Vectorial nature of redox Bohr effects in bovine heart cytochrome c oxidase

AbstractThe vectorial nature of redox Bohr effects (redoxlinked pK shifts) in cytochrome c oxidase from bovine heart incorporated in liposomes has been analyzed. The Bohr effects linked to oxido-reduction of heme a and CuB display membrane vectorial asymmetry. This provides evidence for involvement of redox Bohr effects in the proton pump of the oxidase

The human coronaviruses (HCoVs) and the molecular mechanisms of SARS-CoV-2 infection

In humans, coronaviruses can cause infections of the respiratory system, with damage of varying severity depending on the virus examined: ranging from mild-to-moderate upper respiratory tract diseases, such as the common cold, pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Human coronaviruses known to date, common throughout the world, are seven. The most common-and least harmful-ones were discovered in the 1960s and cause a common cold. Others, more dangerous, identified in the early 2000s and cause more severe respiratory tract infections. Among these the SARS-CoV, isolated in 2003 and responsible for the severe acute respiratory syndrome (the so-called SARS), which appeared in China in November 2002, the coronavirus 2012 (2012-nCoV) cause of the Middle Eastern respiratory syndrome (MERS) from coronavirus, which exploded in June 2012 in Saudi Arabia, and actually SARS-CoV-2. On December 31, 2019, a new coronavirus strain was reported in Wuhan, China, identified as a new coronavirus beta strain ß-CoV from group 2B, with a genetic similarity of approximately 70% to SARS-CoV, the virus responsible of SARS. In the first half of February, the International Committee on Taxonomy of Viruses (ICTV), in charge of the designation and naming of the viruses (i.e., species, genus, family, etc.), thus definitively named the new coronavirus as SARS-CoV-2. This article highlights the main knowledge we have about the biomolecular and pathophysiologic mechanisms of SARS-CoV-2

The Human Respiratory System and its Microbiome at a Glimpse

The recent COVID-19 pandemic promoted efforts to better understand the organizationof the respiratory microbiome and its evolution from birth to adulthood and how it interacts withexternal pathogens and the host immune system. This review aims to deepen understanding of theessential physiological functions of the resident microbiome of the respiratory system on human healthand diseases. First, the general characteristics of the normal microbiota in the different anatomicalsites of the airways have been reported in relation to some factors such as the effect of age, diet andothers on its composition and stability. Second, we analyze in detail the functions and compositionand the correct functionality of the microbiome in the light of current knowledge. Several studiessuggest the importance of preserving the micro-ecosystem of commensal, symbiotic and pathogenicmicrobes of the respiratory system, and, more recently, its relationship with the intestinal microbiome,and how it also leads to the maintenance of human health, has become better understood

An Engineered IFNγ-Antibody Fusion Protein with Improved Tumor-Homing Properties

Interferon-gamma (IFNγ) is one of the central cytokines produced by the innate and adaptive immune systems. IFNγ directly favors tumor growth control by enhancing the immunogenicity of tumor cells, induces IP-10 secretion facilitating (CXCR3+) immune cell infiltration, and can prime macrophages to an M1-like phenotype inducing proinflammatory cytokine release. We had previously reported that the targeted delivery of IFNγ to neoplastic lesions may be limited by the trapping of IFNγ-based products by cognate receptors found in different organs. Here we describe a novel fusion protein consisting of the L19 antibody, specific to the alternatively spliced extra-domain B of fibronectin (EDB), fused to a variant of IFNγ with reduced affinity to its cognate receptor. The product (named L19-IFNγ KRG) selectively localized to tumors in mice, showed favorable pharmacokinetic profiles in monkeys and regained biological activity upon antigen binding. The fusion protein was investigated in two murine models of cancer, both as monotherapy and in combination with therapeutic modalities which are frequently used for cancer therapy. L19-IFNγ KRG induced tumor growth retardation and increased the intratumoral concentration of T cells and NK cells in combination with anti-PD-1

Generation and in vivo validation of an IL-12 fusion protein based on a novel anti-human FAP monoclonal antibody

BACKGROUND In this study, we describe the generation of a fully human monoclonal antibody (named '7NP2') targeting human fibroblast activation protein (FAP), an antigen expressed in the microenvironment of different types of solid neoplasms. METHODS 7NP2 was isolated from a synthetic antibody phage display library and was improved by one round of mutagenesis-based affinity maturation. The tumor recognition properties of the antibody were validated by immunofluorescence procedures performed on cancer biopsies from human patients. A fusion protein consisting of the 7NP2 antibody linked to interleukin (IL)-12 was generated and the anticancer activity of the murine surrogate product (named mIL12-7NP2) was evaluated in mouse models. Furthermore, the safety of the fully human product (named IL12-7NP2) was evaluated in Cynomolgus monkeys. RESULTS Biodistribution analysis in tumor-bearing mice confirmed the ability of the product to selectively localize to solid tumors while sparing healthy organs. Encouraged by these results, therapy studies were conducted in vivo, showing a potent antitumor activity in immunocompetent and immunodeficient mouse models of cancer, both as single agent and in combination with immune checkpoint inhibitors. The fully human product was tolerated when administered to non-human primates. CONCLUSIONS The results obtained in this work provided a rationale for future clinical translation activities using IL12-7NP2

PH DEPENDENCE OF THE VARIOUS PHASES OF THE PROTON PUMP OF CYTOCHROME C OXIDASE

Cytochrome c oxidase(COX)catalyses the reduction of dioxygen to water by ferrocytochrome c.This reaction is coupled to the translocation of up to 1H+/e- 4H+/O2 across the coupling membrane from the inner to the outer aqueous phase (1,2). The catalytic cycle of COX can be divided into two phases: a reductive phase and an oxidative phase.A study is presented on the pHdependence of proton transfer of COX reconstituted in vesicles,associated with the oxidation phase,the reduction phase and the oxidation-rereduction rapid transitio

Protonmotive activity of cytochrome c oxidase: control of oxidoreduction of the heme centers by the protonmotive force in the reconstituted beef heart enzyme

This paper contributes to the characterization of partial steps of electron and proton transfer in mitochondrial cytochrome c oxidase with respect to their membrane arrangement and involvement in energy-linked protonmotive activity. It is shown that delta psi controls electron flow from cytochrome c to heme a is consistent with the view that the latter center is buried in the membrane in a central position. The pressure exerted by delta psi on oxidation of heme alpha 3 by O2 indicates also that this center is buried in the membrane at some distance from the inner side and is consistent with observations showing that protons consumed in the reduction of O2 to H2O derive from the inner space. Electron flow from heme alpha to heme alpha 3 is shown to be specifically controlled by delta pH and in particular by the pH of the inner phase. Analysis of the effect of DCCD treatment of oxidase vesicles reveals that concentrations of this reagent which result in selective modification of subunit III (Prochaska et al., 1981) produce inhibition of redox-linked proton release. Higher concentrations of DCCD which result also in modification of subunits II and IV (Prochaska et al., 1981) cause inhibition of the pH-dependent electron-transfer step from heme alpha to heme alpha 3

A New Look at the Structures of Old Sepsis Actors by Exploratory Data Analysis Tools

Sepsis is a life-threatening condition that accounts for numerous deaths worldwide, usually complications of common community infections (i.e., pneumonia, etc), or infections acquired during the hospital stay. Sepsis and septic shock, its most severe evolution, involve the whole organism, recruiting and producing a lot of molecules, mostly proteins. Proteins are dynamic entities, and a large number of techniques and studies have been devoted to elucidating the relationship between the conformations adopted by proteins and what is their function. Although molecular dynamics has a key role in understanding these relationships, the number of protein structures available in the databases is so high that it is currently possible to build data sets obtained from experimentally determined structures. Techniques for dimensionality reduction and clustering can be applied in exploratory data analysis in order to obtain information on the function of these molecules, and this may be very useful in immunology to better understand the structure-activity relationship of the numerous proteins involved in host defense, moreover in septic patients. The large number of degrees of freedom that characterize the biomolecules requires special techniques which are able to analyze this kind of data sets (with a small number of entries respect to the number of degrees of freedom). In this work we analyzed the ability of two different types of algorithms to provide information on the structures present in three data sets built using the experimental structures of allosteric proteins involved in sepsis. The results obtained by means of a principal component analysis algorithm and those obtained by a random projection algorithm are largely comparable, proving the effectiveness of random projection methods in structural bioinformatics. The usefulness of random projection in exploratory data analysis is discussed, including validation of the obtained clusters. We have chosen these proteins because of their involvement in sepsis and septic shock, aimed to highlight the potentiality of bioinformatics to point out new diagnostic and prognostic tools for the patient

Proton transfer reactions associated with the reaction of the fully reduced, purifierd cytochome c oxidase with molecular oxygen and ferrycianide

A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from ≈3.0 H+/COX at pH 6.2−6.3 to ≈1.2 H+/COX at pH 8.0−8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from ≈1.0 H+/COX at pH 6.2−6.3 to ≈2.8 H+/COX at pH 8.0−8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3−1.6 H+/COX in the pH range 6.2−8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O2 to 2H2O. The results show that the expected stoichiometric proton consumption of 4H+ in the reduction of O2 to 2H2O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H+/COX are initially taken up in the reduction of O2 to two OH- groups bound to the binuclear Fe a3−CuB center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H2O. At alkaline pHs the third and fourth protons are taken up with formation of 2H2O only upon rereduction of COX