New Insights into the Evolution and Gene Structure of the Mitochondrial Carrier Family Unveiled by Analyzing the Frequent and Conserved Intron Positions.

Mitochondrial carriers (MCs) belong to a eukaryotic protein family of transporters that in higher organisms is called the solute carrier family 25 (SLC25). All MCs have characteristic triplicated sequence repeats forming a 3-fold symmetrical structure of a six-transmembrane α-helix bundle with a centrally located substrate-binding site. Biochemical characterization has shown that MCs altogether transport a wide variety of substrates but can be divided into subfamilies, each transporting a few specific substrates. We have investigated the intron positions in the human MC genes and their orthologs of highly diversified organisms. The results demonstrate that several intron positions are present in numerous MC sequences at the same specific points, of which some are 3-fold symmetry related. Many of these frequent intron positions are also conserved in subfamilies or in groups of subfamilies transporting similar substrates. The analyses of the frequent and conserved intron positions in MCs suggest phylogenetic relationships not only between close but also distant homologs as well as a possible involvement of the intron positions in the evolution of the substrate specificity diversification of the MC family members.

Deconstructing SARS-CoV-2 neutralization: A modular molecular framework for computational design and comparison of antibodies and nanobodies targeting the spike RBD.

Since 2020 the COVID-19 pandemic has led scientists to search for strategies to predict the transmissibility and virulence of new severe acute respiratory syndrome coronavirus 2 variants based on the estimation of the affinity of the spike receptor binding domain (RBD) for the human angiotensin-converting enzyme 2 (ACE2) receptor and/or neutralizing antibodies. In this context, our lab developed a computational pipeline to quickly quantify the free energy of interaction at the spike RBD/ACE2 protein-protein interface, reflecting the incidence trend observed in the transmissibility/virulence of the investigated variants. In this new study, we used our pipeline to estimate the free energy of interaction between the RBD from 10 variants, and 14 antibodies (ab), or 5 nanobodies (nb), highlighting the RBD regions preferentially targeted by the investigated ab/nb. Our structural comparative analysis and interaction energy calculations allowed us to propose the most promising RBD regions to be targeted by future ab/nb to be designed by site-directed mutagenesis of existing high-affinity ab/nb, to increase their affinity for the target RBD region, for preventing spike-RBD/ACE2 interactions and virus entry in host cells. Furthermore, we evaluated the ability of the investigated ab/nb to simultaneously interact with the three RBD located on the surface of the trimeric spike protein, which can alternatively be in up- or down- (all-3-up-, all-3-down-, 1-up-/2-down-, 2-up-/1-down-) conformations.

N-acetylaspartate release by glutaminolytic ovarian cancer cells sustains protumoral macrophages.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS-low, glutaminolysis-high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N-acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL-10, enforces GS expression in macrophages. In turn, GS-high macrophages acquire M2-like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2-like macrophage phenotype, IL-10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti-inflammatory, protumoral function of NAA.

Heterozygous RNF13 Gain-of-Function Variants Are Associated with Congenital Microcephaly, Epileptic Encephalopathy, Blindness, and Failure to Thrive.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) initiates a stress response mechanism to clear out the unfolded proteins by either facilitating their re-folding or inducing their degradation. When this fails, an apoptotic cascade is initiated so that the affected cell is eliminated. IRE1α is a critical sensor of the unfolded-protein response, essential for initiating the apoptotic signaling. Here, we report an infantile neurodegenerative disorder associated with enhanced activation of IRE1α and increased apoptosis. Three unrelated affected individuals with congenital microcephaly, infantile epileptic encephalopathy, and profound developmental delay were found to carry heterozygous variants (c.932T>C [p.Leu311Ser] or c.935T>C [p.Leu312Pro]) in RNF13, which codes for an IRE1α-interacting protein. Structural modeling predicted that the variants, located on the surface of the protein, would not alter overall protein folding. Accordingly, the abundance of RNF13 and IRE1α was not altered in affected individuals' cells. However, both IRE1α-mediated stress signaling and stress-induced apoptosis were increased in affected individuals' cells. These results indicate that the RNF13 variants confer gain of function to the encoded protein and thereby lead to altered signaling of the ER stress response associated with severe neurodegeneration in infancy.

Protein structure analysis of the interactions between SARS-CoV-2 spike protein and the human ACE2 receptor: from conformational changes to novel neutralizing antibodies.

The recent severe acute respiratory syndrome, known as Coronavirus Disease 2019 (COVID-19) has spread so much rapidly and severely to induce World Health Organization (WHO) to declare a state of emergency over the new coronavirus SARS-CoV-2 pandemic. While several countries have chosen the almost complete lock-down for slowing down SARS-CoV-2 spread, the scientific community is called to respond to the devastating outbreak by identifying new tools for diagnosis and treatment of the dangerous COVID-19. With this aim, we performed an in silico comparative modeling analysis, which allows gaining new insights into the main conformational changes occurring in the SARS-CoV-2 spike protein, at the level of the receptor-binding domain (RBD), along interactions with human cells angiotensin-converting enzyme 2 (ACE2) receptor, that favor human cell invasion. Furthermore, our analysis provides (1) an ideal pipeline to identify already characterized antibodies that might target SARS-CoV-2 spike RBD, aiming to prevent interactions with the human ACE2, and (2) instructions for building new possible neutralizing antibodies, according to chemical/physical space restraints and complementary determining regions (CDR) mutagenesis of the identified existing antibodies. The proposed antibodies show in silico high affinity for SARS-CoV-2 spike RBD and can be used as reference antibodies also for building new high-affinity antibodies against present and future coronaviruses able to invade human cells through interactions of their spike proteins with the human ACE2. More in general, our analysis provides indications for the set-up of the right biological molecular context for investigating spike RBD-ACE2 interactions for the development of new vaccines, diagnostic kits, and other treatments based on the targeting of SARS-CoV-2 spike protein.

Personalized Medicine in Mitochondrial Health and Disease: Molecular Basis of Therapeutic Approaches Based on Nutritional Supplements and Their Analogs.

Mitochondrial diseases (MDs) may result from mutations affecting nuclear or mitochondrial genes, encoding mitochondrial proteins, or non-protein-coding mitochondrial RNA. Despite the great variability of affected genes, in the most severe cases, a neuromuscular and neurodegenerative phenotype is observed, and no specific therapy exists for a complete recovery from the disease. The most used treatments are symptomatic and based on the administration of antioxidant cocktails combined with antiepileptic/antipsychotic drugs and supportive therapy for multiorgan involvement. Nevertheless, the real utility of antioxidant cocktail treatments for patients affected by MDs still needs to be scientifically demonstrated. Unfortunately, clinical trials for antioxidant therapies using α-tocopherol, ascorbate, glutathione, riboflavin, niacin, acetyl-carnitine and coenzyme Q have met a limited success. Indeed, it would be expected that the employed antioxidants can only be effective if they are able to target the specific mechanism, i.e., involving the central and peripheral nervous system, responsible for the clinical manifestations of the disease. Noteworthily, very often the phenotypes characterizing MD patients are associated with mutations in proteins whose function does not depend on specific cofactors. Conversely, the administration of the antioxidant cocktails might determine the suppression of endogenous oxidants resulting in deleterious effects on cell viability and/or toxicity for patients. In order to avoid toxicity effects and before administering the antioxidant therapy, it might be useful to ascertain the blood serum levels of antioxidants and cofactors to be administered in MD patients. It would be also worthwhile to check the localization of mutations affecting proteins whose function should depend (less or more directly) on the cofactors to be administered, for estimating the real need and predicting the success of the proposed cofactor/antioxidant-based therapy.

CRAT missense variants cause abnormal carnitine acetyltransferase function in an early-onset case of Leigh syndrome.

Leigh syndrome, or subacute necrotizing encephalomyelopathy, is one of the most severe pediatric disorders of the mitochondrial energy metabolism. By performing whole-exome sequencing in a girl affected by Leigh syndrome and her parents, we identified two heterozygous missense variants (p.Tyr110Cys and p.Val569Met) in the carnitine acetyltransferase (CRAT) gene, encoding an enzyme involved in the control of mitochondrial short-chain acyl-CoA concentrations. Biochemical assays revealed carnitine acetyltransferase deficiency in the proband-derived fibroblasts. Functional analyses of recombinant-purified CRAT proteins demonstrated that both missense variants, located in the acyl-group binding site of the enzyme, severely impair its catalytic function toward acetyl-CoA, and the p.Val569Met variant also toward propionyl-CoA and octanoyl-CoA. Although a single recessive variant in CRAT has been recently associated with neurodegeneration with brain iron accumulation (NBIA), this study reports the first kinetic analysis of naturally occurring CRAT variants and demonstrates the genetic basis of carnitine acetyltransferase deficiency in a case of mitochondrial encephalopathy.

SLC25A10 biallelic mutations in intractable epileptic encephalopathy with complex I deficiency.

Mitochondrial diseases are a plethora of inherited neuromuscular disorders sharing defects in mitochondrial respiration, but largely different from one another for genetic basis and pathogenic mechanism. Whole exome sequencing was performed in a familiar trio (trio-WES) with a child affected by severe epileptic encephalopathy associated with respiratory complex I deficiency and mitochondrial DNA depletion in skeletal muscle. By trio-WES we identified biallelic mutations in SLC25A10, a nuclear gene encoding a member of the mitochondrial carrier family. Genetic and functional analyses conducted on patient fibroblasts showed that SLC25A10 mutations are associated with reduction in RNA quantity and aberrant RNA splicing, and to absence of SLC25A10 protein and its transporting function. The yeast SLC25A10 ortholog knockout strain showed defects in mitochondrial respiration and mitochondrial DNA content, similarly to what observed in the patient skeletal muscle, and growth susceptibility to oxidative stress. Albeit patient fibroblasts were depleted in the main antioxidant molecules NADPH and glutathione, transport assays demonstrated that SLC25A10 is unable to transport glutathione. Here, we report the first recessive mutations of SLC25A10 associated to an inherited severe mitochondrial neurodegenerative disorder. We propose that SLC25A10 loss-of-function causes pathological disarrangements in respiratory-demanding conditions and oxidative stress vulnerability.

An overview of combined D-2- and L-2-hydroxyglutaric aciduria: functional analysis of CIC variants.

Combined D-2- and L-2-hydroxyglutaric aciduria (D/L-2-HGA) is a devastating neurometabolic disorder, usually lethal in the first years of life. Autosomal recessive mutations in the SLC25A1 gene, which encodes the mitochondrial citrate carrier (CIC), were previously detected in patients affected with combined D/L-2-HGA. We showed that transfection of deficient fibroblasts with wild-type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels, confirming that deficient CIC is the cause of D/L-2-HGA. We developed and implemented a functional assay and applied it to all 17 missense variants detected in a total of 26 CIC-deficient patients, including eight novel cases, showing reduced activities of varying degrees. In addition, we analyzed the importance of residues affected by these missense variants using our existing scoring system. This allowed not only a clinical and biochemical overview of the D/L-2-HGA patients but also phenotype-genotype correlation studies.

Biochemical characterization of a new mitochondrial transporter of dephosphocoenzyme A in Drosophila melanogaster.

CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.

Single-nucleotide evolution quantifies the importance of each site along the structure of mitochondrial carriers.

Mitochondrial carriers are membrane-embedded proteins consisting of a tripartite structure, a three-fold pseudo-symmetry, related sequences, and similar folding whose main function is to catalyze the transport of various metabolites, nucleotides, and coenzymes across the inner mitochondrial membrane. In this study, the evolutionary rate in vertebrates was screened at each of the approximately 50,000 nucleotides corresponding to the amino acids of the 53 human mitochondrial carriers. Using this information as a starting point, a scoring system was developed to quantify the evolutionary pressure acting on each site of the common mitochondrial carrier structure and estimate its functional or structural relevance. The degree of evolutionary selection varied greatly among all sites, but it was highly similar among the three symmetric positions in the tripartite structure, known as symmetry-related sites or triplets, suggesting that each triplet constitutes an evolutionary unit. Based on evolutionary selection, 111 structural sites (37 triplets) were found to be important. These sites play a key role in structure/function of mitochondrial carriers and are involved in either conformational changes (sites of the gates, proline-glycine levels, and aromatic belts) or in binding and specificity of the transported substrates (sites of the substrate-binding area in between the two gates). Furthermore, the evolutionary pressure analysis revealed that the matrix short helix sites underwent different degrees of selection with high inter-paralog variability. Evidence is presented that these sites form a new sequence motif in a subset of mitochondrial carriers, including the ADP/ATP translocator, and play a regulatory function by interacting with ligands and/or proteins of the mitochondrial matrix.

Prediction of high- and low-affinity quinol-analogue-binding sites in the aa3 and bo3 terminal oxidases from Bacillus subtilis and Escherichia coli1.

Haem-copper oxidases are the terminal enzymes in both prokaryotic and eukaryotic respiratory chains. They catalyse the reduction of dioxygen to water and convert redox energy into a transmembrane electrochemical proton gradient during their catalytic activity. Haem-copper oxidases show substantial structure similarity, but spectroscopic and biochemical analyses indicate that these enzymes contain diverse prosthetic groups and use different substrates (i.e. cytochrome c or quinol). Owing to difficulties in membrane protein crystallization, there are no definitive structural data about the quinol oxidase physiological substrate-binding site(s). In the present paper, we propose an atomic structure model for the menaquinol:O2 oxidoreductase of Bacillus subtilis (QOx.aa3). Furthermore, a multistep computational approach is used to predict residues involved in the menaquinol/menaquinone binding within B. subtilis QOx.aa3 as well as those involved in quinol/quinone binding within Escherichia coli QOx.bo3. Two specific sequence motifs, R70GGXDX4RXQX3PX3FX[D/N/E/Q]X2HYNE97 and G159GSPX2GWX2Y169 (B. subtilis numbering), were highlighted within QOx from Bacillales. Specific residues within the first and the second sequence motif participate in the high- and low-affinity substrate-binding sites respectively. Using comparative analysis, two analogous motifs, R71GFXDX4RXQX8[Y/F]XPPHHYDQ101 and G163EFX3GWX2Y173 (E. coli numbering) were proposed to be involved in Enterobacteriales/Rhodobacterales/Rhodospirillales QOx high- and low-affinity quinol-derivative-binding sites. Results and models are discussed in the context of the literature.

Distance-dependent hydrophobic-hydrophobic contacts in protein folding simulations.

Successful prediction of protein folding from an amino acid sequence is a challenge in computational biology. In order to reveal the geometric constraints that drive protein folding, highlight those constraints kept or missed by distinct lattices and for establishing which class of intra- and inter-secondary structure element interactions is the most relevant for the correct folding of proteins, we have calculated inter-alpha carbon distances in a set of 42 crystal structures consisting of mainly helix, sheet or mixed conformations. The inter-alpha carbon distances were also calculated in several lattice "hydrophobic-polar" models built from the same protein set. We found that helix structures are more prone to form "hydrophobic-hydrophobic" contacts than beta-sheet structures. At a distance lower than or equal to 3.8 Å (very short-range interactions), "hydrophobic-hydrophobic" contacts are almost absent in the native structures, while they are frequent in all the analyzed lattice models. At distances in-between 3.8 and 9.5 Å (short-/medium-range interactions), the best performing lattice for reproducing mainly helix structures is the body-centered-cubic lattice. If protein structures contain sheet portions, lattice performances get worse, with few exceptions observed for double-tetrahedral and body-centered-cubic lattices. Finally, we can observe that ab initio protein folding algorithms, i.e. those based on the employment of lattices and Monte Carlo simulated annealings, can be improved simply and effectively by preventing the generation of "hydrophobic-hydrophobic" contacts shorter than 3.8 Å, by monitoring the "hydrophobic-hydrophobic/polar-polar" contact ratio in short-/medium distance ranges and by using preferentially a body-centered-cubic lattice.

Ultradeep sequencing of a human ultraconserved region reveals somatic and constitutional genomic instability.

Early detection of cancer-associated genomic instability is crucial, particularly in tumour types in which this instability represents the essential underlying mechanism of tumourigenesis. Currently used methods require the presence of already established neoplastic cells because they only detect clonal mutations. In principle, parallel sequencing of single DNA filaments could reveal the early phases of tumour initiation by detecting low-frequency mutations, provided an adequate depth of coverage and an effective control of the experimental error. We applied ultradeep sequencing to estimate the genomic instability of individuals with hereditary non-polyposis colorectal cancer (HNPCC). To overcome the experimental error, we used an ultraconserved region (UCR) of the human genome as an internal control. By comparing the mutability outside and inside the UCR, we observed a tendency of the ultraconserved element to accumulate significantly fewer mutations than the flanking segments in both neoplastic and nonneoplastic HNPCC samples. No difference between the two regions was detectable in cells from healthy donors, indicating that all three HNPCC samples have mutation rates higher than the healthy genome. This is the first, to our knowledge, direct evidence of an intrinsic genomic instability of individuals with heterozygous mutations in mismatch repair genes, and constitutes the proof of principle for the development of a more sensitive molecular assay of genomic instability.

Case report: biallelic DNMT3A mutations in acute myeloid leukemia.

DNMT3A gene mutations, detected in 20-25% of de novo acute myeloid leukemia (AML) patients, are typically heterozygous. Biallelic variants are uncommon, affecting ~3% of cases and identifying a worse prognosis. Indeed, two concomitant DNMT3A mutations were recently associated with shorter event-free survival and overall survival in AML. We present an AML case bearing an unusual DNMT3A molecular status, strongly affecting its function and strangely impacting the global genomic methylation profile. A 56-year-old Caucasian male with a diagnosis of AML not otherwise specified (NOS) presented a complex DNMT3A molecular profile consisting of four different somatic variants mapping on different alleles (in trans). 3D modelling analysis predicted the effect of the DNMT3A mutational status, showing that all the investigated mutations decreased or abolished DNMT3A activity. Although unexpected, DNMT3A's severe loss of function resulted in a global genomic hypermethylation in genes generally involved in cell differentiation. The mechanisms through which DNMT3A contributes to AML remain elusive. We present a unique AML case bearing multiple biallelic DNMT3A variants abolishing its activity and resulting in an unexpected global hypermethylation. The unusual DNMT3A behavior described requires a reflection on its role in AML development and persistence, highlighting the heterogeneity of its deregulation.

Targeting mitochondrial impairment for the treatment of cardiovascular diseases: From hypertension to ischemia-reperfusion injury, searching for new pharmacological targets.

Mitochondria and mitochondrial proteins represent a group of promising pharmacological target candidates in the search of new molecular targets and drugs to counteract the onset of hypertension and more in general cardiovascular diseases (CVDs). Indeed, several mitochondrial pathways result impaired in CVDs, showing ATP depletion and ROS production as common traits of cardiac tissue degeneration. Thus, targeting mitochondrial dysfunction in cardiomyocytes can represent a successful strategy to prevent heart failure. In this context, the identification of new pharmacological targets among mitochondrial proteins paves the way for the design of new selective drugs. Thanks to the advances in omics approaches, to a greater availability of mitochondrial crystallized protein structures and to the development of new computational approaches for protein 3D-modelling and drug design, it is now possible to investigate in detail impaired mitochondrial pathways in CVDs. Furthermore, it is possible to design new powerful drugs able to hit the selected pharmacological targets in a highly selective way to rescue mitochondrial dysfunction and prevent cardiac tissue degeneration. The role of mitochondrial dysfunction in the onset of CVDs appears increasingly evident, as reflected by the impairment of proteins involved in lipid peroxidation, mitochondrial dynamics, respiratory chain complexes, and membrane polarization maintenance in CVD patients. Conversely, little is known about proteins responsible for the cross-talk between mitochondria and cytoplasm in cardiomyocytes. Mitochondrial transporters of the SLC25A family, in particular, are responsible for the translocation of nucleotides (e.g., ATP), amino acids (e.g., aspartate, glutamate, ornithine), organic acids (e.g. malate and 2-oxoglutarate), and other cofactors (e.g., inorganic phosphate, NAD+, FAD, carnitine, CoA derivatives) between the mitochondrial and cytosolic compartments. Thus, mitochondrial transporters play a key role in the mitochondria-cytosol cross-talk by leading metabolic pathways such as the malate/aspartate shuttle, the carnitine shuttle, the ATP export from mitochondria, and the regulation of permeability transition pore opening. Since all these pathways are crucial for maintaining healthy cardiomyocytes, mitochondrial carriers emerge as an interesting class of new possible pharmacological targets for CVD treatments.

Evolution, structure and function of mitochondrial carriers: a review with new insights.

The mitochondrial carriers (MC) constitute a large family (MCF) of inner membrane transporters displaying different substrate specificities, patterns of gene expression and even non-mitochondrial organelle localization. In Arabidopsis thaliana 58 genes encode these six trans-membrane domain proteins. The number in other sequenced plant genomes varies from 37 to 125, thus being larger than that of Saccharomyces cerevisiae and comparable with that of Homo sapiens. In addition to displaying highly similar secondary structures, the proteins of the MCF can be subdivided into subfamilies on the basis of substrate specificity and the presence of specific symmetry-related amino acid triplets. We assessed the predictive power of these triplets by comparing predictions with experimentally determined data for Arabidopsis MCs, and applied these predictions to the not yet functionally characterized mitochondrial carriers of the grass, Brachypodium distachyon, and the alga, Ostreococcus lucimarinus. We additionally studied evolutionary aspects of the plant MCF by comparing sequence data of the Arabidopsis MCF with those of Saccharomyces cerevisiae and Homo sapiens, then with those of Brachypodium distachyon and Ostreococcus lucimarinus, employing intra- and inter-genome comparisons. Finally, we discussed the importance of the approaches of global gene expression analysis and in vivo characterizations in order to address the relevance of these vital carrier proteins.

The Drosophila simulans Genome Lacks the crystal-Stellate System.

The cry-Ste system is a genetic interaction system between heterochromatin and euchromatin in Drosophila melanogaster, regulated via the piRNA pathway. Deregulation of this system leads to meiotic defects and male sterility. Although the cry-Ste system is peculiar to D. melanogaster, ancestors of Ste and Su(Ste) elements are present in the three closely related species, D. simulans, D. sechellia, and D. mauritiana. The birth, evolution, and maintenance of this genetic system in Drosophila melanogaster are of interest. We investigate the presence of sequences homologous to cry and Ste elements in the simulans complex and describe their chromosomal distribution. The organization and expression of cry- and Ste-like sequences were further characterized in the D. simulans genome. Our results allow us to conclude that the cry-Ste genetic interaction system is absent in the D. simulans genome.

Proline/Glycine residues of the PG-levels guide conformational changes along the transport cycle in the mitochondrial carnitine/acylcarnitine carrier (SLC25A20).

Mitochondrial carnitine/acylcarnitine carrier (CAC) is a member of the mitochondrial carrier (MC) family and imports acylcarnitine into the mitochondrial matrix in exchange for carnitine, playing a pivotal role in carnitine shuttle, crucial for fatty acid oxidation. The crystallized structure of CAC has not been solved yet, however, the availability of several in vitro/in silico studies, also based on the crystallized structures of the ADP/ATP carrier in the cytosolic-conformation and in the matrix-conformation, has made possible to confirm the hypothesis of the single-binding centered-gated pore mechanism for all the members of the MC family. In addition, our recent bioinformatics analyses allowed quantifying in silico the importance of protein residues of MC substrate binding region, of those involved in the formation of the matrix and cytosolic gates, and of those belonging to the Pro/Gly (PG) levels, proposed to be crucial for the tilting/kinking/bending of the six MC transmembrane helices, funneling the substrate translocation pathway. Here we present a combined in silico/in vitro analysis employed for investigating the role played by a group of 6 proline residues and 6 glycine residues, highly conserved in CAC, belonging to MC PG-levels. Residues of the PG-levels surround the similarly located MC common substrate binding region, and were proposed to lead conformational changes and substrate translocation, following substrate binding. For our analysis, we employed 3D molecular modeling approaches, alanine scanning site-directed mutagenesis and in vitro transport assays. Our analysis reveals that P130 (H3), G268 (H6) and G220 (H5), mutated in alanine, affect severely CAC transport activity (mutant catalytic efficiency lower than 5 % compared to the wild type CAC), most likely due to their major role in triggering CAC conformational changes, following carnitine binding. Notably, P30A (H1) and G121A (H3) CAC mutants, increase the carnitine uptake up to 217 % and 112 %, respectively, compared to the wild type CAC.

Modeling SARS-CoV-2 spike/ACE2 protein-protein interactions for predicting the binding affinity of new spike variants for ACE2, and novel ACE2 structurally related human protein targets, for COVID-19 handling in the 3PM context.

Aims: The rapid spread of new SARS-CoV-2 variants has highlighted the crucial role played in the infection by mutations occurring at the SARS-CoV-2 spike receptor binding domain (RBD) in the interactions with the human ACE2 receptor. In this context, it urgently needs to develop new rapid tools for quickly predicting the affinity of ACE2 for the SARS-CoV-2 spike RBD protein variants to be used with the ongoing SARS-CoV-2 genomic sequencing activities in the clinics, aiming to gain clues about the transmissibility and virulence of new variants, to prevent new outbreaks and to quickly estimate the severity of the disease in the context of the 3PM. Methods: In our study, we used a computational pipeline for calculating the interaction energies at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface for a selected group of characterized infectious variants of concern/interest (VoC/VoI). By using our pipeline, we built 3D comparative models of the SARS-CoV-2 spike RBD/ACE2 protein complexes for the VoC B.1.1.7-United Kingdom (carrying the mutations of concern/interest N501Y, S494P, E484K at the RBD), P.1-Japan/Brazil (RBD mutations: K417T, E484K, N501Y), B.1.351-South Africa (RBD mutations: K417N, E484K, N501Y), B.1.427/B.1.429-California (RBD mutations: L452R), the B.1.141 (RBD mutations: N439K), and the recent B.1.617.1-India (RBD mutations: L452R; E484Q) and the B.1.620 (RBD mutations: S477N; E484K). Then, we used the obtained 3D comparative models of the SARS-CoV-2 spike RBD/ACE2 protein complexes for predicting the interaction energies at the protein-protein interface. Results: Along SARS-CoV-2 mutation database screening and mutation localization analysis, it was ascertained that the most dangerous mutations at VoC/VoI spike proteins are located mainly at three regions of the SARS-CoV-2 spike "boat-shaped" receptor binding motif, on the RBD domain. Notably, the P.1 Japan/Brazil variant present three mutations, K417T, E484K, N501Y, located along the entire receptor binding motif, which apparently determines the highest interaction energy at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface, among those calculated. Conversely, it was also observed that the replacement of a single acidic/hydrophilic residue with a basic residue (E484K or N439K) at the "stern" or "bow" regions, of the boat-shaped receptor binding motif on the RBD, appears to determine an interaction energy with ACE2 receptor higher than that observed with single mutations occurring at the "hull" region or with other multiple mutants. In addition, our pipeline allowed searching for ACE2 structurally related proteins, i.e., THOP1 and NLN, which deserve to be investigated for their possible involvement in interactions with the SARS-CoV-2 spike protein, in those tissues showing a low expression of ACE2, or as a novel receptor for future spike variants. A freely available web-tool for the in silico calculation of the interaction energy at the SARS-CoV-2 spike RBD/ACE2 protein-protein interface, starting from the sequences of the investigated spike and/or ACE2 variants, was made available for the scientific community at: https://www.mitoairm.it/covid19affinities. Conclusion: In the context of the PPPM/3PM, the employment of the described pipeline through the provided webservice, together with the ongoing SARS-CoV-2 genomic sequencing, would help to predict the transmissibility of new variants sequenced from future patients, depending on SARS-CoV-2 genomic sequencing activities and on the specific amino acid replacement and/or on its location on the SARS-CoV-2 spike RBD, to put in play all the possible counteractions for preventing the most deleterious scenarios of new outbreaks, taking into consideration that a greater transmissibility has not to be necessarily related to a more severe manifestation of the disease. Supplementary Information: The online version contains supplementary material available at 10.1007/s13167-021-00267-w.