Tracking the immune response profiles elicited by the BNT162b2 vaccine in COVID-19 unexperienced and experienced individuals.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection.

Mapping of functional SARS-CoV-2 receptors in human lungs establishes differences in variant binding and SLC1A5 as a viral entry modulator of hACE2.

BACKGROUND: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. METHODS: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. FINDINGS: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. INTERPRETATION: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. FUNDING: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora: Activity, structure and substrate specificity.

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity.

The crystal structure of Erwinia amylovora AmyR, a member of the YbjN protein family, shows similarity to type III secretion chaperones but suggests different cellular functions.

AmyR is a stress and virulence associated protein from the plant pathogenic Enterobacteriaceae species Erwinia amylovora, and is a functionally conserved ortholog of YbjN from Escherichia coli. The crystal structure of E. amylovora AmyR reveals a class I type III secretion chaperone-like fold, despite the lack of sequence similarity between these two classes of protein and lacking any evidence of a secretion-associated role. The results indicate that AmyR, and YbjN proteins in general, function through protein-protein interactions without any enzymatic action. The YbjN proteins of Enterobacteriaceae show remarkably low sequence similarity with other members of the YbjN protein family in Eubacteria, yet a high level of structural conservation is observed. Across the YbjN protein family sequence conservation is limited to residues stabilising the protein core and dimerization interface, while interacting regions are only conserved between closely related species. This study presents the first structure of a YbjN protein from Enterobacteriaceae, the most highly divergent and well-studied subgroup of YbjN proteins, and an in-depth sequence and structural analysis of this important but poorly understood protein family.

Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) from Erwinia amylovora.

Glucose-1-phosphate uridylyltransferase from Erwinia amylovora CFPB1430 was expressed as a His-tag fusion protein in Escherichia coli. After tag removal, the purified protein was crystallized from 100 mM Tris pH 8.5, 2 M ammonium sulfate, 5% ethylene glycol. Diffraction data sets were collected to a maximum resolution of 2.46 Šusing synchrotron radiation. The crystals belonged to the hexagonal space group P62, with unit-cell parameters a = 80.67, b = 80.67, c = 169.18. The structure was solved by molecular replacement using the structure of the E. coli enzyme as a search model.

Unraveling hidden regulatory sites in structurally homologous metalloproteases.

Monitoring enzymatic activity in vivo of individual homologous enzymes such as the matrix metalloproteinases (MMPs) by antagonist molecules is highly desired for defining physiological and pathophysiological pathways. However, the rational design of antagonists targeting enzyme catalytic moieties specific to one of the homologous enzymes often appears to be an extremely difficult task. This is mainly due to the high structural homology at the enzyme active sites shared by members of the protein family. Accordingly, controlling enzymatic activity via alternative allosteric sites has become an attractive proposition for drug design targeting individual homologous enzymes. Yet, the challenge remains to identify such regulatory alternative sites that are often hidden and scattered over different locations on the protein's surface. We have designed branched amphiphilic molecules exhibiting specific inhibitory activity towards individual members of the MMP family. These amphiphilic isomers share the same chemical nature, providing versatile nonspecific binding reactivity that allows to probe hidden regulatory residues on a given protein surface. Using the advantage provided by amphiphilic ligands, here we explore a new approach for determining hidden regulatory sites. This approach includes diverse experimental analysis, such as structural spectroscopic analyses, NMR, and protein crystallography combined with computational prediction of effector binding sites. We demonstrate how our approach works by analyzing members of the MMP family that possess a unique set of such sites. Our work provides a proof of principle for using ligand effectors to unravel hidden regulatory sites specific to members of the structurally homologous MMP family. This approach may be exploited for the design of novel molecular effectors and therapeutic agents affecting protein catalytic function via interactions with structure-specific regulatory sites.

Structural basis for matrix metalloproteinase 1-catalyzed collagenolysis.

The proteolysis of collagen triple-helical structure (collagenolysis) is a poorly understood yet critical physiological process. Presently, matrix metalloproteinase 1 (MMP-1) and collagen triple-helical peptide models have been utilized to characterize the events and calculate the energetics of collagenolysis via NMR spectroscopic analysis of 12 enzyme-substrate complexes. The triple-helix is bound initially by the MMP-1 hemopexin-like (HPX) domain via a four amino acid stretch (analogous to type I collagen residues 782-785). The triple-helix is then presented to the MMP-1 catalytic (CAT) domain in a distinct orientation. The HPX and CAT domains are rotated with respect to one another compared with the X-ray "closed" conformation of MMP-1. Back-rotation of the CAT and HPX domains to the X-ray closed conformation releases one chain out of the triple-helix, and this chain is properly positioned in the CAT domain active site for subsequent hydrolysis. The aforementioned steps provide a detailed, experimentally derived, and energetically favorable collagenolytic mechanism, as well as significant insight into the roles of distinct domains in extracellular protease function.

p120-Ras GTPase activating protein (RasGAP): a multi-interacting protein in downstream signaling.

p120-RasGAP (Ras GTPase activating protein) plays a key role in the regulation of Ras-GTP bound by promoting GTP hydrolysis via its C-terminal catalytic domain. The p120-RasGAP N-terminal part contains two SH2, SH3, PH (pleckstrin homology) and CaLB/C2 (calcium-dependent phospholipid-binding domain) domains. These protein domains allow various functions, such as anti-/pro-apoptosis, proliferation and also cell migration depending of their distinct partners. The p120-RasGAP domain participates in protein-protein interactions with Akt, Aurora or RhoGAP to regulate functions described bellow. Here, we summarize, in angiogenesis and cancer, the various functional roles played by p120-RasGAP domains and their effector partners in downstream signaling.