As peroxirredoxinas (Prxs) são peroxidases dependentes de tiol que catalisam a redução de uma ampla variedade de hidroperóxidos. A atividade catalítica das Prxs é suportada por um resíduo de cisteína catalítico altamente conservado, cuja oxidação pelo hidroperóxido gera o ácido sulfênico (Cys-SOH). Prx1 de Saccharomyces cerevsiae é uma enzima mitocondrial que catalisa a redução do H2O2 gerado no interior da mitocôndria. O mecanismo de redução do ácido sulfênico de Prx1 é uma questão de debate, com a glutarredoxina 2 (Grx2), tiorredoxina 3 (Trx3), tiorredoxina redutase 2 (Trr2) e ascorbato sendo propostos como possíveis redutores. Para avaliar a importância fisiológica de Prx1 na manutenção da homeostase redox mitocondrial, nós investigamos os mecanismos de importação e processamento mitocondrial de Prx1 assim como os de seus possíveis redutores Trr2 e Trx3. Os ensaios de solubilidade e subfracionamento mitocondrial demonstram que Prx1, Trr2 e Trx3 co-localizam na matriz mitocondrial, associadas fracamente com a membrana mitocondrial interna. Além disso, Prx1 apresenta dupla localização, estando presente também no espaço intermembrana mitocondrial possivelmenete na forma solúvel. O mecanismo de importação de Prx1 para o espaço intermembrana envolve a liberação da proteína precursora no interior da bicamada lipídica da membrana interna em decorrência de uma pequena região hidrofóbica localizada imediatamente após a pressequência. Em seguida, a subunidade Imp2 do complexo proteico IMP catalisa a clivagem da região hidrofóbica liberando Prx1 no espaço intermembrana. Durante a importação de Prx1 para a matriz mitocondrial, a enzima é clivada sequencialmente pelas proteases peptidase de processamento mitocondrial (MPP) e octapeptidil aminopeptidase 1 (Oct1). Oct1 catalisa a remoção de oito resíduos de aminoácidos da região N-terminal de Prx1. Esse processamento aumenta a estabilidade de Prx1 no interior da mitocôndria, mas não interfere na sua atividade peroxidásica in vitro. Apesar das enzimas Trr2 e Trx3 não serem clivadas por Oct1, a ausência de Oct1 causa eleavada instabilidade dessas proteínas. O processamento das Prxs por Oct1 parece ser um processo conservado visto que Oct1 de levedura é capaz de clivar a peroxirredoxina mitocondrial humana Prx3 expressa em S. cerevisiae. Estes resultados indicam o envolvimento de Oct1 no processamento das peroxirredoxinas, representando um sistema de controle de qualidade proteico que regula a homeostase das Prxs e, possivelmente, processos redox mitocondriais / Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the reduction of a wide variety of hydroperoxides. The Prxs catalytic activity is provided by the presence of a highly conserved catalytic cysteine residue whose oxidation by hydroperoxide generates sulfenic acid (Cys-SOH). Saccharomyces cerevsiae Prx1 is a mitochondrial enzyme that catalyzes the reduction of the H2O2 generated endogenously by mitochondria. The mechanism of reduction of Prx harboring Cys-SOH is a matter of debate, with glutaredoxin 2 (GRX2), thioredoxin 3 (Trx3), thioredoxin reductase 2 (Trr2), and ascorbate being proposed as possible reducers. To assess the functional role of Prx1 in maintaining the mitochondrial redox homeostasis, we investigated its mechanisms of import and processing, as well as those ones involved with its possible reducers, Trr2 and Trx3. Assays of solubility and mitochondrial sub-fractionation show that Prx1, Trr2 and Trx3 co-localize in the mitochondrial matrix compartment, being marginally associated with the inner mitochondrial membrane. In addition, Prx1 show dual localization, being also present in the mitochondrial intermembrane space, possibly in their soluble form. The import mechanism of Prx1 to the intermembrane space involves the release of protein\'s precursor within the lipid bilayer of the inner membrane due to a small, hydrophobic region located downstream the presequence. Imp2 subunit of the IMP protein complex then catalyzes the cleavage of the hydrophobic region of Prx1, releasing it to the mitochondrial intermembrane space. During its import into the matrix, Prx1 is sequentially cleaved by the mitochondrial processing-peptidase protease (MPP) and by octapeptidil aminopeptidase 1 (Oct1). Oct1 catalyzes the cleavage of eight amino acid residues from the N-terminal region of Prx1. This process increases stability of Prx1 inside the mitochondria, but does not interfere in its peroxidase activity in vitro. Interestingly, absence of Oct1 causes high instability of Trr2 and Trx3, although these proteins are not cleaved by this protease. Remarkably, the processing of Prxs by Oct1 seems to be a conserved process since yeast Oct1 is able to cleave the human mitochondrial peroxiredoxin Prx3 expressed in S. cerevisiae. Altogether, these results indicate the involvement of Oct1 in the processing of peroxiredoxins, representing a protein quality control system that regulates the homeostasis of Prxs and, possibly, mitochondrial redox processes

INTRODUCTION: Damage to the musculoskeletal system through disease, injury, or ageing can have long-lasting, and detrimental effects on one’s overall well-being. By understanding the processes by which the different tissues of the musculoskeletal system function and communicate, we can apply it to a variety of medical interventions that will benefit the patient population. These include reducing the prevalence of injury-inducing ectopic bone formation in muscle and slowing the degeneration of muscle and bone tissue associated with aging. A major focus is the relationship between muscle and bone tissue, specifically the stem cell populations found in each tissue type. Two genes that are thought to mark stem cell populations associated with muscle and bone tissue are Pax7 and Prx1, respectively. OBJECTIVES: Establish the capability and define optimal conditions to culture primary stem cells isolated from the muscle tissue of the reporter animals that fluorescently tag the Pax7 and Prx1 cell populations. Manipulate culture medium conditions to characterize the differentiation potential for multiple lineages, osteogenic, adipogenic, and myogenic. Lastly, assess whether there is more adipogenic cell differentiation in older animal cell cultures. METHODS: The tamoxifen inducible Pax7^tm1(cre/ER2)Gaka/J and Prx1^CreER-GFP were both crossed with B6.Cg-Gt(ROSA)26sor<tm14(CAG-tdTomato)Hze>/J to create the tamoxifen inducible Pax7/Ai14 and Prx1/Ai14 reporter mice. These animals were then crossed to the B6,129S7-Rag1^tm1Mom/J , creating the Pax7/Ai14/Rag and Prx1/Ai14/Rag reporter mice strains. This transgenic mouse model made it possible to fluorescently identify the Pax7 and Prx1 population of cells isolated from the muscle tissue and characterize the differentiation potential to different cell lineages in vitro. Cells were harvested from both male and female mice that received two tamoxifen injections. Cells were then cultured in various culture media conditions. Determination of specific cell densities, culture conditions, and differentiation time points were determined by manipulating these variables, and assessing the levels of proliferation and differentiation. Multiple assays were run in order to quantify and identify the different cell lineages that were grown in culture under different culture conditions. RESULTS: Cells grown on gelatin coated dishes at densities of 2.2x10^4 to 2.2x10^5 showed optimal performance in proliferation and differentiation. Cells grown in Growth Media containing Chick Embryo Extract (GM) and without (GM-) produced myogenic and adipogenic cell types that were positive for Prx1 expression. Prx1 positive cells grown in the Osteo-Inductive Media (GMOI) produced osteogenic cell types validated through tetracycline uptake. Pax7 expression was low in all culture media conditions. Finally, adipogenic cells were present in both younger and older animals. The adipogenic cells for both populations showed positive Prx1 expression. Younger animals showed a larger relative expression of Plin1 in qRT-PCR analysis. CONCLUSIONS: Although Prx1 is thought to be associated with bone tissue, Prx1 positive cells are located within the muscle and able to be cultured. This muscle derived Prx1 cell population is able to differentiate toward the myogenic, adipogenic, and osteogenic cell lineages. By altering the specific components of culture conditions such as extracellular matrices, seeding density, and media constituents, it is possible to force a particular lineage differentiation for Pax7 and Prx1 muscle derived stem cells. Further studies are needed to elucidate the differentiation potential of Prx1 cells isolated from animals of various ages. Additional in vivo studies are needed to understand the mechanisms surrounding the Prx1 and Pax7 population of cells with their roles in healing and regeneration in response to degeneration and trauma.

Understanding the recruitment of multipotent skeletal progenitor cells and the factors that influence their differentiation would be helpful in providing a means for harnessing the regenerative capacity of skeletal progenitor cells in bone tissue engineering. In order to track the recruitment of skeletal stem cells in fracture healing, transgenic mice containing a Tamoxifen-inducible Cre recombinase that had been placed under the control of a 2.4 kb Prx1 promotor were used to induce conditional expression in periosteal skeletal stem cells that express the Prx1 gene. In order to initially see the cells expressing Prx1, a green fluorescent protein gene (GFP) had also been put downstream to the Prx1 promotor. We then crossed these Prx1CreER-GFP transgenic mice with a second strain containing the Beta-galactosidase gene that becomes constitutively expressed after recombination by the Cre recombinase. The enzymatic activity of Beta-galactosidase was then used to generate a colormetric staining reaction that was used to visualize the cells in which recombination had occurred based on a blue staining product. The recombination activity should only be present in Prx1 expressing cells and their progeny. The goal of the present study was to assess several different approaches to optimize the Beta-galactosidase enzymatic staining protocol and to visualize the Prx1-expressing cells during fracture healing. These studies further examined those populations of cells in the fracture calluses that became labeled and arose from the stem cell populations that had expressed Prx1 at post-operative day 7 and 14. The optimization of a staining method for histology will allow this study to track Prx1 cell fates in a fracture model both in response to specific drug treatments, mechanical loading of the fracture during healing and under pathological conditions that effect healing.