Biology subjects

Rasola, A.

Publications and source records attributed to Rasola, A..

9 recordsLinked to original sources

bioRxiv2024

Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity.

The mitochondrial chaperone TRAP1 is a key regulator of cellular homeostasis and its activity has important implications in neurodegeneration, ischemia and cancer. Recent evidence has indicated that TRAP1 mutations are involved in several disorders, even though the structural basis for the impact of point mutations on TRAP1 functions has never been studied. By exploiting a modular structure-based framework and molecular dynamics simulations, we investigated the effect of five TRAP1 mutations on its structure and stability. Each mutation differentially impacts long-range interactions, intra and inter-protomer dynamics and ATPase activity. Changes in these parameters influence TRAP1 functions, as revealed by their effects on the activity of the TRAP1 interactor succinate dehydrogenase (SDH). In keeping with this, TRAP1 point mutations affect the growth and migration of aggressive sarcoma cells, and alter sensitivity to a selective TRAP1 inhibitor. Our work provides new insights on the structure-activity relationship of TRAP1, identifying crucial amino acid residues that regulate TRAP1 proteostatic functions and pro-neoplastic activity.

cancer biology↗

bioRxiv2024

Disulfide-mediated tetramerization of TRAP1 fosters its antioxidant and pro-neoplastic activities.

The mitochondrial chaperone TRAP1 exerts a protective function in cells exposed to diverse stress conditions in both physiological and pathological contexts. In cancer cells, it contributes to neoplastic progression ensuing metabolic rewiring and protection from oxidative insults. TRAP1 works as a homodimer, but recent evidence has indicated that it can form tetramers whose functional effects remain elusive. Here, we find that TRAP1 forms redox-sensitive tetramers via disulfide bonds involving two critical cysteine residues, C261 and C573. TRAP1 tetramerization is elicited by oxidative stress and abrogated upon expression of the double C261S/C573R mutant. In cancer contexts, the expression of the TRAP1 C261S/C573R mutant is unable to inhibit the activity of its client succinate dehydrogenase and to confer protection against oxidative insults, and it hampers invasiveness of aggressive sarcoma cells. Our data indicate that TRAP1 undergoes tetramerization in response to oxidative stress and identify C261 and C573 as critical for TRAP1 structural rearrangement and for functions.

cell biology↗

bioRxiv2021

TRAP1 and cyclophilin D compete at OSCP subunit to regulate enzymatic activity and permeability transition pore opening by F-ATP synthase

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes cell bioenergetic and proteostatic adaptations, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Moreover, TRAP1 inhibits opening of the permeability transition pore (PTP) formed by F-ATP synthase and effectively antagonizes the PTP-inducing effect of CyPD, which elicits mitochondrial depolarization and cell death. Consistently, electrophysiological measurements indicate that TRAP1 and CyPD compete in the modulation of channel activity of purified F-ATP synthase, resulting in PTP inhibition and activation, respectively, and outcompeting each other effect on the channel. Moreover, TRAP1 counteracts PTP induction by CyPD, whereas CyPD reverses TRAP1-mediated PTP inhibition. Our data identify TRAP1 as a F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

cell biology↗

bioRxiv2021

SARS-CoV-2 Spike Protein Mutations and Escape from Antibodies: a Computational Model of Epitope Loss in Variants of Concern

The SARS-CoV-2 spike (S) protein is exposed on the viral surface and is the first point of contact between the virus and the host. For these reasons it represents the prime target for Covid-19 vaccines. In recent months, variants of this protein have started to emerge. Their ability to reduce or evade recognition by S-targeting antibodies poses a threat to immunological treatments and raises concerns for their consequences on vaccine efficacy. To develop a model able to predict the potential impact of S-protein mutations on antibody binding sites, we performed unbiased multi-microsecond molecular dynamics of several glycosylated S-protein variants and applied a straightforward structure-dynamics-energy based strategy to predict potential changes in immunogenic regions on each variant. We recover known epitopes on the reference D614G sequence. By comparing our results, obtained on isolated S-proteins in solution, to recently published data on antibody binding and reactivity in new S variants, we directly show that modifications in the S-protein consistently translate into the loss of potentially immunoreactive regions. Our findings can thus be qualitatively reconnected to the experimentally characterized decreased ability of some of the Abs elicited against the dominant S-sequence to recognize variants. While based on the study of SARS-CoV-2 Spike variants, our computational epitope-prediction strategy is portable and could be applied to study immunoreactivity in mutants of proteins of interest whose structures have been characterized, helping the development/selection of vaccines and antibodies able to control emerging variants.

biophysics↗

bioRxiv2021

Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here, we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion. This provides cells with resistance to pro-oxidants targeting complex I and decreases both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the mitochondrial NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. This anti-neoplastic effect is mimicked both in vitro and in vivo by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3, and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes of these cells. These findings shed light on chemotherapeutic resistance and on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.

cancer biology↗

bioRxiv2020

HIF1a-dependent induction of the mitochondrial chaperone TRAP1 regulates bioenergetic adaptations to hypoxia

The mitochondrial paralog of the Hsp90 chaperone family TRAP1 is often induced in tumors, but the mechanisms controlling its expression, as well as its physiological functions remain poorly understood. Here, we find that TRAP1 is highly expressed in the early stages of Zebrafish development, and its ablation delays embryogenesis while increasing mitochondrial respiration of fish larvae. TRAP1 expression is enhanced by hypoxic conditions both in developing embryos and in cancer models of Zebrafish and mammals. The TRAP1 promoter contains evolutionary conserved hypoxic responsive elements, and HIF1 stabilization increases TRAP1 levels. TRAP1 inhibition by selective compounds or by genetic knock-out maintains a high level of respiration in Zebrafish embryos after exposure to hypoxia. Our data identify TRAP1 as a primary regulator of mitochondrial bioenergetics in highly proliferating cells following reductions in oxygen tension and HIF1 stabilization.

biochemistry↗

bioRxiv2020

The answer lies in the energy: how simple atomistic molecular dynamics simulations may hold the key to epitope prediction on the fully glycosylated SARS-CoV-2 spike protein

Betacoronavirus SARS-CoV-2 is posing a major threat to human health and its diffusion around the world is having dire socioeconomical consequences. Thanks to the scientific communitys unprecedented efforts, the atomic structure of several viral proteins has been promptly resolved. As the crucial mediator of host cell infection, the heavily glycosylated trimeric viral Spike protein (S) has been attracting the most attention and is at the center of efforts to develop antivirals, vaccines, and diagnostic solutions. Herein, we use an energy-decomposition approach to identify antigenic domains and antibody binding sites on the fully glycosylated S protein. Crucially, all that is required by our method are unbiased atomistic molecular dynamics simulations; no prior knowledge of binding properties or ad hoc combinations of parameters/measures extracted from simulations is needed. Our method simply exploits the analysis of energy interactions between all intra-protomer aminoacid and monosaccharide residue pairs, and cross-compares them with structural information (i.e., residueresidue proximity), identifying potential immunogenic regions as those groups of spatially contiguous residues with poor energetic coupling to the rest of the protein. Our results are validated by several experimentally confirmed structures of the S protein in complex with anti- or nanobodies. We identify poorly coupled sub-domains: on the one hand this indicates their role in hosting (several) epitopes, and on the other hand indicates their involvement in large functional conformational transitions. Finally, we detect two distinct behaviors of the glycan shield: glycans with stronger energetic coupling are structurally relevant and protect underlying peptidic epitopes; those with weaker coupling could themselves be poised for antibody recognition. Predicted Immunoreactive regions can be used to develop optimized antigens (recombinant subdomains, synthetic (glyco)peptidomimetics) for therapeutic applications.

biochemistry↗

bioRxiv2019

S-nitrosylation affects TRAP1 structure and ATPase activity and modulates cell response to apoptotic stimuli

The mitochondrial chaperone TRAP1 has been involved in several mitochondrial functions, and modulation of its expression/activity has been suggested to play a role in the metabolic reprogramming distinctive of cancer cells. TRAP1 posttranslational modifications, i.e. phosphorylation, can modify its capability to bind to different client proteins and modulate its oncogenic activity. Recently, it has been also demonstrated that TRAP1 is S-nitrosylated at Cys501, a redox modification associated with its degradation via the proteasome. Here we report molecular dynamics simulations of TRAP1, together with analysis of long-range structural communication, providing a model according to which Cys501 S-nitrosylation induces conformational changes to distal sites in the structure of the protein. The modification is also predicted to alter open and closing motions for the chaperone function. By means of colorimetric assays and site directed mutagenesis aimed at generating C501S variant, we also experimentally confirmed that selective S-nitrosylation of Cys501 decreases ATPase activity of recombinant TRAP1. Coherently, C501S mutant was more active and conferred protection to cell death induced by staurosporine. Overall, our results provide the first in silico, in vitro and cellular evidence of the relevance of Cys501 S-nitrosylation in TRAP1 biology.

biochemistry↗

bioRxiv2019

Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+-dependent death of cancer cells

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (Mitochondria-Associated Membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+-dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signalling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.

cancer biology↗