacity to tolerate mutations, rigorously validating a pervasive idea in the field. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env’s evolution.</p></div

Sites of recurrent cell-culture mutations mapped on Env’s structure.

<p>The 25 sites from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.g002" target="_blank">Fig 2B</a> where the mutation frequency increased >3-fold in at least two replicates after cell-culture passage. <b>(A)</b> Trimeric Env (PDB 5FYK [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.ref073" target="_blank">73</a>]) with one monomer in grey and the others in white, oriented so the membrane-proximal region is at the bottom. Sites of cell-culture mutations are shown as spheres, colored red-to-blue according to primary sequence. Most of these sites fall in one of three clusters. Mutations in the first cluster disrupt potential glycosylation sites at Env’s apex. The second cluster includes or is adjacent to sites where mutations are known to affect Env’s conformational dynamics [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.ref074" target="_blank">74</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.ref075" target="_blank">75</a>]. <b>(B)</b> The third cluster is near the co-receptor binding surface. This panel shows an apex-down view of monomeric gp120 (grey) in complex with CD4 (green) (PDB 3JWD [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.ref048" target="_blank">48</a>]). Sites of recurrent cell-culture mutations are shown as spheres colored according to primary sequence as in panel A. The black bar indicates cropping of CD4. <b>(C)</b> The same view as panel B, but the spheres now show sites known to affect binding to CXCR4 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.ref010" target="_blank">10</a>] or CCR5 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006114#ppat.1006114.ref076" target="_blank">76</a>]. Note the extensive overlap between the spheres in this panel and panel B.</p