1,3,8-Triazaspiro[4.5]decane Derivatives Inhibit Permeability Transition Pores through a FO-ATP Synthase c Subunit Glu119-Independent Mechanism That Prevents Oligomycin A-Related Side Effects.

Permeability transition pore (PTP) molecular composition and activity modulation have been a matter of research for several years, especially due to their importance in ischemia reperfusion injury (IRI). Notably, c subunit of ATP synthase (Csub) has been identified as one of the PTP-forming proteins and as a target for cardioprotection. Oligomycin A is a well-known Csub interactor that has been chemically modified in-depth for proposed new pharmacological approaches against cardiac reperfusion injury. Indeed, by taking advantage of its scaffold and through focused chemical improvements, innovative Csub-dependent PTP inhibitors (1,3,8-Triazaspiro[4.5]decane) have been synthetized in the past. Interestingly, four critical amino acids have been found to be involved in Oligomycin A-Csub binding in yeast. However, their position on the human sequence is unknown, as is their function in PTP inhibition. The aims of this study are to (i) identify for the first time the topologically equivalent residues in the human Csub sequence; (ii) provide their in vitro validation in Oligomycin A-mediated PTP inhibition and (iii) understand their relevance in the binding of 1,3,8-Triazaspiro[4.5]decane small molecules, as Oligomycin A derivatives, in order to provide insights into Csub interactions. Notably, in this study we demonstrated that 1,3,8-Triazaspiro[4.5]decane derivatives inhibit permeability transition pores through a FO-ATP synthase c subunit Glu119-independent mechanism that prevents Oligomycin A-related side effects.

Translation termination codons in protein synthesis and disease.

Fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense as well as stop codons (UGA, UAG, UAA), which are usually localized at the 3' of mRNA and drive the release of the polypeptide chain. However, either natural (NTCs) or premature (PTCs) termination codons, the latter arising from nucleotide changes, can undergo a recoding process named ribosome or translational readthrough, which insert specific amino acids (NTCs) or subset(s) depending on the stop codon type (PTCs). This process is particularly relevant for nonsense mutations, a relatively frequent cause of genetic disorders, which impair gene expression at different levels by potentially leading to mRNA degradation and/or synthesis of truncated proteins. As a matter of fact, many efforts have been made to develop efficient and safe readthrough-inducing compounds, which have been challenged in several models of human disease to provide with a therapy. In this view, the dissection of the molecular determinants shaping the outcome of readthrough, namely nucleotide and protein contexts as well as their interplay and impact on protein structure/function, is crucial to identify responsive nonsense mutations resulting in functional full-length proteins. The interpretation of experimental and mechanistic findings is also important to define a possibly clear picture of potential readthrough-favorable features useful to achieve rescue profiles compatible with therapeutic thresholds typical of each targeted disorder, which is of primary importance for the potential translatability of readthrough into a personalized and mutation-specific, and thus patient-oriented, therapeutic strategy.

An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency.

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5' splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

Translational readthrough of GLA nonsense mutations suggests dominant-negative effects exerted by the interaction of wild-type and missense variants.

Nonsense mutations are relatively frequent in the rare X-linked lysosomal α-galactosidase A (α-Gal) deficiency (Fabry disease; FD), but have been poorly investigated. Here, we evaluated the responsiveness of a wide panel (n = 14) of GLA premature termination codons (PTCs) to the RNA-based approach of drug-induced readthrough through expression of recombinant α-Gal (rGal) nonsense and missense variants.We identified four high-responders to the readthrough-inducing aminoglycoside G418 in terms of full-length protein (C56X/W209X, ≥10% of wild-type rGal) and/or activity (Q119X/W209X/Q321X, ~5-7%), resulting in normal (Q119X/Q321X) or reduced (C56X, 0.27 ± 0.11; W209X, 0.35 ± 0.1) specific activity.To provide mechanistic insights we investigated the predicted amino acid substitutions mediated by readthrough (W209C/R, C56W/R), which resulted in correct lysosomal localization and appreciable protein/activity levels for the W209C/R variants. Differently, the C56W/R variants, albeit appreciably produced and localized into lysosomes, were inactive, thus indicating detrimental effects of substitutions at this position.Noticeably, when co-expressed with the functional W209C or W209R variants, the wild-type rGal displayed a reduced specific activity (0.5 ± 0.2 and 0.6 ± 0.2, respectively) that, considering the dimeric features of the α-Gal enzyme, suggested dominant-negative effects of missense variants through their interaction with the wild-type.Overall, we provide a novel mechanism through which amino acids inserted during readthrough might impact on the functional protein output. Our findings may also have implications for the interpretation of pathological phenotypes in heterozygous FD females, and for other human disorders involving dimeric or oligomeric proteins.

Tailoring the CRISPR system to transactivate coagulation gene promoters in normal and mutated contexts.

Engineered transcription factors (TF) have expanded our ability to modulate gene expression and hold great promise as bio-therapeutics. The first-generation TF, based on Zinc Fingers or Transcription-Activator-like Effectors (TALE), required complex and time-consuming assembly protocols, and were indeed replaced in recent years by the CRISPR activation (CRISPRa) technology. Here, with coagulation F7/F8 gene promoters as models, we exploited a CRISPRa system based on deactivated (d)Cas9, fused with a transcriptional activator (VPR), which is driven to its target by a single guide (sg)RNA. Reporter gene assays in hepatoma cells identified a sgRNA (sgRNAF7.5) triggering a ~35-fold increase in the activity of F7 promoter, either wild-type, or defective due to the c.-61T>G mutation. The effect was higher (~15-fold) than that of an engineered TALE-TF (TF4) targeting the same promoter region. Noticeably, when challenged on the endogenous F7 gene, the dCas9-VPR/sgRNAF7.5 combination was more efficient (~6.5-fold) in promoting factor VII (FVII) protein secretion/activity than TF4 (~3.8-fold). The approach was translated to the promoter of F8, whose reduced expression causes hemophilia A. Reporter gene assays in hepatic and endothelial cells identified sgRNAs that, respectively, appreciably increased F8 promoter activity (sgRNAF8.1, ~8-fold and 3-fold; sgRNAF8.2, ~19-fold and 2-fold) with synergistic effects (~38-fold and 2.7-fold). Since modest increases in F7/F8 expression would ameliorate patients' phenotype, the CRISPRa-mediated transactivation extent might approach the low therapeutic threshold. Through this pioneer study we demonstrated that the CRISPRa system is easily tailorable to increase expression, or rescue disease-causing mutations, of different promoters, with potential intriguing implications for human disease models.

Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth.

Although mitochondria play a multifunctional role in cancer progression and Ca2+ signaling is remodeled in a wide variety of tumors, the underlying mechanisms that link mitochondrial Ca2+ homeostasis with malignant tumor formation and growth remain elusive. Here, we show that phosphorylation at the N-terminal region of the mitochondrial calcium uniporter (MCU) regulatory subunit MICU1 leads to a notable increase in the basal mitochondrial Ca2+ levels. A pool of active Akt in the mitochondria is responsible for MICU1 phosphorylation, and mitochondrion-targeted Akt strongly regulates the mitochondrial Ca2+ content. The Akt-mediated phosphorylation impairs MICU1 processing and stability, culminating in reactive oxygen species (ROS) production and tumor progression. Thus, our data reveal the crucial role of the Akt-MICU1 axis in cancer and underscore the strategic importance of the association between aberrant mitochondrial Ca2+ levels and tumor development.

Secretion of wild-type factor IX upon readthrough over F9 pre-peptide nonsense mutations causing hemophilia B.

Pre-peptide regions of secreted proteins display wide sequence variability, even among highly homologous proteins such as coagulation factors, and are intracellularly removed, thus potentially favoring secretion of wild-type proteins upon suppression of nonsense mutations (translational readthrough). As models we selected F9 nonsense mutations with readthrough-favorable features affecting the pre-peptide and pro-peptide regions of coagulation factor IX (FIX), which cause hemophilia B (HB). Only the p.Gly21Ter (c.61G > T) in the variable pre-peptide hydrophobic core significantly responded (secretion, 4.1 ± 0.5% of wild-type; coagulant activity, 4.0 ± 0.3%) to the readthrough-inducer geneticin. Strikingly, for the p.Gly21Ter mutation, the resulting specific coagulant activity (0.96 ± 0.11) was compatible with normal function, thus suggesting secretion of FIX with wild-type features upon readthrough and removal of pre-peptide. Expression of the predicted readthrough-deriving missense variants (Gly21Trp/Cys/Arg) revealed a preserved specific activity (ranging from 0.84 to 0.98), thus supporting our observation. Conversely, rescue of the p.Cys28Ter (c.84T > A) and p.Lys45Ter (c.133A > T) was prevented by constraints of adjacent cleavage sites, a finding consistent with the association of most missense mutations affecting these regions with severe or moderate HB. Overall, our data indicate that suppression of nonsense mutations in the pre-peptide core preserves mature protein features, thus making this class of mutations preferred candidates for therapeutic readthrough.

Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity.

Dissection of pleiotropic effects of missense mutations, rarely investigated in inherited diseases, is fundamental to understanding genotype-phenotype relationships. Missense mutations might impair mRNA processing in addition to protein properties. As a model for hemophilia A, we investigated the highly prevalent F8 c.6046c>t/p.R2016W (exon 19) mutation. In expression studies exploiting lentiviral vectors, we demonstrated that the amino acid change impairs both Factor VIII (FVIII) secretion (antigen 11.0±0.4% of wild-type) and activity (6.0±2.9%). Investigations in patients' ectopic F8 mRNA and with minigenes showed that the corresponding nucleotide change also decreases correct splicing to 70±5%, which is predicted to lower further FVIII activity (4.2±2%), consistently with patients' levels (<1-5%). Masking the mutated exon 19 region by antisense U7snRNA supported the presence of a splicing regulatory element, potentially affected by several missense mutations causing hemophilia A. Among these, the c.6037g>a (p.G2013R) reduced exon inclusion to 41±3% and the c.6053a>g (p.E2018G) to 28±2%, similarly to a variant affecting the 5' splice site (c.6113a>g, p.N2038S, 26±2%), which displayed normal protein features upon recombinant expression. The p.G2013R reduced both antigen (7.0±0.9%) and activity (8.4±0.8%), while the p.E2018G produced a dysfunctional molecule (antigen: 69.0±18.1%; activity: 19.4±2.3%). In conclusion, differentially altered mRNA and protein patterns produce a gradient of residual activity, and clarify genotype-phenotype relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Taken together we provide a clear example of interplay between mRNA and protein mechanisms of disease that operate in shaping many other inherited disorders.

An engineered tale-transcription factor rescues transcription of factor VII impaired by promoter mutations and enhances its endogenous expression in hepatocytes.

Tailored approaches to restore defective transcription responsible for severe diseases have been poorly explored. We tested transcription activator-like effectors fused to an activation domain (TALE-TFs) in a coagulation factor VII (FVII) deficiency model. In this model, the deficiency is caused by the -94C > G or -61T > G mutation, which abrogate the binding of Sp1 or HNF-4 transcription factors. Reporter assays in hepatoma HepG2 cells naturally expressing FVII identified a single TALE-TF (TF4) that, by targeting the region between mutations, specifically trans-activated both the variant (>100-fold) and wild-type (20-40-fold) F7 promoters. Importantly, in the genomic context of transfected HepG2 and transduced primary hepatocytes, TF4 increased F7 mRNA and protein levels (2- to 3-fold) without detectable off-target effects, even for the homologous F10 gene. The ectopic F7 expression in renal HEK293 cells was modestly affected by TF4 or by TALE-TF combinations. These results provide experimental evidence for TALE-TFs as gene-specific tools useful to counteract disease-causing promoter mutations.