RESUMO

Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm-1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.

Assuntos

RESUMO

AIMS: Mitochondrial (mt) DNA replication is strongly associated with oxidative stress, a condition triggered by aging and hyperglycemia, both of which contribute to mitophagy disruption and inflammation. This observational exploratory study evaluated mtDNA-copy number (mtDNA-CN) and expression of genes involved in mitochondriogenesis (PPARGC1A, TFAM, TFB1M, TFB2M), mitophagy (PINK1, PRKN), and inflammatory pathways triggered by hyperglycemia (TXNIP, NLRP3, NFKB1), in the postcentral gyrus of adults and older individuals with and without type 2 diabetes mellitus (T2D). MAIN METHODS: Quantitative real-time PCR was employed to evaluate mtDNA-CN and gene expression; tissue autofluorescence, a marker of aging and of cells with damaged organelles, was also quantified. KEY FINDINGS: No correlation was found between age and mtDNA-CN, but a direct correlation was observed for cases with mtDNA-CN >1000 (r = 0.41). The mtDNA-CN >1000 group had greater tissue autofluorescence and higher body mass index compared to the mtDNA-CN <1000 group (BMI; 25.7 vs 22.0 kg/m2, respectively). mtDNA-CN correlated with tissue autofluorescence in the overall sample (r = 0.55) and in the T2D group (r = 0.64). PINK and PRKN expressions were inversely correlated with age. Mitochondriogenesis genes and TXNIP expressions were higher in the T2D group, and correlations among the mitochondriogenesis genes were also stronger in this group, relative to the subgroup with mtDNA-CN >1000.

Assuntos

RESUMO

The effects of UV light on the skin have been extensively investigated. However, systematic information about how the exposure to ultraviolet-A (UVA) light, the least energetic but the most abundant UV radiation reaching the Earth, shapes the subcellular organization of proteins is lacking. Using subcellular fractionation, mass-spectrometry-based proteomics, machine learning algorithms, immunofluorescence, and functional assays, we mapped the subcellular reorganization of the proteome of human keratinocytes in response to UVA light. Our workflow quantified and assigned subcellular localization for over 1,600 proteins, of which about 200 were found to redistribute upon UVA exposure. Reorganization of the proteome affected modulators of signaling pathways, cellular metabolism, and DNA damage response. Strikingly, mitochondria were identified as one of the main targets of UVA-induced stress. Further investigation demonstrated that UVA induces mitochondrial fragmentation, up-regulates redox-responsive proteins, and attenuates respiratory rates. These observations emphasize the role of this radiation as a potent metabolic stressor in the skin.

RESUMO

Epidermal photoaging contributes to skin fragility over time and it is a risk factor for skin cancer. Photoaging has been associated for a long time with exposure to Ultraviolet-A (UVA) light, the predominant component of the solar ultraviolet radiation. While the cellular mechanisms underlying UVA-induced photoaging in the dermis have been well characterized, UVA's action on the epidermis remains elusive. Here, proteomic analysis was conducted to derive the cellular responses induced by an environmentally relevant dose of UVA in primary human keratinocytes. We also investigated the effects of UVA on non-transformed immortalized keratinocytes (HaCaT cells), bearing potentially oncogenic mutations. We showed that UVA induces proteome remodeling and senescence in primary keratinocytes, eliciting potent antioxidant and pro-inflammatory responses. Additionally, we showed that UVA modulates the secretory phenotype of these cells to the extent of inducing paracrine oxidative stress and immune system activation in pre-malignant keratinocytes. These observations offer insights into the cellular mechanisms by which UVA drives photoaging in the skin.

Assuntos

RESUMO

AMP-activated protein kinase (AMPK) regulates many different metabolic pathways in eukaryote cells including mitochondria biogenesis and energy homeostasis. Here we identify a patient with hypotonia, weakness, delayed milestones and neurological impairment since birth harbouring a novel homozygous mutation in the AMPK catalytic α-subunit 1, encoded by the PRKAA1 gene. The homozygous mutation p.S487L in isoform 1 present in the patient is in a cryptic residue for AMPK activity. In the present study, we performed the characterization of mitochondrial respiratory properties of the patient, in comparison to healthy controls, through the culture of skin fibroblasts in order to understand some of the cellular consequences of the PRKAA1 mutation. In these assays, mitochondrial respiratory complex I showed lower activity, which was followed by a decrement in the mtDNA copy number, which is a probable consequence of the lower expression of PGC-1α and PRKAA1 itself as measured in our quantitative PCRs experiments. Confirming the effect of the patient mutation in respiration, transfection of patient fibroblasts with wild type PRKAA1 partially restore complex I level. The preliminary clinic evaluations of the patient suggested a metabolic defect related to the mitochondrial respiratory function, therefore treatment with CoQ10 supplementation dose started four years ago and a clear improvement in motor skills and strength has been achieved with this treatment.

Assuntos

Assuntos

RESUMO

O número de indivíduos que sofrem com patologias associadas ao consumo abusivo de etanol tem aumentado significativamente no último século. Como consequência desse fato, os custos associados ao tratamento do alcoolismo, bem como das doenças associadas a ele também têm aumentado, onerando o sistema de saúde e se tornando um problema de saúde pública de grande relevância atualmente. Os mecanismos moleculares que desencadeiam muitas dessas doenças não estão completamente esclarecidos. O tecido hepático é o mais afetado pelo etanol e as mitocôndrias têm sido apontadas como alvos cruciais na toxicidade hepática induzida pelo álcool. Logo, o objetivo desse trabalho foi investigar como o consumo de etanol afeta o estado redox e o metabolismo mitocondriais no fígado. Ratos Wistar machos adultos jovens e de meia-idade receberam ad libitum solução alcoólica 25% (v/v) como única fonte de líquido. Os grupos controle receberam somente água. Ambos os grupos receberam ração ad libitum. Mitocôndrias hepáticas foram isoladas usando técnicas padrão. O consumo de ração e de líquidos foi significativamente menor em animais que ingeriram álcool, resultando em menor ganho de massa corpórea nos protocolos utilizados. As mitocôndrias dos animais que consumiram etanol apresentaram menores níveis de respiração em condição basal e quando energizadas com substratos respiratórios. A atividade e os níveis protéicos de citocromo c oxidase foi menor nos grupos tratados com etanol. Independente da duração do período de tratamento, mitocôndrias hepáticas de animais que ingeriram álcool foram menos susceptíveis à transição de permeabilidade mitocondrial induzida por cálcio, quando comparadas às mitocôndrias dos animais do grupo controle...

The number of people suffering from alcoholism has increased significantly over the last century. As a result, costs associated with treating the addiction itself as well as the associated pathologies have also increased, such that this is considered as public health issue. Furthermore, the molecular events leading to several of these diseases are not yet clearly understood. Hepatic tissue is the most affected by alcohol, and mitochondria have been suggested to be a crucial target in alcohol-induced liver toxicity. Thus, the aim of our study was to investigate how ethanol consumption affects the redox state and mitochondrial metabolism in the liver. Young adult and middle-aged male Wistar rats were given a 25 % (v/v) ethanol solution as the only source of drinking water. Control groups received water only. Liver mitochondria were isolated using standard techniques. Food and water intake was significantly lower in alcohol-drinking rats, resulting in lower weight gain during the treatment regimes. Mitochondria from the alcohol-drinking group had lower respiration under levels in basal condition, when energized by substrates feeding electrons into complexes I and IV. Cytochrome c oxidase activity and protein levels were lower in the alcohol group as well. Additionally, regardless of the length of the treatment, liver mitochondria from the alcohol-treated animals were more resistant to Ca2+-induced mitochondrial permeability transition (MPT), when compared to mitochondria from control animals. This effect was abrogated by oxidizing agents of pyridine nucleotides (acetoacetate, diamide or tert butylhydroperoxide) or in uncoupled mitochondria. We also found that liver mitochondria from the alcohol-drinking rats had a more reduced pyridine nucleotide pool and higher NAD(P)H/NAD(P)+ ratios. In addition, Nampt (an enzyme of the NAD+ synthetic pathway) protein levels did not differ after alcohol consumption. ..

Assuntos