Structured Tandem Repeats in Protein Interactions.

Tandem repeats (TRs) in protein sequences are consecutive, highly similar sequence motifs. Some types of TRs fold into structural units that pack together in ensembles, forming either an (open) elongated domain or a (closed) propeller, where the last unit of the ensemble packs against the first one. Here, we examine TR proteins (TRPs) to see how their sequence, structure, and evolutionary properties favor them for a function as mediators of protein interactions. Our observations suggest that TRPs bind other proteins using large, structured surfaces like globular domains; in particular, open-structured TR ensembles are favored by flexible termini and the possibility to tightly coil against their targets. While, intuitively, open ensembles of TRs seem prone to evolve due to their potential to accommodate insertions and deletions of units, these evolutionary events are unexpectedly rare, suggesting that they are advantageous for the emergence of the ancestral sequence but are early fixed. We hypothesize that their flexibility makes it easier for further proteins to adapt to interact with them, which would explain their large number of protein interactions. We provide insight into the properties of open TR ensembles, which make them scaffolds for alternative protein complexes to organize genes, RNA and proteins.

PluriBAC: A Versatile Baculovirus-Based Modular System to Express Heterologous Genes in Different Biotechnological Platforms.

Baculoviruses are insect-specific pathogens widely used in biotechnology. In particular, the Autographa californica nucleopolyhedrovirus (AcMNPV) has been exploited as a platform for bio-inputs production. This is why the improvement of the technologies used for the production of recombinant baculoviruses takes on particular relevance. To achieve this goal, we developed a highly versatile baculoviral transfer vector generation system called PluriBAC. The PluriBAC system consists of three insert entry levels using Golden Gate assembly technology. The wide availability of vectors and sticky ends allows enough versatility to combine more than four different promoters, genes of interest, and terminator sequences. Here, we report not only the rational design of the PluriBAC system but also its use for the generation of baculoviral reporter vectors applied to different fields of biotechnology. We demonstrated that recombinant AcMNPV baculoviruses generated with the PluriBAC system were capable of infecting Spodoptera frugiperda larvae. On the other hand, we found that the recombinant budded virions (BV) generated using our system were capable of transducing different types of tumor and normal cells both in vitro and in vivo. Our findings suggest that the PluriBAC system could constitute a versatile tool for the generation of insecticide and gene therapy vectors.

Mitochondrial Peptide Humanin Facilitates Chemoresistance in Glioblastoma Cells.

Humanin (HN) is a mitochondrial-derived peptide with robust cytoprotective effects in many cell types. Although the administration of HN analogs has been proposed to treat degenerative diseases, its role in the pathogenesis of cancer is poorly understood. Here, we evaluated whether HN affects the chemosensitivity of glioblastoma (GBM) cells. We found that chemotherapy upregulated HN expression in GBM cell lines and primary cultures derived from GBM biopsies. An HN analog (HNGF6A) boosted chemoresistance, increased the migration of GBM cells and improved their capacity to induce endothelial cell migration and proliferation. Chemotherapy also upregulated FPR2 expression, an HN membrane-bound receptor, and the HNGF6A cytoprotective effects were inhibited by an FPR2 receptor antagonist (WRW4). These effects were observed in glioma cells with heterogeneous genetic backgrounds, i.e., glioma cells with wild-type (wtIDH) and mutated (mIDH) isocitrate dehydrogenase. HN silencing using a baculoviral vector that encodes for a specific shRNA for HN (BV.shHN) reduced chemoresistance, and impaired the migration and proangiogenic capacity of GBM cells. Taken together, our findings suggest that HN boosts the hallmark characteristics of GBM, i.e., chemoresistance, migration and endothelial cell proliferation. Thus, strategies that inhibit the HN/FPR2 pathway may improve the response of GBM to standard therapy.

Evaluation of Baculoviruses as Gene Therapy Vectors for Brain Cancer.

We aimed to assess the potential of baculoviral vectors (BV) for brain cancer gene therapy. We compared them with adenoviral vectors (AdV), which are used in neuro-oncology, but for which there is pre-existing immunity. We constructed BVs and AdVs encoding fluorescent reporter proteins and evaluated their transduction efficiency in glioma cells and astrocytes. Naïve and glioma-bearing mice were intracranially injected with BVs to assess transduction and neuropathology. Transgene expression was also assessed in the brain of BV-preimmunized mice. While the expression of BVs was weaker than AdVs in murine and human glioma cell lines, BV-mediated transgene expression in patient-derived glioma cells was similar to AdV-mediated transduction and showed strong correlation with clathrin expression, a protein that interacts with the baculovirus glycoprotein GP64, mediating BV endocytosis. BVs efficiently transduced normal and neoplastic astrocytes in vivo, without apparent neurotoxicity. BV-mediated transgene expression was stable for at least 21 days in the brain of naïve mice, but it was significantly reduced after 7 days in mice systemically preimmunized with BVs. Our findings indicate that BVs efficiently transduce glioma cells and astrocytes without apparent neurotoxicity. Since humans do not present pre-existing immunity against BVs, these vectors may constitute a valuable tool for the delivery of therapeutic genes into the brain.

Exploring the Role of the Inhibitor of Apoptosis BIRC6 in Breast Cancer: A Database Analysis.

PURPOSE: The aim of the present work was to investigate the role of apoptosis inhibitor BIRC6 (baculoviral IAP repeat-containing protein 6) in breast cancer (BC), focusing particularly on its involvement in the metastatic cascade. METHODS: We analyzed BIRC6 mRNA expression levels and copy number variations in three BC databases from The Cancer Genome Atlas comparing clinical and molecular attributes. Genomic analysis was performed using the cBioPortal platform, whereas transcriptomic studies (mRNA expression levels, correlation heatmaps, survival plots, and gene ontology) were performed using USC Xena and R. Statistical significance was set at P < .05. RESULTS: Our bioinformatic analyses showed that there was a differential expression of BIRC6 in cancer samples when compared with normal samples. Copy number variations that involve amplification and gain of BIRC6 gene were correlated with negative hormone receptor tumors, higher prognostic indexes, younger age at diagnosis, and both chemotherapy and radiotherapy administration. Transcriptomic and gene ontology analyses showed that, under conditions of high BIRC6 mRNA levels, there are differential expression patterns in apoptotic, proliferation, and metastatic pathways. CONCLUSION: In summary, our in silico data suggest that BIRC6 plays an antiapoptotic, pro-proliferative, and apparent prometastatic role and could be a relevant molecular target for treatment of BC tumors.