Biology subjects

Negro, A.

Publications and source records attributed to Negro, A..

4 recordsLinked to original sources

bioRxiv2022

A detailed landscape of genomic alterations in malignant peripheral nerve sheath tumor cell lines challenges the current MPNST diagnosis

BackgroundMalignant peripheral nerve sheath tumors (MPNSTs) are soft tissue sarcomas that arise from the peripheral nervous system. Half of the tumors develop in the context of the genetic disease Neurofibromatosis type 1 (NF1) and the rest are sporadic sarcomas. MPNSTs have a dismal prognosis due to their aggressiveness and tendency to metastasize, and new treatment options are needed. The diagnosis of MPNSTs can be challenging, especially outside of the NF1 context since specific histological criteria have not been completely established. Genomic analysis may both facilitate differential diagnoses and suggest precision medicine strategies. MethodsWe generated a complete genomic resource of a set of widely used human NF1-related and sporadic MPNST cell lines by applying ploidy analysis, whole genome and whole exome sequencing and SNP-array analysis, complemented by methylome-based classification and immunofluorescence of cell identity markers (SOX9, SOX10, S100B). ResultsNF1 MPNST cell lines faithfully recapitulated the genomic copy number profile of primary MPNSTs. Structural variants were key players in the complete inactivation of most recurrently altered tumor suppressor genes (TSGs) (NF1, CDKN2A, SUZ12/EED), while small variants played a minor role in the NF1 context, both concerning TSG inactivation and due to the absence of gain-of-function mutations. In clear contrast, the sporadic cell lines (STS-26T, HS-Sch-2, HS-PSS) did not recapitulate the copy number profile of primary MPNSTs. They carried different TSG inactivation and exhibited gain-of-function mutations by predicted kinase activation or generation of fusion genes. Mutational frequencies and signatures emerged as promising informative tools for aiding in MPNST differential diagnosis. Due to the multiple genomic differences exhibited, we complemented their characterization using a methylome-based classifier. All NF1-related cell lines were assigned within the MPNST group, while sporadic cell lines clustered either with melanomas or with an uncertain MPNST-like sarcoma group. The staining of cell identity markers reinforced the idea of a potential misdiagnose of the MPNSTs used to derive the sporadic cell lines analyzed. ConclusionsDeep genomic analysis, together with methylome-based sarcoma classification and cell identity marker analysis, challenged the MPNST identity of sporadic cell lines. Results presented here open an opportunity to revise MPNST differential diagnosis and classification.

genomics↗

bioRxiv2022

Antisense oligonucleotides targeting exon 11 are able to partially rescue the Neurofibromatosis Type 2 phenotype in vitro

Neurofibromatosis type 2 (NF2) is an autosomal dominant condition caused by loss of function variants in the NF2 gene, which codes for the protein Merlin, and characterized by the development of multiple tumours of the nervous system. The clinical presentation of the disease is variable and related to the type of the inherited germline variant. Here, we tested if PMOs could be used to correct the splice signalling caused by variants at +/-13 within the intron-exon boundary region. Here we show that the PMOs designed for these variants do not constitute a therapeutic approach. Furthermore, we evaluated the use of phosphorodiamidate morpholino oligomers (PMOs) to reduce the severity of the effects of NF2 truncating variants with the aim of generating milder hypomorphic isoforms in vitro through the induction of the in-frame deletion of the exon-carrying variant. We were able to specifically induce the skipping of exons 4, 8 and 11 maintaining the NF2 gene reading frame at cDNA level. Only the skipping of exon 11 produced a hypomorphic Merlin (Merlin-e11), able to partially rescue the observed phenotype in primary fibroblast cultures from NF2 patients, being encouraging for the treatment of patients harbouring truncating variants located in exon 11.

genetics↗

bioRxiv2021

TRAP1 and cyclophilin D compete at OSCP subunit to regulate enzymatic activity and permeability transition pore opening by F-ATP synthase

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes cell bioenergetic and proteostatic adaptations, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Moreover, TRAP1 inhibits opening of the permeability transition pore (PTP) formed by F-ATP synthase and effectively antagonizes the PTP-inducing effect of CyPD, which elicits mitochondrial depolarization and cell death. Consistently, electrophysiological measurements indicate that TRAP1 and CyPD compete in the modulation of channel activity of purified F-ATP synthase, resulting in PTP inhibition and activation, respectively, and outcompeting each other effect on the channel. Moreover, TRAP1 counteracts PTP induction by CyPD, whereas CyPD reverses TRAP1-mediated PTP inhibition. Our data identify TRAP1 as a F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

cell biology↗

bioRxiv2021

Human -Synuclein Inhibits Platelets Aggregation in vitro by Interfering with the -Thrombin/Protease-Activated Receptor 1 Functional Axis

-Synuclein (Syn) is a small (140 amino acids) disordered, acidic (pI: 4.7) protein, highly conserved in vertebrates and implicated in the pathogenesis of Parkinsons disease (PD), a neurodegenerative disease characterized by the deposition of Syn amyloid fibrils in dopaminergic neurons. Beyond the central nervous system, significant expression of Syn has also been measured in the blood (~1 M), where platelets are the main cellular hosts of Syn. Although the pathological implication of Syn in PD is widely accepted, the physiological role of blood Syn is still elusive. Starting from the notion that platelets are either the major cellular reservoir of Syn in the blood and, concomitantly, act as key players in hemostasis, being activated also by -thrombin (T) via cleavage of protease-activated receptors (PARs), we decided to investigate the possibility that Syn could modulate platelet activation by interfering with the T-PAR functional axis. Using multiple electrode aggregometry, i.e. a fast and specific platelet-function-testing method, as well as steady-state fluorescence spectroscopy, surface plasmon resonance, and fluorescence microscopy, we show here that monomeric Syn functions as a negative regulator of T-mediated platelets activation. Syn acts either directly, via competitive inhibition of PAR1 activation by T and TRAP6 agonist, and indirectly, by scavenging T on the platelet plasma membrane. A simple electrostatic model of Syn platelet antiaggregating effect is proposed and the possible role of the protein at the interplay of amyloidosis and thrombosis is discussed.

biochemistry↗