CHCHD4 binding affects the active site of apoptosis inducing factor (AIF): Structural determinants for allosteric regulation.

Apoptosis-inducing factor (AIF), which is confined to mitochondria of normal healthy cells, is the first identified caspase-independent cell death effector. Moreover, AIF is required for the optimal functioning of the respiratory chain machinery. Recent findings have revealed that AIF fulfills its pro-survival function by interacting with CHCHD4, a soluble mitochondrial protein which promotes the entrance and the oxidative folding of different proteins in the inner membrane space. Here, we report the crystal structure of the ternary complex involving the N-terminal 27-mer peptide of CHCHD4, NAD+, and AIF harboring its FAD (flavin adenine dinucleotide) prosthetic group in oxidized form. Combining this information with biophysical and biochemical data on the CHCHD4/AIF complex, we provide a detailed structural description of the interaction between the two proteins, validated by both chemical cross-linking mass spectrometry analysis and site-directed mutagenesis.

Head or tail? A molecular dynamics approach to the complex structure of TNF-associated factor TRAF2.

Tumor necrosis factor receptor-associated factor proteins (TRAFs) are trimeric proteins that play a fundamental role in signaling, acting as intermediaries between the tumor necrosis factor (TNF) receptors and the proteins that transmit the downstream signal. The monomeric subunits of all the TRAF family members share a common tridimensional structure: a C-terminal globular domain and a long coiled-coil tail characterizing the N-terminal section. In this study, the dependence of the TRAF2 dynamics on the length of its tail was analyzed in silico. In particular, we used the available crystallographic structure of a C-terminal fragment of TRAF2 (168 out of 501 a.a.), TRAF2-C, and that of a longer construct, addressed as TRAF2-plus, that we have re-constructed using the AlphaFold2 code. The results indicate that the longer N-terminal tail of TRAF2-plus has a strong influence on the dynamics of the globular regions in the protein C-terminal head. In fact, the quaternary interactions among the TRAF2-C subunits change asymmetrically in time, while the movements of TRAF2-plus monomers are rather limited and more ordered than those of the shorter construct. Such findings shed a new light on the dynamics of TRAF subunits and on the protein mechanism in vivo, since TRAF monomer-trimer equilibrium is crucial for several reasons (receptor recognition, membrane binding, hetero-oligomerization).

New Class of Benzodiazepinone Derivatives as Pro-Death Agents Targeting BIR Domains in Cancer Cells.

Inhibitor of Apoptosis Proteins (IAPs) are validated targets for cancer therapy, and the deregulation of their activities within the NF-κB pathway correlates with chemoresistance events, even after treatment with IAPs-antagonists in the clinic (Smac-mimetics). The molecule FC2 was identified as a NF-κB pathway modulator in MDA-MB-231 adenocarcinoma cancer cells after virtual screening of the Chembridge library against the Baculoviral IAP Repeat 1 (BIR1) domain of cIAP2 and XIAP. An improved cytotoxic effect is observed when FC2 is combined with Smac-mimetics or with the cytokine Tumor Necrosis Factor (TNF). Here, we propose a library of 22 derivatives of FC2, whose scaffold was rationally modified starting from the position identified as R1. The cytotoxic effect of FC2 derivatives was evaluated in MDA-MB-231 and binding to the cIAP2- and XIAP-BIR1 domains was assessed in fluorescence-based techniques and virtual docking. Among 22 derivatives, 4m and 4p display improved efficacy/potency in MDA-MB-231 cells and low micromolar binding affinity vs the target proteins. Two additional candidates (4b and 4u) display promising cytotoxic effects in combination with TNF, suggesting the connection between this class of molecules and the NF-κB pathway. These results provide the rationale for further FC2 modifications and the design of novel IAP-targeting candidates supporting known therapies.

Structure-based identification of a new IAP-targeting compound that induces cancer cell death inducing NF-κB pathway.

Inhibitors of apoptosis proteins (IAPs) are validated onco-targets, as their overexpression correlates with cancer onset, progression, diffusion and chemoresistance. IAPs regulate cell death survival pathways, inflammation, and immunity. Targeting IAPs, by impairing their protein-protein interaction surfaces, can affect events occurring at different stages of cancer development. To this purpose, we employed a rational virtual screening approach to identify compounds predicted to interfere with the assembly of pro-survival macromolecular complexes. One of the candidates, FC2, was shown to bind in vitro the BIR1 domains of both XIAP and cIAP2. Moreover, we demonstrated that FC2 can induce cancer cell death as a single agent and, more potently, in combination with the Smac-mimetic SM83 or with the cytokine TNF. FC2 determined a prolonged activation of the NF-κB pathway, accompanied to a stabilization of XIAP-TAB1 complex. This candidate molecule represents a valuable lead compound for the development of a new class of IAP-antagonists for cancer treatment.

Ligand Binding in Allosteric Flavoproteins: Part 1. Quantitative Analysis of the Interaction with NAD+ of the Apoptosis Inducing Factor (AIF) Harboring FAD in the Reduced State.

To perform their action, flavoproteins usually interact with a variety of low molecular weight partners, including electron transporters, yielding transient complexes whose tightness is often controlled by the redox state of the bound flavin cofactor. As a case study, here we describe the quantitative analysis of the redox-dependent interaction of the mammalian apoptosis inducing factor (AIF) with its NAD+ ligand. In particular, we report a protocol for the spectrophotometric titration of AIF in its reduced state under anaerobic conditions with NAD+, in order to determine the dissociation constant of the resulting complex.

[The investigation of non-verbal behavior through Nonverbal Behavior Analysis Matrix in the forensic setting]. / L'indagine del comportamento non verbale attraverso la Nonverbal Behavior Analysis Matrix in ambito forense.

The application of the neuroscience in forensic sciences has long opened up new scenarios within the legal world. While on one hand we tend to emphasize the benefits of the new research methods based on the recent neuroscience knowledge, on the other hand the use of these new tools has sparked a justified debate at the international level that touches all the areas that directly or indirectly approach forensics. Factors like lack of responsability or mitigation provided by science are part of those topics that, thanks to neurosciences, are back in the spotlight on a highly empirical basis. At the same time, these factors mentioned above influence our way to interpret reality. NBAM takes place within this debate as a new scientifically proven protocol and technologically supported, making way for an objective development of the neuroscience research.

Computational and Experimental Characterization of NF023, A Candidate Anticancer Compound Inhibiting cIAP2/TRAF2 Assembly.

Protein-protein interactions are the basis of many important physiological processes and are currently promising, yet difficult, targets for drug discovery. In this context, inhibitor of apoptosis proteins (IAPs)-mediated interactions are pivotal for cancer cell survival; the interaction of the BIR1 domain of cIAP2 with TRAF2 was shown to lead the recruitment of cIAPs to the TNF receptor, promoting the activation of the NF-κB survival pathway. In this work, using a combined in silico-in vitro approach, we identified a drug-like molecule, NF023, able to disrupt cIAP2 interaction with TRAF2. We demonstrated in vitro its ability to interfere with the assembly of the cIAP2-BIR1/TRAF2 complex and performed a thorough characterization of the compound's mode of action through 248 parallel unbiased molecular dynamics simulations of 300 ns (totaling almost 75 µs of all-atom sampling), which identified multiple binding modes to the BIR1 domain of cIAP2 via clustering and ensemble docking. NF023 is, thus, a promising protein-protein interaction disruptor, representing a starting point to develop modulators of NF-κB-mediated cell survival in cancer. This study represents a model procedure that shows the use of large-scale molecular dynamics methods to typify promiscuous interactors.

Structure-Activity Relationship of NF023 Derivatives Binding to XIAP-BIR1.

Inhibitors of Apoptosis Proteins (IAPs) are conserved E3-ligases that ubiquitylate substrates to prevent apoptosis and activate the NF-kB survival pathway, often deregulated in cancer. IAPs-mediated regulation of NF-kB signaling is based on the formation of protein complexes by their type-I BIR domains. The XIAP-BIR1 domain dimerizes to bind two TAB1 monomers, leading to downstream NF-kB activation. Thus, impairment of XIAP-BIR1 dimerization could represent a novel strategy to hamper cell survival in cancer. To this aim, we previously reported NF023 as a potential inhibitor of XIAP-BIR1 dimerization. Here we present a thorough analysis of NF023 binding to XIAP-BIR1 through biochemical, biophysical and structural data. The results obtained indicate that XIAP-BIR1 dimerization interface is involved in NF023 binding, and that NF023 overall symmetry and the chemical features of its central moiety are essential for an efficient interaction with the protein. Such strategy provides original hints for the development of novel BIR1-specific compounds as pro-apoptotic agents.

Targeting the BIR Domains of Inhibitor of Apoptosis (IAP) Proteins in Cancer Treatment.

Inhibitor of apoptosis (IAP) proteins are characterized by the presence of the conserved baculoviral IAP repeat (BIR) domain that is involved in protein-protein interactions. IAPs were initially thought to be mainly responsible for caspase inhibition, acting as negative regulators of apoptosis, but later works have shown that IAPs also control a plethora of other different cellular pathways. As X-linked IAP (XIAP), and other IAP, levels are often deregulated in cancer cells and have been shown to correlate with patients' prognosis, several approaches have been pursued to inhibit their activity in order to restore apoptosis. Many small molecules have been designed to target the BIR domains, the vast majority being inspired by the N-terminal tetrapeptide of Second Mitochondria-derived Activator of Caspases/Direct IAp Binding with Low pI (Smac/Diablo), which is the natural XIAP antagonist. These compounds are therefore usually referred to as Smac mimetics (SMs). Despite the fact that SMs were intended to specifically target XIAP, it has been shown that they also interact with cellular IAP-1 (cIAP1) and cIAP2, promoting their proteasome-dependent degradation. SMs have been tested in combination with several cytotoxic compounds and are now considered promising immune modulators which can be exploited in cancer therapy, especially in combination with immune checkpoint inhibitors. In this review, we give an overview of the structural hot-spots of BIRs, focusing on their fold and on the peculiar structural patches which characterize the diverse BIRs. These structures are exploited/exploitable for the development of specific and active IAP inhibitors.

Structure-based design and molecular profiling of Smac-mimetics selective for cellular IAPs.

Inhibitor of Apoptosis Proteins (IAPs) is highly conserved negative regulators of apoptosis overexpressed in many cancer cells. Based on their endogenous antagonist, Smac/DIABLO, mimic compounds (Smac-mimetics, SMs) have been developed to inhibit IAPs prosurvival activity, showing promising effects in advanced phases of clinical trials. Since different IAP homologs play distinctive roles in cancer cell survival and immunomodulation, SM-induced apoptosis proceeds through diverse mechanisms. After binding to their BIR3 domain, SMs have been shown to rapidly induce auto-ubiquitylation and degradation of cellular IAPs (cIAPs), thus leading to cell death mainly by activation of the noncanonical NF-κB pathway. For this reason, we started the BIR3-driven design of compounds selective for cIAP1 and with reduced affinity for X-linked IAP (XIAP), in order to focus SMs antitumor activity on cIAPs degradation. In this work, we describe the crystal structures of the BIR3 domains of cIAP1 and XIAP, each in complex with a cIAP1-selective SM (SM130 and SM114, respectively). The two SMs displayed 23- and 32-fold higher affinity for cIAP1-BIR3 over XIAP-BIR3 in molecular displacement experiments based on fluorescence polarization. In vitro cell-based assays confirmed that both selective SMs triggered apoptosis in cancer cells with different efficiencies by inducing caspases-3, -8, and -9-independent cIAP1 degradation. The design of cIAPs-selective compounds represents an innovative approach in the field of anticancer drugs development, being useful to elucidate different prosurvival mechanisms and to reduce the adverse effects of pan-IAPs compounds in cancer therapy. DATABASE: Structural data are available in the Protein Data Bank database under the accession codes 6EXW (cIAP1-BIR3/SM130 complex) and 6EY2 (XIAP-BIR3/SM114 complex).

Cellulose production is coupled to sensing of the pyrimidine biosynthetic pathway via c-di-GMP production by the DgcQ protein of Escherichia coli.

Production of cellulose, a stress response-mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c-di-GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N-carbamoyl-aspartate appears to be favoured by protein-protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N-carbamoyl-aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.

Structural bases of the altered catalytic properties of a pathogenic variant of apoptosis inducing factor.

The apoptosis-inducing factor (AIF) is a FAD-containing protein playing critical roles in caspase-independent apoptosis and mitochondrial respiratory chain biogenesis and maintenance. While its lethal role is well known, the details of its mitochondrial function remain elusive. So far, nineteen allelic variants of AIF have been associated to human diseases, mainly affecting the nervous system. A strict correlation is emerging between the degree of impairment of its ability to stabilize the charge-transfer (CT) complex between FAD and NAD+ and the severity of the resulting pathology. Recently, we demonstrated that the G307E replacement in murine AIF (equivalent to the pathogenic G308E in the human protein) dramatically decreases the rate of CT complex formation through the destabilization of the flavoprotein interaction with NAD(H). To provide further insights into the structural bases of its altered functional properties, here we report the first crystal structure of an AIF pathogenic mutant variant in complex with NAD+ (murine AIF-G307ECT) in comparison with its oxidized form. With respect to wild type AIF, the mutation leads to an altered positioning of NAD+ adenylate moiety, which slows down CT complex formation. Moreover, the altered balance between the binding of the adenine/nicotinamide portions of the coenzyme determines a large drop in AIF-G307E ability to discriminate between NADH and NADPH.

The activator of apoptosis Smac-DIABLO acts as a tetramer in solution.

Smac-DIABLO in its mature form (20.8 kDa) binds to baculoviral IAP repeat (BIR) domains of inhibitor of apoptosis proteins (IAPs) releasing their inhibitory effects on caspases, thus promoting cell death. Despite its apparent molecular mass (∼100 kDa), Smac-DIABLO was held to be a dimer in solution, simultaneously targeting two distinct BIR domains. We report an extensive biophysical characterization of the protein alone and in complex with the X-linked IAP (XIAP)-BIR2-BIR3 domains. Our data show that Smac-DIABLO adopts a tetrameric assembly in solution and that the tetramer is able to bind two BIR2-BIR3 pairs of domains. Our small-angle x-ray scattering-based tetrameric model of Smac-DIABLO/BIR2-BIR3 highlights some conformational freedom of the complex that may be related to optimization of IAPs binding.

NF023 binding to XIAP-BIR1: searching drugs for regulation of the NF-κB pathway.

Inhibitor of Apoptosis Proteins (IAPs) are the target of extensive research in the field of cancer therapy since they regulate apoptosis and cell survival. Smac-mimetics, the most promising IAP-targeting compounds specifically recognize the IAP-BIR3 domain and promote apoptosis, competing with caspases for IAP binding. Furthermore, Smac-mimetics interfere with the NF-κB survival pathway, inducing cIAP1 and cIAP2 degradation through an auto-ubiquitination process. It has been shown that the XIAP-BIR1 (X-BIR1) domain is involved in the interaction with TAB1, an upstream adaptor for TAK1 kinase activation, which in turn couples with the NF-κB survival pathway. Preventing X-BIR1 dimerization abolishes XIAP-mediated NF-κB activation, thus implicating a proximity-induced mechanism for TAK1 activation. In this context, in a systematic search for a molecule capable of impairing X-BIR1/TAB1 assembly, we identified the compound NF023. Here we report the crystal structure of the human X-BIR1 domain in the absence and in the presence of NF023, as a starting concept for the design of novel BIR1-specific compounds acting synergistically with existing pro-apoptotic drugs in cancer therapy.

TREX1 C-terminal frameshift mutations in the systemic variant of retinal vasculopathy with cerebral leukodystrophy.

Retinal vasculopathy with cerebral leukodystrophy (RVCL) is an adult-onset disorder caused by C-terminal heterozygous frameshift (fs) mutations in the human 3'-5' DNA exonuclease TREX1. Hereditary systemic angiopathy (HSA) is considered a variant of RVCL with systemic involvement of unknown genetic cause, described in a unique family so far. Here we describe the second case of RVCL with systemic involvement, characterized by cerebral calcifications and pseudotumoral lesions, retinopathy, osteonecrosis, renal and hepatic failure. The genetic screening of TREX1 in this patient revealed the novel heterozygous T270fs mutation on the C-terminal region. On the same gene, we found the V235fs mutation, formerly shown in RVCL, in one patient previously reported with HSA. These mutations lead to important alterations of the C-terminal of the protein, with the loss of the transmembrane helix (T270fs) and the insertion of a premature stop codon, resulting in a truncated protein (V235fs). Functional analysis of T270fs-mutated fibroblasts showed a prevalent localization of the protein in the cytosol, rather than in the perinuclear region. RVCL with systemic involvement is an extremely rare condition, whose diagnosis is complex due to multiorgan manifestations, unusual radiological and histopathological findings, not easily attributable to a single disease. It should be suspected in young adults with systemic microangiopathy involving retina, liver, kidney, bones and brain. Here we confirm the causative role played by TREX1 autosomal dominant fs mutations disrupting the C-terminal of the protein, providing a model for the study of stroke in young adults.

Novel neurofibromatosis type 2 mutation presenting with status epilepticus.

Neurofibromatosis type 2 (NF2) is a dominantly inherited syndrome caused by mutations of the tumour-suppressor NF2, which encodes the merlin protein. Mutations are associated with a predisposition to development of benign tumours in the central nervous system. Even though cerebral cortical lesions are frequently associated with seizures, epilepsy is rarely described in NF2. Here, we describe an adult case of NF2 in which the onset of symptoms was characterised by status epilepticus. In this patient, we identified the novel c.428_430delCTTdel mutation in NF2, involving the amino-terminal FERM domain, which is fundamental for the correct tumour suppressor function of the protein. Bioinformatic analyses revealed an important structural perturbation of the FERM domain, with a predicted impairment of the anti-tumour activity.

Kinetic and structural evidences on human prolidase pathological mutants suggest strategies for enzyme functional rescue.

Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients' fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.

Structural insight into inhibitor of apoptosis proteins recognition by a potent divalent smac-mimetic.

Genetic alterations enhancing cell survival and suppressing apoptosis are hallmarks of cancer that significantly reduce the efficacy of chemotherapy or radiotherapy. The Inhibitor of Apoptosis Protein (IAP) family hosts conserved proteins in the apoptotic pathway whose over-expression, frequently found in tumours, potentiates survival and resistance to anticancer agents. In humans, IAPs comprise eight members hosting one or more structural Baculoviral IAP Repeat (BIR) domains. Cellular IAPs (cIAP1 and 2) indirectly inhibit caspase-8 activation, and regulate both the canonical and the non-canonical NF-κB signaling pathways. In contrast to cIAPs, XIAP (X chromosome-linked Inhibitor of Apoptosis Protein) inhibits directly the effector caspases-3 and -7 through its BIR2 domain, and initiator caspase-9 through its BIR3 domain; molecular docking studies suggested that Smac/DIABLO antagonizes XIAP by simultaneously targeting both BIR2 and BIR3 domains. Here we report analytical gel filtration, crystallographic and SAXS experiments on cIAP1-BIR3, XIAP-BIR3 and XIAP-BIR2BIR3 domains, alone and in the presence of compound 9a, a divalent homodimeric Smac mimetic. 9a is shown to bind two BIR domains inter- (in the case of two BIR3) and intra-molecularly (in the case of XIAP-BIR2BIR3), with higher affinity for cIAP1-BIR3, relative to XIAP-BIR3. Despite the different crystal lattice packing, 9a maintains a right handed helical conformation in both cIAP1-BIR3 and XIAP-BIR3 crystals, that is likely conserved in solution as shown by SAXS data. Our structural results demonstrate that the 9a linker length, its conformational degrees of freedom and its hydrophobicity, warrant an overall compact structure with optimal solvent exposure of its two active moieties for IAPs binding. Our results show that 9a is a good candidate for pre-clinical and clinical studies, worth of further investigations in the field of cancer therapy.

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization.

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head-head (8), tail-tail (9) or head-tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2-BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail-tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.

A NMR and computational study of Smac mimics targeting both the BIR2 and BIR3 domains in XIAP protein.

In this paper we report an extensive NMR analysis of small ligands (Smac mimics) complexed with different constructs of XIAP. The mimics-binding site of XIAP is known as the BIR3 domain - primary, and the linker BIR2 region - secondary site. Interactions between the BIR3 domain and Smac mimics have been extensively studied by X-ray but, as of today, there are scarce data about the interaction between BIR2, or the whole linker-BIR2-BIR3 construct, and Smac mimics. In order to characterize our Smac mimics, we performed a STD NMR study between our 4-substituted, 1-aza-2-oxobicyclo[5.3.0]decane scaffold-based molecules and three different XIAP fragments: single BIR2 and BIR3 domains, and bifunctional linker-BIR2-BIR3. The results were integrated with docking calculations and molecular dynamics simulations. NMR data, which are consistent with biological tests, indicated that the two BIR subunits interact differently with our Smac mimics and suggest that the ligands enter into more intimate contact with the linker-BIR2-BIR3. In conclusion, we observe that the SMAC mimics showed with the construct linker-BIR2-BIR3 a series of NOE contacts that were not observed in the mono-domain ligand:BIR2 or :BIR3 complexes. So, in agreement with the computational models we believe that the linker moieties of the binding site play a key role in the stability of the protein complex.