Serum Albumin: A Multifaced Enzyme

Human serum albumin (HSA) is the most abundant protein in plasma, contributing actively to oncotic pressure maintenance and fluid distribution between body compartments. HSA acts as the main carrier of fatty acids, recognizes metal ions, affects pharmacokinetics of many drugs, provides the metabolic modification of some ligands, renders potential toxins harmless, accounts for most of the anti-oxidant capacity of human plasma, and displays esterase, enolase, glucuronidase, and peroxidase (pseudo)-enzymatic activities. HSA-based catalysis is physiologically relevant, affecting the metabolism of endogenous and exogenous compounds including proteins, lipids, cholesterol, reactive oxygen species (ROS), and drugs. Catalytic properties of HSA are modulated by allosteric effectors, competitive inhibitors, chemical modifications, pathological conditions, and aging. HSA displays anti-oxidant properties and is critical for plasma detoxification from toxic agents and for pro-drugs activation. The enzymatic properties of HSA can be also exploited by chemical industries as a scaffold to produce libraries of catalysts with improved proficiency and stereoselectivity for water decontamination from poisonous agents and environmental contaminants, in the so called “green chemistry” field. Here, an overview of the intrinsic and metal dependent (pseudo-)enzymatic properties of HSA is reported to highlight the roles played by this multifaced protein.

Complexity and Specificity of Sec61-Channelopathies: Human Diseases Affecting Gating of the Sec61 Complex

The rough endoplasmic reticulum (ER) of nucleated human cells has crucial functions in protein biogenesis, calcium (Ca2+) homeostasis, and signal transduction. Among the roughly one hundred components, which are involved in protein import and protein folding or assembly, two components stand out: The Sec61 complex and BiP. The Sec61 complex in the ER membrane represents the major entry point for precursor polypeptides into the membrane or lumen of the ER and provides a conduit for Ca2+ ions from the ER lumen to the cytosol. The second component, the Hsp70-type molecular chaperone immunoglobulin heavy chain binding protein, short BiP, plays central roles in protein folding and assembly (hence its name), protein import, cellular Ca2+ homeostasis, and various intracellular signal transduction pathways. For the purpose of this review, we focus on these two components, their relevant allosteric effectors and on the question of how their respective functional cycles are linked in order to reconcile the apparently contradictory features of the ER membrane, selective permeability for precursor polypeptides, and impermeability for Ca2+. The key issues are that the Sec61 complex exists in two conformations: An open and a closed state that are in a dynamic equilibrium with each other, and that BiP contributes to its gating in both directions in cooperation with different co-chaperones. While the open Sec61 complex forms an aqueous polypeptide-conducting- and transiently Ca2+-permeable channel, the closed complex is impermeable even to Ca2+. Therefore, we discuss the human hereditary and tumor diseases that are linked to Sec61 channel gating, termed Sec61-channelopathies, as disturbances of selective polypeptide-impermeability and/or aberrant Ca2+-permeability.

Fatty Acid Allosteric Regulation of C-H Activation in Plant and Animal Lipoxygenases

Lipoxygenases (LOXs) catalyze the (per) oxidation of fatty acids that serve as important mediators for cell signaling and inflammation. These reactions are initiated by a C-H activation step that is allosterically regulated in plant and animal enzymes. LOXs from higher eukaryotes are equipped with an N-terminal PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain that has been implicated to bind to small molecule allosteric effectors, which in turn modulate substrate specificity and the rate-limiting steps of catalysis. Herein, the kinetic and structural evidence that describes the allosteric regulation of plant and animal lipoxygenase chemistry by fatty acids and their derivatives are summarized.

Rapid exploration of the epitope coverage produced by an Ebola survivor to guide the discovery of therapeutic antibody cocktails

ABSTRACT Background Development of successful neutralizing antibodies is dependent upon broad epitope coverage to increase the likelihood of achieving therapeutic function. Recent advances in synthetic biology have allowed us to conduct an epitope binning study on a large panel of antibodies identified to bind to Ebola virus glycoprotein with only published sequences. Methods and Results A rapid, first-pass epitope binning experiment revealed seven distinct epitope families that overlapped with known structural epitopes from the literature. A focused set of antibodies was selected from representative clones per bin to guide a second-pass binning that revealed previously unassigned epitopes, confirmed epitopes known to be associated with neutralizing antibodies, and demonstrated asymmetric blocking of EBOV GP from allosteric effectors reported from literature. Conclusions Critically, this workflow allows us to probe the epitope landscape of EBOV GP without any prior structural knowledge of the antigen or structural benchmark clones. Incorporating epitope binning on hundreds of antibodies during early stage antibody characterization ensures access to a library’s full epitope coverage, aids in the identification of high quality reagents within the library that recapitulate this diversity for use in other studies, and ultimately enables the rational development of therapeutic cocktails that take advantage of multiple mechanisms of action such as cooperative synergistic effects to enhance neutralization function and minimize the risk of mutagenic escape. The use of high-throughput epitope binning during new outbreaks such as the current COVID-19 pandemic is particularly useful in accelerating timelines due to the large amount of information gained in a single experiment.

SynBio and the Boundaries between Functional and Pathogenic RepA-WH1 Bacterial Amyloids

ABSTRACT Amyloids are protein polymers that were initially linked to human diseases. Across the whole Tree of Life, many disease-unrelated proteins are now emerging for which amyloids represent distinct functional states. Most bacterial amyloids described are extracellular, contributing to biofilm formation. However, only a few have been found in the bacterial cytosol. This paper reviews from the perspective of synthetic biology (SynBio) our understanding of the subtle line that separates functional from pathogenic and transmissible amyloids (prions). In particular, it is focused on RepA-WH1, a functional albeit unconventional natural amyloidogenic protein domain that participates in controlling DNA replication of bacterial plasmids. SynBio approaches, including protein engineering and the design of allosteric effectors such as diverse ligands and an optogenetic module, have enabled the generation in RepA-WH1 of an intracellular cytotoxic prion-like agent in bacteria. The synthetic RepA-WH1 prion has the potential to develop into novel antimicrobials.

Modulation of the bacterial CobB sirtuin deacylase activity by N-terminal acetylation

In eukaryotic cells, the N-terminal amino moiety of many proteins is modified by N-acetyltransferases (NATs). This protein modification can alter the folding of the target protein; can affect binding interactions of the target protein with substrates, allosteric effectors, or other proteins; or can trigger protein degradation. In prokaryotes, only ribosomal proteins are known to be N-terminally acetylated, and the acetyltransferases responsible for this modification belong to the Rim family of proteins. Here, we report that, inSalmonella enterica, the sirtuin deacylase CobB long isoform (CobBL) is N-terminally acetylated by the YiaC protein of this bacterium. Results of in vitro acetylation assays showed that CobBLwas acetylated by YiaC; liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm these results. Results of in vitro and in vivo experiments showed that CobBLdeacetylase activity was negatively affected when YiaC acetylated its N terminus. We report 1) modulation of a bacterial sirtuin deacylase activity by acetylation, 2) that the Gcn5-related YiaC protein is the acetyltransferase that modifies CobBL, and 3) that YiaC is an NAT. Based on our data, we propose the name of NatA (N-acyltransferase A) in lieu of YiaC to reflect the function of the enzyme.

AlloSigMA 2: paving the way to designing allosteric effectors and to exploring allosteric effects of mutations

The AlloSigMA 2 server provides an interactive platform for exploring the allosteric signaling caused by ligand binding and/or mutations, for analyzing the allosteric effects of mutations and for detecting potential cancer drivers and pathogenic nsSNPs. It can also be used for searching latent allosteric sites and for computationally designing allosteric effectors for these sites with required agonist/antagonist activity. The server is based on the implementation of the Structure-Based Statistical Mechanical Model of Allostery (SBSMMA), which allows one to evaluate the allosteric free energy as a result of the perturbation at per-residue resolution. The Allosteric Signaling Map (ASM) providing a comprehensive residue-by-residue allosteric control over the protein activity can be obtained for any structure of interest. The Allosteric Probing Map (APM), in turn, allows one to perform the fragment-based-like computational design experiment aimed at finding leads for potential allosteric effectors. The server can be instrumental in elucidating of allosteric mechanisms and actions of allosteric mutations, and in the efforts on design of new elements of allosteric control. The server is freely available at: http://allosigma.bii.a-star.edu.sg