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

Abstract

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. ATP binding and hydrolysis; a middle-domain (MD), that contains that contains part of the ATP-binding pocket [5], and the binding site for client proteins; a C-terminal domain (CTD) that constitutes the interface for homodimerization [6]. Differently form the other Hsp90 orthologs, TRAP1 lacks the linker domain between MD and CTD, and exhibits a N-terminal extension that acts as thermal regulator of its chaperone activity [7].The ATPase cycle of TRAP1 has been characterized in detail, with both protomers assisting the folding of the target proteins through structural modifications associated with repeated cycles of ATP binding, hydrolysis and release [2,8,9]. During the ATPase cycle, TRAP1 can be present under three different states: i) an open conformation (called apo); ii) a close conformation with the NTD placed between the two protomers; iii) an intermediate coiled-coil conformation with both NTD in close proximity. In the absence of substrates, TRAP1 is present in an open state, while the binding of two 2 molecules of ATP induces structural changes leading to a closed asymmetric conformation [8], this being distinctive of TRAP1 among Hsp90 family members. ATP binding provides the energy required for substrate remodelling which takes place by a two-step reaction. The hydrolysis of the first ATP produces symmetric changes in protomers, establishing a structural rearrangement in the substrate binding site. The hydrolysis of the second ATP

Targeting the mitochondrial chaperone TRAP1: strategies and therapeutic perspectives

“…By downregulating SDH enzymatic activity, TRAP1 causes succinate accumulation in cells, establishing a proneoplastic, pseudohypoxic phenotype via succinate-dependent induction of the transcription factor HIF1α [32]. Further layers of complexity are provided by a variety of post-translational modifications (PTMs) of TRAP1, including phosphorylations, S-nitrosylations, and acetylations [19,[34][35][36][37], with multifaceted effects on its chaperone activity that are only partially understood.…”

“…For instance, in a different tumor cell model, SIRT3 activation recapitulates the effect of TRAP1 inhibition in increasing SDH activity [40], suggesting the existence of further layers of complexity. Reversible acetylation is part of a wider picture of TRAP1 PTMs that also include nitrosylation [36,37] and phosphorylation [19,35], opening the innovative perspective of targeting TRAP1 interactions with the PTM-inducing enzymes to obtain highly specific functional effects.…”

Dynamically Shaping Chaperones. Allosteric Modulators of HSP90 Family as Regulatory Tools of Cell Metabolism in Neoplastic Progression

“…PHD inhibition stabilizes the transcription factor HIF1α, increasing invasiveness, angiogenesis and further metabolic changes in tumor cells ( 98 ), whereas inhibition of KDMs, which hydroxylate lysine residue on histones, and of TETs, which induce DNA demethylation of CpG islands near gene promoters, prompts complex epigenetic rearrangements in neoplastic cells ( Figure 3 ). Oncogenic kinase pathways directly target TRAP1, as it is both Tyr-phosphorylated in a Src-dependent way and Ser-phosphorylated by ERK1/2, favoring cytochrome oxidase inhibition and enhancing TRAP1 inhibition of SDH activity, respectively ( 78 , 81 ), whereas S-nitrosylation elicits TRAP1 degradation ( 76 ) and decreases its ATPase activity ( 77 ) ( Table 2 ).…”

PyInteraph2 and PyInKnife2 to analyze networks in protein structural ensembles

“…The copyright holder for this preprint this version posted November 23, 2020. ; https://doi.org/10.1101/2020.11.22.381616 doi: bioRxiv preprint translational modifications (Faienza et al, 2020;Lambrughi, De Gioia, et al, 2016). More broadly, PyInteraph has been used to study protein dynamics and allostery (Lambrughi, De Gioia, et al, 2016;Sora and Papaleo, 2019;Marino andDell'Orco, 2016, 2019;Galochkina et al, 2019;Faienza et al, 2020;Abbas et al, 2019;Bonì et al, 2020;Borsatto et al, 2019). PyInteraph is also included in planned protocols from scientific consortia to study the genetics and the molecular mechanisms at the base of different diseases (Borges et al, 2020).…”

“…In 2014, we developed PyInteraph (Tiberti et al ., 2014)for the study of protein structure networks (PSNs) from structural ensembles, especially suited to work on trajectories from atomistic simulations such as Molecular Dynamics (MD). Examples of PyInteraph applications includes: i) the study of the effects of mutations in disease-related proteins (Nygaard et al ., 2016; Kønig et al ., 2019; Marino et al ., 2015; Pantsar et al ., 2018; Lambrughi, Lucchini, et al ., 2016; Kumar and Papaleo, 2020; Fas et al ., 2019; Endo et al ., 2020; Michelini et al ., 2020; Di Stazio et al ., 2020), ii) to characterize or design variants for enzymes of industrial interest (Jónsdóttir et al ., 2014; Papaleo, Parravicini, et al ., 2014; Michetti et al ., 2017; Óskarsson et al ., 2016; Singh et al ., 2016), to study the binding of biomolecules to a target protein (Di Rita et al ., 2018), and to disclose the effect of post-translational modifications (Faienza et al ., 2020; Lambrughi, De Gioia, et al ., 2016). More broadly, PyInteraph has been used to study protein dynamics and allostery (Lambrughi, De Gioia, et al ., 2016; Sora and Papaleo, 2019; Marino and Dell’Orco, 2016, 2019; Galochkina et al ., 2019; Faienza et al ., 2020; Abbas et al ., 2019; Bonì et al ., 2020; Borsatto et al ., 2019).…”