Microproteins in skeletal muscle: hidden keys in muscle physiology.

Recent advances in the transcriptomics, translatomics, and proteomics have led us to the exciting new world of functional endogenous microproteins. These microproteins have a small size and are derived from small open reading frames (smORFs) of RNAs previously annotated as non-coding (e.g. lncRNAs and circRNAs) as well as from untranslated regions and canonical mRNAs. The presence of these microproteins reveals a much larger translatable portion of the genome, shifting previously defined dogmas and paradigms. These findings affect our view of organisms as a whole, including skeletal muscle tissue. Emerging evidence demonstrates that several smORF-derived microproteins play crucial roles during muscle development (myogenesis), maintenance, and regeneration, as well as lipid and glucose metabolism and skeletal muscle bioenergetics. These microproteins are also involved in processes including physical activity capacity, cellular stress, and muscular-related diseases (i.e. myopathy, cachexia, atrophy, and muscle wasting). Given the role of these small proteins as important key regulators of several skeletal muscle processes, there are rich prospects for the discovery of new microproteins and possible therapies using synthetic microproteins.

DDX6 Helicase Behavior and Protein Partners in Human Adipose Tissue-Derived Stem Cells during Early Adipogenesis and Osteogenesis.

DDX6 helicase is an RNA-binding protein involved in different aspects of gene expression regulation. The roles played by DDX6 depend on the complexes associated with it. Here, for the first time, we characterize the protein complexes associated with DDX6 in human adipose tissue-derived stem cells (hASCs) and analyze the dynamics of this helicase under different conditions of translational activity and differentiation. The results obtained demonstrated that the DDX6 helicase is associated with proteins involved in the control of mRNA localization, translation and metabolism in hASCs. DDX6 complexes may also assemble into more complex structures, such as RNA-dependent granules, the abundance and composition of which change upon inhibited translational activity. This finding supports the supposition that DDX6 is possibly involved in the regulation of the mRNA life cycle in hASCs. Although there was no significant variation in the protein composition of these complexes during early adipogenic or osteogenic induction, there was a change in the distribution pattern of DDX6: the number of DDX6 granules per cell was reduced during adipogenesis and was enhanced during osteogenesis.

Cell cycle genes are downregulated after adipogenic triggering in human adipose tissue-derived stem cells by regulation of mRNA abundance.

The adipogenic process is characterized by the expression of adipocyte differentiation markers that lead to changes in cell metabolism and to the accumulation of lipid droplets. Moreover, during early adipogenesis, cells undergo a strong downregulation of translational activity with a decrease in cell size, proliferation and migration. In the present study, we identified that after 24 hours of adipogenic induction, human adipose tissue-derived stem cells (hASCs) undergo a G1-cell cycle arrest consistent with reduced proliferation, and this effect was correlated with a shift in polysome profile with an enrichment of the monosomal fraction and a reduction of the polysomal fraction. Polysome profiling analysis also revealed that this change in the monosomal/polysomal ratio was related to a strong downregulation of cell cycle and proliferation genes, such as cyclins and cyclin-dependent kinases (CDKs). Comparing total and polysome-associated mRNA sequencing, we also observed that this downregulation was mostly due to a reduction of cell cycle and proliferation transcripts via control of total mRNA abundance, rather than by translational control.

Human adipose-derived stem cells (ADSC) and human periodontal ligament stem cells (PDLSC) as cellular substrates of a toxicity prediction assay.

With the increasing need to develop in vitro assays to replace animal use, human stem cell-derived methods are emerging and showing outstanding contributions to the toxicological screening of substances. Adult human stem cells such as adipose-derived stem cells (ADSC) and periodontal ligament stem cells (PDLSC) were used as cell substrates for a cytotoxicity assay and toxicity prediction using the neutral red uptake (NRU) assay. First, primary cell cultures from three independent donors, from each tissue source, were characterized as mesenchymal stem cells (MSC) by plastic adherence and appropriate immunophenotype for MSC markers (positive for CD90, CD73, and CD105 and negative for CD11b, CD34, CD45, HLADR, and CD19). Furthermore, ADSC and PDLSC were able to differentiate into adipocytes and osteoblasts when maintained under the same culture conditions previously established for the NRU assay. NRU assays for three reference test substances were performed. R2 was higher than 0.85 for all conditions, showing the feasibility to calculate IC50 values. The IC50 values were then used to predict the LD50 of the test substances, which were comparable to previous results and the ICCVAM standard test report. Primary ADSC and PDLSC showed the potential to be considered as additional models for use in cytotoxicity assays.

Downregulation of the protein synthesis machinery is a major regulatory event during early adipogenic differentiation of human adipose-derived stromal cells.

Commitment of adult stem cells involves the activation of specific gene networks regulated from transcription to protein synthesis. Here, we used ribosome profiling to identify mRNAs regulated at the translational level, through both differential association to polysomes and modulation of their translational rates. We observed that translational regulation during the differentiation of human adipose-derived stromal cells (hASCs, also known as adipose-derived mesenchymal stem cells), a subset of which are stem cells, to adipocytes was a major regulatory event. hASCs showed a significant reduction of whole protein synthesis after adipogenic induction and a downregulation of the expression and translational efficiency of ribosomal proteins. Additionally, focal adhesion and cytoskeletal proteins were downregulated at the translational level. This negative regulation of the essential biological functions of hASCs resulted in a reduction in cell size and the potential of hASCs to migrate. We analyzed whether the inactivation of key translation initiation factors was involved in this observed major repression of translation. We showed that there was an increase in the hypo phosphorylated forms of 4E-BP1, a negative regulator of translation, during early adipogenesis. Our results showed that extensive translational regulation occurred during the early stage of the adipogenic differentiation of hASCs.

Metabolic switches during the first steps of adipogenic stem cells differentiation.

The understanding of metabolism during cell proliferation and commitment provides a greater insight into the basic biology of cells, allowing future applications. Here we evaluated the energy and oxidative changes during the early adipogenic differentiation of human adipose tissue-derived stromal cells (hASCs). hASCs were maintained under differentiation conditions during 3 and 7days. Oxygen consumption, mitochondrial mass and membrane potential, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) and catalase activities, non-protein thiols (NPT) concentration and lipid peroxidation were analyzed. We observed that 7days of adipogenic induction are required to stimulate cells to consume more oxygen and increase mitochondrial activity, indicating organelle maturation and a transition from glycolytic to oxidative energy metabolism. ROS production was only increased after 3days and may be involved in the differentiation commitment. ROS source was not only the mitochondria and we suggest that NOX proteins are related to ROS generation and therefore adipogenic commitment. ROS production did not change after 7days, but an increased activity of catalase and NPT concentration as well as a decreased lipid peroxidation were observed. Thus, a short period of differentiation induction is able to change the energetic and oxidative metabolic profile of hASCs and stimulate cytoprotection processes.

Ribonomic analysis of human DZIP1 reveals its involvement in ribonucleoprotein complexes and stress granules.

BACKGROUND: DZIP1 (DAZ-interacting protein 1) has been described as a component of the Hh signaling pathway with a putative regulatory role in ciliogenesis. DZIP1 interacts with DAZ RNA binding proteins in embryonic stem cells and human germ cells suggesting a role in mRNA regulation. RESULTS: We investigated DZIP1 function in HeLa cells and its involvement in ribonucleoprotein complexes. DZIP1 was predominantly located in granules in the cytoplasm. Under oxidative stress conditions, DZIP1 re-localized to stress granules. DZIP appears to be important for the formation of stress granules during the stress response. We used immunoprecipitation assays with antibodies against DZIP1 and microarray hybridization to identify mRNAs associated with DZIP1. The genetic networks formed by the DZIP1-associated mRNAs were involved in cell cycle and gene expression regulation. DZIP1 is involved in the Hedgehog signaling pathway. We used cyclopamine, a specific inhibitor of this pathway, to analyze the expression of DZIP1 and its associated mRNAs. The abundance of DZIP1-associated mRNAs increased with treatment; however, the silencing or overexpression of DZIP1 in HeLa cells had no effect on the accumulation of the associated mRNAs. Polysomal profile analysis by sucrose gradient centrifugation demonstrated the presence of DZIP1 in the polysomal fraction. CONCLUSIONS: Our results suggest that DZIP1 is part of an RNP complex that occupies various subcellular locations. The diversity of the mRNAs associated with DZIP1 suggests that this protein is a component of different RNPs associated with translating polysomes and with RNA granules.

mRNA localization mechanisms in Trypanosoma cruzi.

Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

Polysome profiling shows extensive posttranscriptional regulation during human adipocyte stem cell differentiation into adipocytes.

Adipocyte stem cells (hASCs) can proliferate and self-renew and, due to their multipotent nature, they can differentiate into several tissue-specific lineages, making them ideal candidates for use in cell therapy. Most attempts to determine the mRNA profile of self-renewing or differentiating stem cells have made use of total RNA for gene expression analysis. Several lines of evidence suggest that self-renewal and differentiation are also dependent on the control of protein synthesis by posttranscriptional mechanisms. We used adipogenic differentiation as a model, to investigate the extent to which posttranscriptional regulation controlled gene expression in hASCs. We focused on the initial steps of differentiation and isolated both the total mRNA fraction and the subpopulation of mRNAs associated with translating ribosomes. We observed that adipogenesis is committed in the first days of induction and three days appears as the minimum time of induction necessary for efficient differentiation. RNA-seq analysis showed that a significant percentage of regulated mRNAs were posttranscriptionally controlled. Part of this regulation involves massive changes in transcript untranslated regions (UTR) length, with differential extension/reduction of the 3'UTR after induction. A slight correlation can be observed between the expression levels of differentially expressed genes and the 3'UTR length. When we considered association to polysomes, this correlation values increased. Changes in the half lives were related to the extension of the 3'UTR, with longer UTRs mainly stabilizing the transcripts. Thus, changes in the length of these extensions may be associated with changes in the ability to associate with polysomes or in half-life.

The overexpression of the trypanosomatid-exclusive TcRBP19 RNA-binding protein affects cellular infection by Trypanosoma cruzi

To characterise the trypanosomatid-exclusive RNA-binding protein TcRBP19, we analysed the phenotypic changes caused by its overexpression. Although no evident changes were observed when TcRBP19 was ectopically expressed in epimastigotes, the metacyclogenesis process was affected. Notably, TcRBP19 overexpression also led to a decrease in the number of infected mammalian cells. These findings suggest that TcRBP19 may be involved in the life cycle progression of the Trypanosoma cruzi parasite.

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis.

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.

The zinc finger protein TcZFP2 binds target mRNAs enriched during Trypanosoma cruzi metacyclogenesis

Trypanosomes are parasitic protozoa in which gene expression is primarily controlled through the regulation of mRNA stability and translation. This post-transcriptional control is mediated by various families of RNA-binding proteins, including those with zinc finger CCCH motifs. CCCH zinc finger proteins have been shown to be essential to differentiation events in trypanosomatid parasites. Here, we functionally characterise TcZFP2 as a predicted post-transcriptional regulator of differentiation in Trypanosoma cruzi. This protein was detected in cell culture-derived amastigotes and trypomastigotes, but it was present in smaller amounts in metacyclic trypomastigote forms of T. cruzi. We use an optimised recombinant RNA immunopreciptation followed by microarray analysis assay to identify TcZFP2 target mRNAs. We further demonstrate that TcZFP2 binds an A-rich sequence in which the adenosine residue repeats are essential for high-affinity recognition. An analysis of the expression profiles of the genes encoding the TcZFP2-associated mRNAs throughout the parasite life cycle by microarray hybridisation showed that most of the associated mRNAs were upregulated in the metacyclic trypomastigote forms, also suggesting a role for TcZFP2 in metacyclic trypomastigote differentiation. Knockdown of the orthologous Trypanosoma brucei protein levels showed ZFP2 to be a positive regulator of specific target mRNA abundance.

Distinct subcellular localization of tRNA-derived fragments in the infective metacyclic forms of Trypanosoma cruzi

Small non-coding RNAs derived from transfer RNAs have been identified as a broadly conserved prokaryotic and eukaryotic response to stress. Their presence coincides with changes in developmental state associated with gene expression regulation. In the epimastigote form of Trypanosoma cruzi, tRNA fragments localize to posterior cytoplasmic granules. In the infective metacyclic form of the parasite, we found tRNA-derived fragments to be abundant and evenly distributed within the cytoplasm. The fragments were not associated with polysomes, suggesting that the tRNA-derived fragments may not be directly involved in translation control in metacyclics.

PUMILIO-2 is involved in the positive regulation of cellular proliferation in human adipose-derived stem cells.

Stem cells can either differentiate into more specialized cells or undergo self-renewal. Several lines of evidence from different organisms suggest that these processes depend on the post-transcriptional regulation of gene expression. The presence of the PUF [Pumilio/FBF (fem-3 binding factor)] domain defines a conserved family of RNA binding proteins involved in repressing gene expression. It has been suggested that a conserved function of PUF proteins is to repress differentiation and sustain the mitotic proliferation of stem cells. In humans, Pumilio-2 (PUM2) is expressed in embryonic stem cells and adult germ cells. Here we show that PUM2 is expressed in a subpopulation of adipose-derived stem cell (ASC) cultures, with a granular pattern of staining in the cytoplasm. Protein levels of PUM2 showed no changes during the differentiation of ASCs into adipocytes. Moreover, RNAi knockdown of pum2 did not alter the rate of adipogenic differentiation compared with wild-type control cells. A ribonomic approach was used to identify PUM2-associated mRNAs. Microarray analysis showed that PUM2-bound mRNAs are part of gene networks involved in cell proliferation and gene expression control. We studied pum2 expression in cell cultures with low or very high levels of proliferation and found that changes in pum2 production were dependent on the proliferation status of the cell. Transient knockdown of pum2 expression by RNAi impaired proliferation of ASCs in vitro. Our results suggest that PUM2 does not repress differentiation of ASCs but rather is involved in the positive control of ASCs division and proliferation.

Human cardiac explant-conditioned medium: soluble factors and cardiomyogenic effect on mesenchymal stem cells.

The use of conditioned medium (CM) from human cardiac explants (HCEs) as a potential source of paracrine factors for adult stem cell signaling has never been evaluated. We hypothesized that HCEs might provide a source of soluble factors triggering the differentiation of mesenchymal stem cells (MSCs) into cardiomyocyte-like cells. By using two-dimensional electrophoresis (2-DE) gels/mass spectrometry and antibody macroarray assays, we found that HCEs release macromolecules, including cytokines, growth factors and myocardial and metabolism-related proteins into the culture medium. We identified a total of 20 proteins in the HCE-CM. However, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-DE, these 20 proteins account for only a fraction of the total number of proteins present in the HCE-CM. We also found that CM increased the proliferation of bone marrow-derived-MSCs (BM-MSCs) in vitro. Unlike the other effects, this effect was most evident after 48 h of culture. Moreover, we examined the effect of HCE-CM on levels of mRNA and protein for specific cardiac markers. We showed that a surprisingly big fraction of BM-MSCs (3.4-5.0%) treated in vitro with HCE-CM became elongated and began to express cardiac markers, consistent with their possible differentiation into cardiomyocyte-like cells. Our in vitro model may be useful not only per se, but also for studies of the mechanisms of action of soluble factors involved in cell differentiation, paving the way for possible new protein-based treatments in the future.

Are purified or expanded cord blood-derived CD133+ cells better at improving cardiac function?

Endothelial progenitor cells (EPCs), which express the CD133 marker, can differentiate into mature endothelial cells (ECs) and create new blood vessels. Normal angiogenesis is unable to repair the injured tissues that result from myocardial infarction (MI). Patients who have high cardiovascular risks have fewer EPCs and their EPCs exhibit greater in vitro senescence. Human umbilical cord blood (HUCB)-derived EPCs could be an alternative to rescue impaired stem cell function in the sick and elderly. The aim of this study was to purify HUCB-derived CD133(+) cells, expand them in vitro and evaluate the efficacy of the purified and expanded cells in treating MI in rats. CD133(+) cells were selected for using CD133-coupled magnetic microbeads. Purified cells stained positive for EPC markers. The cells were expanded and differentiated in media supplemented with fetal calf serum and basic fibroblast growth factor, insulin-like growth factor-I and vascular endothelial growth factor (VEGF). Differentiation was confirmed by lack of staining for EPC markers. These expanded cells exhibited increased expression of mature EC markers and formed tubule-like structures in vitro. Only the expanded cells expressed VEGF mRNA. Cells were expanded up to 70-fold during 60 days of culture, and they retained their functional activity. Finally, we evaluated the therapeutic potential of purified and expanded CD133(+) cells in treating MI by intramyocardially injecting them into a rat model of MI. Rats were divided into three groups: A (purified CD133(+) cells-injected); B (expanded CD133(+) cells-injected) and C (saline buffer-injected). We observed a significant improvement in left ventricular ejection fraction for groups A and B. In summary, CD133(+) cells can be purified from HUCB, expanded in vitro without loosing their biological activity, and both purified and expanded cells show promising results for use in cellular cardiomyoplasty. However, further pre-clinical testing should be performed to determine whether expanded CD133(+) cells have any clinical advantages over purified CD133(+) cells.

Expression of cardiac function genes in adult stem cells is increased by treatment with nitric oxide agents.

Mesenchymal stem cells (MSCs) have received special attention for cardiomyoplasty because several studies have shown that they differentiate into cardiomyocytes both in vitro and in vivo. Nitric oxide (NO) is a free radical signaling molecule that regulates several differentiation processes including cardiomyogenesis. Here, we report an investigation of the effects of two NO agents (SNAP and DEA/NO), able to activate both cGMP-dependent and -independent pathways, on the cardiomyogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs). The cells were isolated, cultured and treated with NO agents. Cardiac- and muscle-specific gene expression was analyzed by indirect immunofluorescence, flow cytometry, RT-PCR and real-time PCR. We found that untreated (control) ADSCs and BM-MSCs expressed some muscle markers and NO-derived intermediates induce an increased expression of some cardiac function genes in BM-MSCs and ADSCs. Moreover, NO agents considerably increased the pro-angiogenic potential mostly of BM-MSCs as determined by VEGF mRNA levels.

Formação in vitro de túbulos capilares a partir de células de sangue de cordão umbilical humano com perspectivas para aplicação terapêutica / In vitro formation of capillary tubules from human umbilical cord blood cells with perspectives for therapeutic application

OBJETIVO: As células progenitoras endoteliais (CPE), caracterizadas pelo marcador CD133+, contribuem para a neovascularização, e o aumento no número dessas células pode ser uma ferramenta terapêutica promissora. O sangue de cordão umbilical humano contém um número significante de CPE, sugerindo a possibilidade do uso destas células para a revascularização de tecidos isquêmicos. O objetivo desse trabalho foi analisar a funcionalidade das células CD133+ diferenciadas in vitro. MÉTODOS: As células diferenciadas foram caracterizadas por citometria de fluxo; a expressão do mRNA de VEGF foi avaliada por RT-PCR e a funcionalidade, por meio de ensaios de formação de túbulos capilares. RESULTADOS: As células diferenciadas perderam os marcadores de CPE, mantiveram em níveis baixos os marcadores das linhagens hematopoética e monocíticas e aumentaram a expressão dos marcadores de células endoteliais adultas. As células diferenciadas apresentaram transcritos no mRNA de VEGF e mostraram-se capazes de formar túbulos capilares in vitro. CONCLUSÃO: As células CD133+ diferenciadas in vitro em células endoteliais demonstraram serem funcionalmente ativas, abrindo perspectiva para seu uso futuro em aplicações terapêuticas.

OBJECTIVE: Endothelial progenitor cells (EPC) caracterized by the CD133+ marker, contribute to the neovascularization. Increasing EPC number in vitro could be a promising therapeutic tool. Human umbilical cord blood maintains a significant number of EPC, suggesting the possibility to use these cells to induce the revascularization of ischemic tissues. The aim of this study was to analize the in vitro function of differentiated CD133+ cells. METHODS: Cells were characterized by flow cytometry, VEGF mRNA expression was evaluated by the RT-PCR analysis and the functionally by essays of capillary tubes formation. RESULTS: Differentiated cells lost EPC markers, maintained low levels of markers for hematopoietic and monocytic cell lines and increased the expression of adult endothelial cell markers. Differentiated cells expressed VEGF mRNA and were capable to induce in vitro capillary tubules formation. CONCLUSION: CD133+ cells differentiated into endothelial cells in vitro are functionally active initiating the possibility of their use in future therapeutic applications.

Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress.

Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.

Trypanosoma cruzi: a stage-specific calpain-like protein is induced after various kinds of stress

Calpains are calcium-dependent cysteine proteinases found in all living organisms and are involved in diverse cellular processes. Calpain-like proteins have been reported after in silico analysis of the Tritryps genome and are believed to play important roles in cell functions of trypanosomatids. We describe the characterization of a member of this family, which is differentially expressed during the life-cycle of Trypanosoma cruzi.