Molecular mechanisms behind the generation of pro-oncogenic HIV-1 matrix protein p17 variants.

HIV-1 matrix protein p17 variants (vp17s), characterized by amino acid insertions at the COOH-terminal region of the viral protein, have been recently identified and studied for their biological activity. Different from their wild-type counterpart (refp17), vp17s display a potent B cell growth and clonogenic activity. Recent data have highlighted the higher prevalence of vp17s in people living with HIV-1 (PLWH) with lymphoma compared with those without lymphoma, suggesting that vp17s may play a key role in lymphomagenesis. Molecular mechanisms involved in vp17 development are still unknown. Here we assessed the efficiency of HIV-1 Reverse Transcriptase (RT) in processing this genomic region and highlighted the existence of hot spots of mutation in Gag, at the end of the matrix protein and close to the matrix-capsid junction. This is possibly due to the presence of inverted repeats and palindromic sequences together with a high content of Adenine in the 322-342 nucleotide portion, which constrain HIV-1 RT to pause on the template. To define the recombinogenic properties of hot spots of mutation in the matrix gene, we developed plasmid vectors expressing Gag and a minimally modified Gag variant, and measured homologous recombination following cell co-nucleofection by next-generation sequencing. Data obtained allowed us to show that a wide range of recombination events occur in concomitance with the identified hot spots of mutation and that imperfect events may account for vp17s generation.

Detection of HIV-1 matrix protein p17 in sera of viremic and aviremic patients.

People living with human immunodeficiency virus type 1 (HIV-1), even if successfully treated with a combined antiretroviral therapy, display a persistent inflammation and chronic immune activation, and an increasing risk of developing cardiovascular and thrombotic events, cancers, and neurologic disorders. Accumulating evidence reveals that biologically active HIV-1 proteins may play a role in the development of these HIV-1-associated conditions. The HIV-1 matrix protein p17 (p17) is released and accumulates in different organs and tissue where it may exert multiple biological activities on different target cells. To assess a role of p17 in different HIV-1-related pathological processes, it is central to definitively ascertain and quantitate its expression in a large number of sera obtained from HIV-1-infected (HIV-1+) patients. To this aim, we developed a specific and highly sensitive p17 capture immunoenzymatic assay. Data obtained highlight a heterogeneous expression of p17 in blood of tested patients, with patients who were negative or displayed from low to relatively high p17 blood concentrations (range from 0.05 to 7.29 nM). Moreover, we found that blood p17 concentration was totally independent from the viremic status of the patient. This finding calls for monitoring HIV-1+ patients in order to evaluate a possible correlation between p17 amount in blood and the likelihood of developing HIV-1-related pathological conditions.

Germline NUP98 Variants in Two Siblings with a Rothmund-Thomson-Like Spectrum: Protein Functional Changes Predicted by Molecular Modeling.

Two adult siblings born to first-cousin parents presented a clinical phenotype reminiscent of Rothmund-Thomson syndrome (RTS), implying fragile hair, absent eyelashes/eyebrows, bilateral cataracts, mottled pigmentation, dental decay, hypogonadism, and osteoporosis. As the clinical suspicion was not supported by the sequencing of RECQL4, the RTS2-causative gene, whole exome sequencing was applied and disclosed the homozygous variants c.83G>A (p.Gly28Asp) and c.2624A>C (p.Glu875Ala) in the nucleoporin 98 (NUP98) gene. Though both variants affect highly conserved amino acids, the c.83G>A looked more intriguing due to its higher pathogenicity score and location of the replaced amino acid between phenylalanine-glycine (FG) repeats within the first NUP98 intrinsically disordered region. Molecular modeling studies of the mutated NUP98 FG domain evidenced a dispersion of the intramolecular cohesion elements and a more elongated conformational state compared to the wild type. This different dynamic behavior may affect the NUP98 functions as the minor plasticity of the mutated FG domain undermines its role as a multi-docking station for RNA and proteins, and the impaired folding can lead to the weakening or the loss of specific interactions. The clinical overlap of NUP98-mutated and RTS2/RTS1 patients, accounted by converging dysregulated gene networks, supports this first-described constitutional NUP98 disorder, expanding the well-known role of NUP98 in cancer.

Virtual Drug Repositioning as a Tool to Identify Natural Small Molecules That Synergize with Lumacaftor in F508del-CFTR Binding and Rescuing.

Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.

In silico drug repositioning on F508del-CFTR: A proof-of-concept study on the AIFA library.

Computational drug repositioning is of growing interest to academia and industry, for its ability to rapidly screen a huge number of candidates in silico (exploiting comprehensive drug datasets) together with reduced development cost and time. The potential of drug repositioning has not been fully evaluated yet for cystic fibrosis (CF), a disease mainly caused by deletion of Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. F508del-CFTR is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. CF is still a fatal disease. Nowadays, it is treatable by some CFTR-rescuing drugs, but new-generation drugs with stronger therapeutic benefits and fewer side effects are still awaited. In this manuscript we report about the results of a pilot computational drug repositioning screening in search of F508del-CFTR-targeted drugs performed on AIFA library by means of a dedicated computational pipeline and surface plasmon resonance binding assay to experimentally validate the computational findings.

Evolution toward beta common chain receptor usage links the matrix proteins of HIV-1 and its ancestors to human erythropoietin.

The HIV-1 matrix protein p17 (p17) is a pleiotropic molecule impacting on different cell types. Its interaction with many cellular proteins underlines the importance of the viral protein as a major determinant of human specific adaptation. We previously showed the proangiogenic capability of p17. Here, by integrating functional analysis and receptor binding, we identify a functional epitope that displays molecular mimicry with human erythropoietin (EPO) and promotes angiogenesis through common beta chain receptor (ßCR) activation. The functional EPO-like epitope was found to be present in the matrix protein of HIV-1 ancestors SIV originated in chimpanzees (SIVcpz) and gorillas (SIVgor) but not in that of HIV-2 and its ancestor SIVsmm from sooty mangabeys. According to biological data, evolution of the EPO-like epitope showed a clear differentiation between HIV-1/SIVcpz-gor and HIV-2/SIVsmm branches, thus highlighting this epitope on p17 as a divergent signature discriminating HIV-1 and HIV-2 ancestors. P17 is known to enhance HIV-1 replication. Similarly to other ßCR ligands, p17 is capable of attracting and activating HIV-1 target cells and promoting a proinflammatory microenvironment. Thus, it is tempting to speculate that acquisition of an epitope on the matrix proteins of HIV-1 ancestors capable of triggering ßCR may have represented a critical step to enhance viral aggressiveness and early human-to-human SIVcpz/gor dissemination. The hypothesis that the p17/ßCR interaction and ßCR abnormal stimulation may also play a role in sustaining chronic activation and inflammation, thus marking the difference between HIV-1 and HIV-2 in term of pathogenicity, needs further investigation.

Recent Strategic Advances in CFTR Drug Discovery: An Overview.

Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transformed cystic fibrosis (CF) from a fatal disease to a treatable chronic condition. However, new-generation drugs able to bind CFTR with higher specificity/affinity and to exert stronger therapeutic benefits and fewer side effects are still awaited. Computational methods and biosensors have become indispensable tools in the process of drug discovery for many important human pathologies. Instead, they have been used only piecemeal in CF so far, calling for their appropriate integration with well-tried CF biochemical and cell-based models to speed up the discovery of new CFTR-rescuing drugs. This review will give an overview of the available structures and computational models of CFTR and of the biosensors, biochemical and cell-based assays already used in CF-oriented studies. It will also give the reader some insights about how to integrate these tools as to improve the efficiency of the drug discovery process targeted to CFTR.

Heparin and heparan sulfate proteoglycans promote HIV-1 p17 matrix protein oligomerization: computational, biochemical and biological implications.

p17 matrix protein released by HIV+ cells interacts with leukocytes heparan sulfate proteoglycans (HSPGs), CXCR1 and CXCR2 exerting different cytokine-like activities that contribute to AIDS pathogenesis. Since the bioactive form of several cytokines is represented by dimers/oligomers and oligomerization is promoted by binding to heparin or HSPGs, here we evaluated if heparin/HSPGs also promote p17 oligomerization. Heparin favours p17 dimer, trimer and tetramer assembly, in a time- and biphasic dose-dependent way. Heparin-induced p17 oligomerization is of electrostatic nature, being it prevented by NaCl, by removing negative sulfated groups of heparin and by neutralizing positive lysine residues in the p17 N-terminus. A new computational protocol has been implemented to study heparin chains up to 24-mer accommodating a p17 dimer. Molecular dynamics show that, in the presence of heparin, two p17 molecules undergo conformational modifications creating a continuous "electropositive channel" in which heparin sulfated groups interact with p17 basic amino acids, promoting its dimerization. At the cell surface, HSPGs induce p17 oligomerization, as demonstrated by using B-lymphoblastoid Namalwa cells overexpressing the HSPG Syndecan-1. Also, HSPGs on the surface of BJAB and Raji human B-lymphoblastoid cells are required to p17 to induce ERK1/2 activation, suggesting that HS-induced oligomerization plays a role in p17-induced lymphoid dysregulation during AIDS.