Biology subjects

Uguccioni, M.

Publications and source records attributed to Uguccioni, M..

6 recordsLinked to original sources

bioRxiv2026

GRK-dependent ACKR3 endocytosis and chemokine scavenging is independent of receptor phosphorylation and beta-arrestin

Desensitization and internalization of most G protein-coupled receptors (GPCRs) depend on phosphorylation by GPCR kinases (GRKs), promoting {beta}-arrestin recruitment. Atypical chemokine receptors (ACKRs), including ACKR3, are structurally related to classical chemokine receptors but do not activate heterotrimeric G proteins. ACKR3 signaling and trafficking have been proposed to depend on GRK5-mediated phosphorylation and {beta}-arrestin interaction. However, the respective roles of {beta}-arrestins, GRKs, and receptor phosphorylation in chemokine scavenging and in constitutive or ligand-induced trafficking remain debated. Using bioluminescence resonance energy transfer (BRET)-based biosensors and immunofluorescence imaging with fluorescently labeled receptors and chemokines, we examined ACKR3 interaction with {beta}-arrestin1/2 and assessed chemokine scavenging and receptor trafficking in {beta}-arrestin-deficient ({Delta}{beta}arr1/2) cells. We also evaluated the contribution of GRK-mediated phosphorylation. {beta}-arrestins supported agonist-independent receptor internalization but were dispensable for chemokine-induced internalization and chemokine scavenging. In contrast, GRKs were required for ligand-promoted endocytosis, with either GRK2/3 or GRK5/6 being sufficient. Mutation of ACKR3 phosphorylation sites impaired {beta}-arrestin recruitment but did not completely block internalization and scavenging, whereas complete C-terminal truncation abolished both processes. Consistently, kinase-dead GRK2 rescued ACKR3 endocytosis in {Delta}GRK2/3/5/6 cells, indicating a scaffolding role partially independent of kinase activity. Moreover, G{beta}{gamma} was not required for GRK2-mediated ACKR3 endocytosis, as a PH-domain-deleted GRK2 mutant restored internalization in {Delta}GRK2/3/5/6 cells, and G{beta}{gamma} sequestration by {beta}ARKct-CAAX did not inhibit this process consistent with the notion that ACKR3 does not promote G protein activation. Thus, ligand-promoted ACKR3 internalization and chemokine scavenging occur independently of {beta}-arrestins but requires GRKs. One-sentence summaryGRKs are essential for ACKR3 endocytosis and chemokine scavenging, whereas {beta}-arrestins and receptor phosphorylation are dispensable.

cell biology↗

bioRxiv2025

A Potent Nonapeptide Inhibitor for the CXCL12/HMGB1 heterocomplex: A Computational and Experimental Approach

Inflammation is a vital defense mechanism activated in response to injury or infection, which induces the release of cytokines and chemokines to promote tissue repair. However, persistent inflammation may result in the development of autoimmune diseases. During pathological conditions such as rheumatoid arthritis, HMGB1 is kept in its reduced isoform and can complex with CXCL12 enhancing cell migration and exacerbating the immune responses. Small organic compounds selective for HMGB1 have been previously reported to be able to disrupt the CXCL12/HMGB1 heterocomplex, but due to their low affinity, they are unsuitable for further development as novel anti-inflammatory drugs. We previously reported a peptide (HBP08) that binds to HMGB1 with high affinity (Kd = 0.8 {micro}M), and blocks the activity of the heterocomplex, in line with a wide literature that supports the use of peptides to design protein-protein interaction inhibitors. In the present work, we computationally optimized the HBP08 peptide sequence, finding new analogues endowed with improved affinity for HMGB1. In particular, HBP08-2 inhibited the activity of the CXCL12/HMGB1 heterocomplex with an IC50 10-fold lower (3.31 {micro}M) and displayed a Kd 28-fold lower (28.1 {+/-} 7.0 nM) than the parent peptide HBP08.

biochemistry↗

bioRxiv2022

Human neutralizing antibodies to cold linear epitopes and to subdomain 1 of SARS-CoV-2

Emergence of SARS-CoV-2 variants diminishes the efficacy of vaccines and antiviral monoclonal antibodies. Continued development of immunotherapies and vaccine immunogens resilient to viral evolution is therefore necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. Antibody fp.006 binds the fusion peptide and cross-reacts against coronaviruses of the four genera, including the nine human coronaviruses, through recognition of a conserved motif that includes the S2 site of proteolytic cleavage. Antibody hr2.016 targets the stem helix and neutralizes SARS-CoV-2 variants. Antibody sd1.040 binds to subdomain 1, synergizes with antibody rbd.042 for neutralization and, like fp.006 and hr2.016, protects mice when present as bispecific antibody. Thus, coldspot-guided antibody discovery reveals donor-derived neutralizing antibodies that are cross-reactive with Orthocoronavirinae, including SARS-CoV-2 variants. One sentence summaryBroadly cross-reactive antibodies that protect from SARS-CoV-2 variants are revealed by virus coldspot-driven discovery.

immunology↗

bioRxiv2022

Anti-chemokine antibodies after SARS-CoV-2 infection correlate with favorable disease course

Infection by SARS-CoV-2 leads to diverse symptoms, which can persist for months. While antiviral antibodies are protective, those targeting interferons and other immune factors are associated with adverse COVID-19 outcomes. Instead, we discovered that antibodies against specific chemokines are omnipresent after COVID-19, associated with favorable disease, and predictive of lack of long COVID symptoms at one year post infection. Anti-chemokine antibodies are present also in HIV-1 infection and autoimmune disorders, but they target different chemokines than those in COVID-19. Monoclonal antibodies derived from COVID- 19 convalescents that bind to the chemokine N-loop impair cell migration. Given the role of chemokines in orchestrating immune cell trafficking, naturally arising anti-chemokine antibodies associated with favorable COVID-19 may be beneficial by modulating the inflammatory response and thus bear therapeutic potential. One-Sentence SummaryNaturally arising anti-chemokine antibodies associate with favorable COVID-19 and predict lack of long COVID.

immunology↗

bioRxiv2019

Systematic development of peptide inhibitors targeting the CXCL12/HMGB1 interaction

During inflammatory reactions, the production and release of chemotactic factors guide the recruitment of selective leukocyte subpopulations. HMGB1 and the chemokine CXCL12, both released in the microenvironment, form a heterocomplex, which exclusively acts on the chemokine receptor CXCR4, enhancing cell migration and, in some pathological conditions such as Rheumatoid Arthritis, exacerbating the immune response. An excessive cell influx at the inflammatory site can be diminished by disrupting the heterocomplex. Here, we report the computationally driven identification of a novel peptide (HBP08), which binds HMGB1 with the highest affinity reported so far (Kd of 0.8 {+/-} 0.1 M), able to selectively inhibit the activity of the CXCL12/HMGB1 heterocomplex. The identification of this peptide represents an important step towards the development of innovative pharmacological tools for the treatment of severe chronic inflammatory conditions characterized by an uncontrolled immune response.

biochemistry↗

bioRxiv2019

Oxidation state dependent conformational changes of HMGB1 regulates the formation of the CXCL12/HMGB1 heterocomplex

High-mobility Group Box 1 (HMGB1) is an abundant protein present in all mammalian cells and involved in several processes. During inflammation or tissue damage, HMGB1 is released in the extracellular space and, depending on its redox state, can form a heterocomplex with CXCL12. The heterocomplex acts exclusively on the chemokine receptor CXCR4 enhancing leukocyte recruitment. Here, we used multi-microsecond molecular dynamics (MD) simulations to elucidate the effect of the disulfide bond on the structure and dynamics of HMGB1. The results of the MD simulations show that the presence or lack of the disulfide bond between Cys23 and Cys45 modulates the conformational space explored by HMGB1, making the reduced protein more suitable to form a complex with CXCL12.

biochemistry↗