Characterization and functional analysis of cis-acting elements of the human farnesyl diphosphate synthetase (FDPS) gene 5′ flanking region

AbstractFarnesyl diphosphate synthetase (FDPS) is a key enzyme in the isoprenoid pathway responsible for cholesterol biosynthesis, post-translational protein modifications and synthesis of steroid hormones, whose expression is regulated by phorbol esters and polyunsaturated fatty acids.Genomic comparison of the 5′ upstream sequence of the FDPS genes identifies conserved binding sites for NF-Y, SP1, SRE3, and YY1 regulatory elements in rat, mouse, dog and chimpanzee. Two additional specific consensus sequences, upstream of the core promoter that had not been analysed previously, are shared only by human and chimpanzee genomes.The work presented here aimed at characterizing these genomic sequence elements in the human FDPS promoter region and their contribution to gene expression. We have characterized functionally the minimal basal promoter of the human FDPS gene by means of deletion mutants and we have identified two cis-acting elements which modulate the FDPS gene expression and are recognized by Pax5 and OCT-1 transcription factors

Introductory Chapter: Malaria in 2022 – Promises and Unmet Needs

In this chapter were described: biological cycle of Plasmodia and diagnostic assays for malaria: microscopic examination, Rapid Diagnostic Test (RDT) based on antigen, Indirect Fluorescent Antibody Test (IFA test), and also, recently introduced and accepted, the nucleic-acid detection method by PCR or LAMP technologie

IKKepsilon involvement in Tax-mediated activation of INF pathway

HTLV-1 Tax de-regulates several cellular signaling pathways leading to cell transformation by altering gene expression, intracellular protein distribution and cell proliferation. Tax-1 induces persistent activation of several transcriptional factors and signal transduction pathways, including NF-\u3baB and CREB/ATF. It is known that Tax-1 constitutively activates TAK1 (transforming growth factor-\u3b2-activated kinase 1) and modifies the interferon (INF) regulatory signals by controlling the expression of INF transcription factors 3 (INF3) and INF4. We have recently reported that HTLV-1 and HTLV-2 Tax proteins interact with TAK1-binding protein 2 (TAB2) of the NF-\u3baB pathway and that both Tax proteins transactivate NF-\u3baB promoters [1]. TAB2 functions as an adaptor protein to recruit TAK1 to TRAF2 (TNF-\u3b1 receptor-associated factor) in TNF-\u3b1 signaling pathways. In the present study we have investigated Tax-1 and Tax-2 role in modifying INF and NF-\u3baB activation through the recruitment of IKKepsilon, an I\u3baB kinase homologue involved in NF-\u3baB and INF3 signaling pathways. By co-immunoprecipitation experiments, we have found that both IKKepsilon and Tax-1, but not Tax-2, are present in protein complexes in transfected cells. IKKepsilon and Tax-1 or Tax-2 role in the activation of INF responsive elements or NF-\u3baB containing promoters have been analyzed after transfecting the protein genes in 293T cells and measuring the effect by luciferase assay. Co-expression of Tax-1 and IKKepsilon resulted in an increased IRF activation mediated by IKKepsilon. Interaction of IKKepsilon with Tax-1 and Tax-2 and their possible effects in the de-regulation of the IRF3 pathways will be discussed

Comparison of Tax-1 and Tax-2B post-translational modifications using specific lysine mutants in relation to activation of NF-κB and intracellular localization

ost-translational modifications of HTLV-1 and HTLV-2 Tax-1 and Tax-2 proteins have been shown to play a critical role in their cellular localization, transactivation and protein interactions. Five of ten lysine residues were found to be major targets for Tax-1 modifications: Lys189(K4); Lys197(K5), Lys263(K6), Lys280(K7) and Lys284(K8), are essential for ubiquitination, while sumoylation takes place on Lys280 (K7) and Lys284(K8). Tax-2 contains four additional lysine residues, namely at position Lys100(K2i), Lys149(K3i), Lys185(K3ii), and Lys356(K10i).Very few studies have been so far performed on Tax-2 lysine mutants. We have previously demonstrated that Tax-2B is ubiquitinated and sumoylated similarly to Tax-1. To identify the Tax-2 lysine residues which are directly involved in post-translational modifications, we have constructed a series of Tax-2B mutants with substitutions of lysine (K) residues by arginines (R) and analyzed them for NF-kB and CREB/ATF transactivation, intracellular distribution and extent of ubiquitination and sumoylation. We have found that Tax-2 K7-8R mutant, contrary to its Tax-1 homologue, is only partially affected in its capacity to transactivate NF-\u3baB pathway, is regularly sumoylated and presents formation of nuclear bodies by confocal analysis. However, Tax-2 mutants with extended (K3ii-8R) and/or total (K1-10iR) mutation rate were severely affected for NF-kB transactivation and sumoylation. By comparing Tax-2 WT with mutants K7-8R and K3ii-8R, we observed that the reduction of NF-\u3baB activity is correlated to a parallel decrease in sumoylation. These results suggest that the target for Tax-2 ubiquitination and sumoylation differs from that described for Tax-1

Comparison of the Genetic Organization, Expression Strategies and Oncogenic Potential of HTLV-1 and HTLV-2

Human T cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) are genetically related complex retroviruses that are capableof immortalizing human T-cells in vitro and establish life-long persistent infections in vivo. In spite of these apparent similarities,HTLV-1 and HTLV-2 exhibit a significantly different pathogenic potential. HTLV-1 is recognized as the causative agent of adult Tcell eukemia/lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associatedmyelopathy (TSP/HAM). In contrast,HTLV- 2 has not been causally linked to human malignancy, although it may increase the risk of developing inflammatory neuropathies and infectious diseases. The present paper is focused on the studies aimed at defining the viral genetic determinants of the pathobiology of HTLV-1 and HTLV-2 through a comparison of the expression strategies and functional properties of the different gene products of the two viruses

Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein.

BACKGROUND:Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway.RESULTS:The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1.CONCLUSIONS:Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway

Molecular characterization of HIV-1 Nef and ACOT8 interaction: insights from in silico structural predictions and in vitro functional assays

HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identi cation of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immuno uorescence analyses showed that ACOT8 Arg45-Phe55 and Arg86-Pro93 regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys91 plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These ndings improve the comprehensionof the association between HIV-1 Nef and ACOT8, helping elucidating the biological e ect of their interaction

M48U1 and Tenofovir combination synergistically inhibits HIV infection in activated PBMCs and human cervicovaginal histocultures

Microbicides are considered a promising strategy for preventing human immunodeficiency virus (HIV-1) transmission and disease. In this report, we first analyzed the antiviral activity of the miniCD4 M48U1 peptide formulated in hydroxyethylcellulose (HEC) hydrogel in activated peripheral blood mononuclear cells (PBMCs) infected with R5-and X4-Tropic HIV-1 strains. The results demonstrate that M48U1 prevented infection by several HIV-1 strains including laboratory strains, and HIV-1 subtype B and C strains isolated from the activated PBMCs of patients. M48U1 also inhibited infection by two HIV-1 transmitted/founder infectious molecular clones (pREJO.c/2864 and pTHRO.c/2626). In addition, M48U1 was administered in association with tenofovir, and these two antiretroviral drugs synergistically inhibited HIV-1 infection. In the next series of experiments, we tested M48U1 alone or in combination with tenofovir in HEC hydrogel with an organ-like structure mimicking human cervicovaginal tissue. We demonstrated a strong antiviral effect in absence of significant tissue toxicity. Together, these results indicate that co-Treatment with M48U1 plus tenofovir is an effective antiviral strategy that may be used as a new topical microbicide to prevent HIV-1 transmission

Evaluation of saliva and nasopharyngeal swab sampling for genomic detection of SARS-CoV-2 in children accessing a pediatric emergency department during the second pandemic wave

SARS-CoV-2 infection is mainly detected by multiplex real-time RT-PCR from upper respiratory specimens, which is considered the gold-standard technique for SARS-CoV-2 infection diagnosis. A nasopharyngeal (NP) swab represents the clinical sample of choice, but NP swabbing can be uncomfortable to the patients, especially for pediatric-age participants, requires trained healthcare personnel, and may generate an aerosol, increasing the intrinsic exposure risk of healthcare workers. The objective of this study was to compare paired NP and saliva samples (SS) collected from pediatric patients to evaluate whether the saliva collection procedure may be considered a valuable alternative to the classical NP swab (NPS) sampling in children. In this study, we describe a SARS-CoV-2 multiplex real-time RT-PCR protocol for SS, comparing the results with the paired NPS specimens from 256 pediatric patients (mean age 4.24 ± 4.40 years) admitted to the hospital emergency room of Azienda Ospedaliera Universitaria Integrata (AOUI), Verona, and randomly enrolled between September 2020 and December 2020. The saliva sampling demonstrated consistent results when compared to NPS use. The SARS-CoV-2 genome was detected in 16 out of 256 (6.25%) NP samples, among which 13 (5.07%) were positive even when paired SS were analyzed. Moreover, SARS-CoV-2-negative NPS and SS were consistent, and the overall concordances between NPS and SS were detected in 253 out of 256 samples (98.83%). Our results suggest that saliva samples may be considered a valuable alternative to NPS for SARS-CoV-2 direct diagnosis with multiplex real-time RT-PCR in pediatric patients