SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

BACKGROUND: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.

HIV-1 Low-Frequency Variants Identified in Antiretroviral-Naïve Subjects with Virologic Failure after 12 Months of Follow-Up in Panama.

Low-frequency mutations associated with drug resistance have been related to virologic failure in subjects with no history of pre-treatment and recent HIV diagnosis. In total, 78 antiretroviral treatment (ART)-naïve subjects with a recent HIV diagnosis were selected and followed by CD4+ T lymphocytes and viral load tests to detect virologic failure. We sequenced the basal samples retrospectively using next-generation sequencing (NGS), looking for low-frequency mutations that had not been detected before using the Sanger sequencing method (SSM) and describing the response to ART. Twenty-two subjects developed virologic failure (VF), and thirteen of them had at least one drug-resistance mutation associated with Reverse Transcriptase Inhibitors (RTI) and Protease Inhibitors (PIs) at frequency levels ≤ 1%, not detected previously in their basal genotyping test. No resistance mutations were observed to Integrase Strand Transfer Inhibitors (INSTIs). We identified a possible cause of VF in ART-naïve subjects with low-frequency mutations detected. To our knowledge, this is the first evaluation of pre-existing drug resistance for HIV-1 minority variants carried out on ART-naïve people living with HIV/AIDS (PLWHA) by analyzing the HIV-1 pol gene using NGS in the country.

HIV-1 Antiretroviral Drug Resistance Mutations in Treatment Naïve and Experienced Panamanian Subjects: Impact on National Use of EFV-Based Schemes.

The use of antiretroviral therapy in HIV infected subjects prevents AIDS-related illness and delayed occurrence of death. In Panama, rollout of ART started in 1999 and national coverage has reached 62.8% since then. The objective of this study was to determine the level and patterns of acquired drug resistance mutations of clinical relevance (ADR-CRM) and surveillance drug resistance mutations (SDRMs) from 717 HIV-1 pol gene sequences obtained from 467 ARV drug-experienced and 250 ARV drug-naïve HIV-1 subtypes B infected subjects during 2007-2013, respectively. The overall prevalence of SDRM and of ADR-CRM during the study period was 9.2% and 87.6%, respectively. The majority of subjects with ADR-CRM had a pattern of mutations that confer resistance to at least two classes of ARV inhibitors. The non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations K103N and P225H were more prevalent in both ARV drug-naïve and ARV drug-experienced subjects. The nucleoside reverse transcriptase inhibitor (NRTI) mutation M184V was more frequent in ARV drug-experienced individuals, while T215YFrev and M41L were more frequent in ARV drug-naïve subjects. Prevalence of mutations associated to protease inhibitors (PI) was lower than 4.1% in both types of subjects. Therefore, there is a high level of resistance (>73%) to Efavirenz/Nevirapine, Lamivudine and Azidothymidine in ARV drug-experienced subjects, and an intermediate to high level of resistance (5-10%) to Efavirenz/Nevirapine in ARV drug-naïve subjects. During the study period, we observed an increasing trend in the prevalence of ADR-CRM in subjects under first-line schemes, but not significant changes in the prevalence of SDRM. These results reinforce the paramount importance of a national surveillance system of ADR-CRM and SDRM for national management policies of subjects living with HIV.

Human immunodeficiency virus type 1 (HIV-1) subtype B epidemic in Panama is mainly driven by dissemination of country-specific clades.

The Human immunodeficiency virus type-1 (HIV-1) subtype B is the most predominant clade in Central America; but information about the evolutionary history of this virus in this geographic region is scarce. In this study, we reconstructed the spatiotemporal and population dynamics of the HIV-1 subtype B epidemic in Panama. A total of 761 HIV-1 subtype B pol sequences obtained in Panama between 2004 and 2013 were combined with subtype B pol sequences from the Americas and Europe. Maximum Likelihood phylogenetic analyses revealed that HIV-1 subtype B infections in Panama derived from the dissemination of multiple founder viruses. Most Panamanian subtype B viruses (94.5%) belong to the pandemic viral strain proposed as originated in the US, whereas others (5.5%) were intermixed among non-pandemic Caribbean strains. The bulk (76.6%) of subtype B sequences from Panama grouped within 12 country-specific clades that were not detected in other Central American countries. Bayesian coalescent-based analyses suggest that most Panamanian clades probably originated between the early 1970s and the early 1980s. The root location of major Panamanian clades was traced to the most densely populated districts of Panama province. Major Panamanian clades appear to have experienced one or two periods of exponential growth of variable duration between the 1970s and the 2000s, with median growth rates from 0.2 to 0.4 year(-1). Thus, the HIV-1 subtype B epidemic in Panama is driven by the expansion of local viral strains that were introduced from the Caribbean and other American countries at an early stage of the AIDS pandemic.

Molecular epidemiology of HIV-1 in Panama: origin of non-B subtypes in samples collected from 2007 to 2013.

Phylogenetic studies have suggested that the HIV-1 epidemic in the Americas is mainly dominated by HIV subtype B. However, countries of South America and the Caribbean have recently reported changes in their circulating HIV-1 genetic profiles. The aim of this study was to characterize the molecular profile of the HIV-1 epidemic in Panama by the analysis of 655 polymerase gene (pol) sequences that were obtained from HIV-infected Panamanians diagnosed between 1987 and 2013. Blood samples were collected from recently infected, antiretroviral drug-naïve and treatment-experienced subjects since mid-2007 to 2013. Viral RNA from plasma was extracted and sequences of HIV protease and reverse transcriptase genes were obtained. Bootscanning and phylogenetic methods were used for HIV subtyping and to trace the putative origin of non-B subtype strains. Our results showed that HIV-1 infections in Panama are dominated by subtype B (98.9%). The remaining 1.1% is represented by a diverse collection of recombinant variants including: three URFs_BC, one CRF20_BG, and one CRF28/29_BF, in addition to one subtype F1 and one subtype C, none of which were previously reported in Panama. The non-B subtype variants detected in Panama were probably introduced from Brazil (subtype F1 and CRF28/29_BF), Cuba (CRF20_BG), Dominican Republic (URFs_BC) and India (subtype C). Panama is the geographical vertex that connects the North with South America and the Caribbean through trade and cultural relations, which may explain the observed introductions of non-B subtype HIV-1 variants from both the Caribbean and South America into this Central American country.

Beginning of personalized medicine in Panama: Molecular and pathological characteristics of gastrointestinal stromal tumors from archival paraffin-embedded tissue.

This is the first study from Central America to analyze genetic mutations and histopathological features associated with gastrointestinal stromal tumors (GIST). Mutations found in the tyrosine kinase membrane receptors c-kit and pdgfra are associated with clinical and pathological characteristics of GIST. New drugs that inhibit the expression of these oncogenes at the molecular level substantially improve the quality of life for patients with this tumor. It is therefore essential for patient care in Panama that genetic analysis of GIST tumors continues to develop from the pilot study presented herein into routine clinical use. This study evaluated 39 cases of GIST in Panama, using samples archived at the Instituto Oncológico Nacional from 1994 to 2004. DNA from paraffin­embedded tumor tissues was isolated and amplified for the exons of c-kit and pdgfra associated with a high frequency of mutations. Direct PCR sequencing of specific exons was performed, and those with different alleles were cloned and re-sequenced. Amino acid sequences were inferred from DNA and aligned to Genbank reference sequences to determine the position and type of mutation. The highest frequency of mutations was found in exon 11 of the c-kit gene (70%). Mutations found in this exon were heterogeneous, while only one type of mutation (p.A502_Y503dup) was observed in c-kit exon 9. Mutations in the pdgfra gene constituted several substitutions, with the deletion p.D842V being observed most frequently. The observed GIST-associated mutations were previously described. Four patients with mutations associated with familial GIST were also found. The majority (66%) of patients with mutations in exon 11 (residues 550-591) were considered to be at high risk and 75% of patients with mutations specifically within residues 556-560 (exon 11) were considered to have high-risk GIST. This is the first molecular study of GIST in Central America. It was performed to gain a better understanding of the cancer-associated mutations of KIT and platelet­derived growth factor receptor α (PDGFRA) receptors. This may aid in the prediction of clinical evolution and guide the use of specific drug treatments in patients with GIST in Panama.