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.

Pathogenic Variants in NUP214 Cause "Plugged" Nuclear Pore Channels and Acute Febrile Encephalopathy.

We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleoporin NUP214 causing acute febrile encephalopathy. Clinical symptoms include neurodevelopmental regression, seizures, myoclonic jerks, progressive microcephaly, and cerebellar atrophy. NUP214 and NUP88 protein levels were reduced in primary skin fibroblasts derived from affected individuals, while the total number and density of nuclear pore complexes remained normal. Nuclear transport assays exhibited defects in the classical protein import and mRNA export pathways in affected cells. Direct surface imaging of fibroblast nuclei by scanning electron microscopy revealed a large increase in the presence of central particles (known as "plugs") in the nuclear pore channels of affected cells. This observation suggests that large transport cargoes may be delayed in passage through the nuclear pore channel, affecting its selective barrier function. Exposure of fibroblasts from affected individuals to heat shock resulted in a marked delay in their stress response, followed by a surge in apoptotic cell death. This suggests a mechanistic link between decreased cell survival in cell culture and severe fever-induced brain damage in affected individuals. Our study provides evidence by direct imaging at the single nuclear pore level of functional changes linked to a human disease.

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.

Effect of cariporide on ram sperm pH regulation and motility: possible role of NHE1.

Sperm motility, a feature essential for in vivo fertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHi regulation, among which sodium-hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+ for H+ across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparent Km for external Na+ of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHi recovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation. In vitro fertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHi and motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+ exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.

Nitro-substituted tetrahydroindolizines and homologs: Design, kinetics, and mechanism of α-glucosidase inhibition.

A series of 1-[(methylsulfonyl)methyl]-2-nitro-5,6,7,8-tetrahydroindolizines and homologs were designed, prepared, and evaluated as non-sugar-type α-glucosidase inhibitors. The inhibitory activity appeared to be related to cyclo homologation with the best congeners being tetrahydroindolizines. The introduction of a methoxycarbonyl group as an additional hydrogen bond acceptor into the exocyclic methylene group was beneficial affording the most potent congener 3e (half maximal inhibitory concentration, IC50=8.0±0.1µM) which displayed 25-fold higher inhibitory activity than 1-deoxynojirimycin (2, IC50=203±9µM)-the reference compound. Kinetic analysis indicated that compound 3e is a mixed inhibitor with preference for the free enzyme over the α-glucosidase-substrate complex (Ki,free=3.6µM; Ki,bound=7.6µM). Molecular docking experiments were in agreement with kinetic results indicating reliable interactions with both the catalytic cleft and other sites. Circular dichroism spectroscopy studies suggested that the inhibition exerted by 3e may involve changes in the secondary structure of the enzyme. Considering the relatively low molecular weight of 3e together with its high fraction of sp3 hybridized carbon atoms, this nitro-substituted tetrahydroindolizine may be considered as a good starting point towards new leads in the area of α-glucosidase inhibitors.

Pathogenic potential of SLC25A15 mutations assessed by transport assays and complementation of Saccharomyces cerevisiae ORT1 null mutant.

HHH syndrome is an autosomal recessive urea cycle disorder caused by alterations in the SLC25A15 gene encoding the mitochondrial ornithine carrier 1, which catalyzes the transport of cytosolic ornithine into the mitochondria in exchange for intramitochondrial citrulline. In this study the functional effects of several SLC25A15 missense mutations p.G27R, p.M37R, p.N74A, p.F188L, p.F188Y, p.S200K, p.R275Q and p.R275K have been tested by transport assays in reconstituted liposomes and complementation of Saccharomyces cerevisiae ORT1 null mutant in arginine-less synthetic complete medium. The HHH syndrome-causing mutations p.G27R, p.M37R, p.F188L and p.R275Q had impaired transport and did not complement ORT1∆ cells (except p.M37R slightly after 5 days in solid medium). The experimentally produced mutations p.N74A, p.S200K and p.R275K exhibited normal or considerable transport activity and complemented ORT1∆ cells after 3 days (p.N74A, p.S200K) or 5 days (p.R275K) incubation. Furthermore, the experimentally produced p.F188Y mutation displayed a substantial transport activity but did not complement the ORT1∆ cells in both liquid and solid media. In view of the disagreement in the results obtained between the two methods, it is recommended that the method of complementing the S. cerevisiae ORT1 knockout strain is used complimentary with the measurement of the catalytic activity, in order to distinguish HHH syndrome-causing mutations from isomorphisms.

A novel mutation in the SLC25A15 gene in a Turkish patient with HHH syndrome: functional analysis of the mutant protein.

The hyperornithinemia-hyperammonemia-homocitrullinuria syndrome is a rare autosomal recessive disorder caused by the functional deficiency of the mitochondrial ornithine transporter 1 (ORC1). ORC1 is encoded by the SLC25A15 gene and catalyzes the transport of cytosolic ornithine into mitochondria in exchange for citrulline. Although the age of onset and the severity of the symptoms vary widely, the disease usually manifests in early infancy. The typical clinical features include protein intolerance, lethargy, episodic confusion, cerebellar ataxia, seizures and mental retardation. In this study, we identified a novel p.Ala15Val (c.44C>T) mutation by genomic DNA sequencing in a Turkish child presenting severe tantrum, confusion, gait disturbances and loss of speech abilities in addition to hyperornithinemia, hyperammonemia and homocitrullinuria. One hundred Turkish control chromosomes did not possess this variant. The functional effect of the novel mutation was assessed by both complementation of the yeast ORT1 null mutant and transport assays. Our study demonstrates that the A15V mutation dramatically interferes with the transport properties of ORC1 since it was shown to inhibit ornithine transport nearly completely.

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.

Targeting the mitochondrial trifunctional protein restrains tumor growth in oxidative lung carcinomas.

Metabolic reprogramming is a common hallmark of cancer, but a large variability in tumor bioenergetics exists between patients. Using high-resolution respirometry on fresh biopsies of human lung adenocarcinoma, we identified 2 subgroups reflected in the histologically normal, paired, cancer-adjacent tissue: high (OX+) mitochondrial respiration and low (OX-) mitochondrial respiration. The OX+ tumors poorly incorporated [18F]fluorodeoxy-glucose and showed increased expression of the mitochondrial trifunctional fatty acid oxidation enzyme (MTP; HADHA) compared with the paired adjacent tissue. Genetic inhibition of MTP altered OX+ tumor growth in vivo. Trimetazidine, an approved drug inhibitor of MTP used in cardiology, also reduced tumor growth and induced disruption of the physical interaction between the MTP and respiratory chain complex I, leading to a cellular redox and energy crisis. MTP expression in tumors was assessed using histology scoring methods and varied in negative correlation with [18F]fluorodeoxy-glucose incorporation. These findings provide proof-of-concept data for preclinical, precision, bioenergetic medicine in oxidative lung carcinomas.

Molecular modeling of antibodies for the treatment of TNFα-related immunological diseases.

Therapeutic monoclonal antibodies (mAbs) have high efficacy in treating TNF α-related immunological diseases. Other than neutralizing TNF α, these IgG1 antibodies exert Fc receptor-mediated effector functions such as the complement-dependent cytotoxicity (CDC) and antibody-dependent cell cytotoxicity (ADCC). The crystallizable fragment (Fc) of these IgG1 contains a single glycosylation site at Asn 297/300 that is essential for the CDC and ADCC. Glycosylated antibodies lacking core fucosylation showed an improved ADCC. However, no structural data are available concerning the ligand-binding interaction of these mAbs used in TNF α-related diseases and the role of the fucosylation. We therefore used comparative modeling for generating complete 3D mAb models that include the antigen-binding fragment (Fab) portions of infliximab, complexed with TNF α (4G3Y.pdb), the Fc region of the human IGHG1 fucosylated (3SGJ) and afucosylated (3SGK) complexed with the Fc receptor subtype Fcγ RIIIA, and the Fc region of a murine immunoglobulin (1IGT). After few thousand steps of energy minimization on the resulting 3D mAb models, minimized final models were used to quantify interactions occurring between Fcγ RIIIA and the fucosylated/afucosylated Fc fragments. While fucosylation does not affect Fab-TNF α interactions, we found that in the absence of fucosylation the Fc-mAb domain and Fcγ RIIIA are closer and new strong interactions are established between G129 of the receptor and S301 of the Chimera 2 Fc mAb; new polar interactions are also established between the Chimera 2 Fc residues Y299, N300, and S301 and the Fcγ RIIIA residues K128, G129, R130, and R155. These data help to explain the reduced ADCC observed in the fucosylated mAbs suggesting the specific AA residues involved in binding interactions.

Identification of new highly selective inhibitors of the human ADP/ATP carriers by molecular docking and in vitro transport assays.

Mitochondrial carriers are proteins that shuttle a variety of metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. The mitochondrial ADP/ATP carriers (AACs) specifically translocate the ATP synthesized within mitochondria to the cytosol in exchange for the cytosolic ADP, playing a key role in energy production, in promoting cell viability and regulating mitochondrial permeability transition pore opening. In Homo sapiens four genes code for AACs with different tissue distribution and expression patterns. Since AACs are dysregulated in several cancer types, the employment of known and new AAC inhibitors might be crucial for inducing mitochondrial-mediated apoptosis in cancer cells. Albeit carboxyatractyloside (CATR) and bongkrekic acid (BKA) are known to be powerful and highly selective AAC inhibitors, able to induce mitochondrial dysfunction at molecular level and poisoning at physiological level, we estimated here for the first time their affinity for the human recombinant AAC2 by in vitro transport assays. We found that the inhibition constants of CATR and BKA are 4 nM and 2.0 µM, respectively. For finding new AAC inhibitors we also performed a docking-based virtual screening of an in-house developed chemical library and we identified about 100 ligands showing high affinity for the AAC2 binding region. By testing 13 commercially available molecules, out of the 100 predicted candidates, we found that 2 of them, namely suramin and chebulinic acid, are competitive AAC2 inhibitors with inhibition constants 0.3 µM and 2.1 µM, respectively. We also demonstrated that chebulinic acid and suramin are "highly selective" AAC2 inhibitors, since they poorly inhibit other human mitochondrial carriers (namely ORC1, APC1 and AGC1).

Overexpression in E. coli and purification of the L. pneumophila Lpp2981 protein.

The Lpp2981 gene from Legionella pneumophila, the causative agent of Legionnaire's disease, was cloned into the pMWT7 plasmid. The construct was used to express this gene in Escherichia coli. Five different bacterial strains were tested to overexpress the gene but without success. Sequence analysis revealed a cluster of four rare codons near the 5'-end of the gene. These codons were replaced with those commonly used in E. coli. The mutated Lpp2981 gene was successfully expressed in all the E. coli strains tested. The expressed protein (with an apparent molecular mass of 30 kDa) was collected in the insoluble fraction of the cell lysate, purified as inclusion bodies and functionally reconstituted into liposomes. The highest level of overexpression was obtained in E. coli C0214 after 6 h of induction with isopropyl-ß-D-thiogalactopyranoside at 37 °C, yielding 74 mg of purified protein per liter of culture. We conclude that the clustering of rare codons at the 5'-end of the open-reading frame is a critical factor for the heterologous expression of Lpp2981 in E. coli.