Steroids Up-Regulate p66Shc Longevity Protein in Growth Regulation by Inhibiting Its Ubiquitination

p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism.In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway

Pathological mitophagy disrupts mitochondrial homeostasis in Leber's hereditary optic neuropathy

Leber's hereditary optic neuropathy (LHON), a disease associated with a mitochondrial DNA mutation, is characterized by blindness due to degeneration of retinal ganglion cells (RGCs) and their axons, which form the optic nerve. We show that a sustained pathological autophagy and compartment-specific mitophagy activity affects LHON patient-derived cells and cybrids, as well as induced pluripotent-stem-cell-derived neurons. This is variably counterbalanced by compensatory mitobiogenesis. The aberrant quality control disrupts mitochondrial homeostasis as reflected by defective bioenergetics and excessive reactive oxygen species production, a stress phenotype that ultimately challenges cell viability by increasing the rate of apoptosis. We counteract this pathological mechanism by using autophagy regulators (clozapine and chloroquine) and redox modulators (idebenone), as well as genetically activating mitochondrial biogenesis (PGC1-α overexpression). This study substantially advances our understanding of LHON pathophysiology, providing an integrated paradigm for pathogenesis of mitochondrial diseases and druggable targets for therapy

Novel Expression of Zona Pellucida 3 Protein in Normal Testis; Potential Functional Implications

The expression of the zona pellucida glycoprotein 3 (ZP3), originally thought to be specific for oocytes, was recently extended to ovarian, prostate, colorectal and lung cancers. Earlier successful ZP3 immunization of a transgenic mouse model carrying a ZP3 positive ovarian tumor emphasized the suitability of ZP3 for cancer immunotherapy. This study was carried out to determine whether any other normal tissues besides the ovary in healthy human and mouse tissues may express ZP3, considered important to exclude off-target effects of ZP3 cancer immunotherapy. Strong ZP3 expression was found in normal human and mouse testis. ZP3 protein and mRNA transcripts were localized in spermatogonia, spermatocytes and round and elongated spermatids of both human and mouse testis, as well as in a mouse spermatogonial cell line, but absent in testicular Sertoli, Leydig, spermatogonial stem and progenitor cells. All other normal human and mouse tissues were ZP3 negative. This surprising testicular ZP3 expression has implications for the development of ZP3 cancer immunotherapies, and it also alludes to the potential of using ZP3 as a target for the development of a male immunocontraceptive.</p

The Lipid Transfer Protein CERT Interacts with the Chlamydia Inclusion Protein IncD and Participates to ER-Chlamydia Inclusion Membrane Contact Sites

Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development

Identification of tetrahydrocarbazoles as novel multifactorial drug candidates for treatment of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder and the most frequent cause of dementia. To date, there are only a few approved drugs for AD, which show little or no effect on disease progression. Impaired intracellular calcium homeostasis is believed to occur early in the cascade of events leading to AD. Here, we examined the possibility of normalizing the disrupted calcium homeostasis in the endoplasmic reticulum (ER) store as an innovative approach for AD drug discovery. High-throughput screening of a small-molecule compound library led to the identification of tetrahydrocarbazoles, a novel multifactorial class of compounds that can normalize the impaired ER calcium homeostasis. We found that the tetrahydrocarbazole lead structure, first, dampens the enhanced calcium release from ER in HEK293 cells expressing familial Alzheimer's disease (FAD)-linked presenilin 1 mutations. Second, the lead structure also improves mitochondrial function, measured by increased mitochondrial membrane potential. Third, the same lead structure also attenuates the production of amyloid-beta (A beta) peptides by decreasing the cleavage of amyloid precursor protein (APP) by beta-secretase, without notably affecting alpha- and gamma-secretase cleavage activities. Considering the beneficial effects of tetrahydrocarbazoles addressing three key pathological aspects of AD, these compounds hold promise for the development of potentially effective AD drug candidates

Integrated high-content quantification of intracellular ROS levels and mitochondrial morphofunction

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-contentmicroscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow, and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells

Variations of niacin content in saltwater fish and their relation with dietary RDA in Polish subjects grouped by age

Introduction. A rich and natural source of readily assimilated dietary protein together with invaluable vitamins and minerals are fish, particularly the saltwater species. The quality of any given foodstuff is determined by its nutritional value, which in turn depends on the food type and methods used for manufacture, processing and storage. Many fish products contain fewer water soluble vitamins than the source foodstuff as a result of using various technologies during food processing, such as smoking or deep freezing, where vitamins are often either degraded or leached out. In the case of niacin it is relatively easy to make good such losses by eating niacin-rich foods or by taking dietary supplements e.g. the essential amino acid L-tryptophan. Objectives. To determine niacin content in sea fish that are commonly available on the Polish market and to assess whether this dietary source is sufficient to satisfy the RDA requirements for various age groups of selected subjects living in Poland. Material and methods. Niacin levels were measured firstly in 10 saltwater fish species together with butterfish and Norwegian salmon that formed a separate group. Altogether, 15 types of fish products were analysed in all. They consisted of smoked fish: whitefish, butterfish, sprat, trout, herring (kippers) and mackerel, and frozen fish: butterfish, Norwegian salmon, sole, grenadier and panga. Each product was measured as ten replicates, thus in total 150 analyses were performed. A microbiologically-based method was used for the niacin determination, with enzyme hydrolysis by 40 mg papain and diastase on a 2 g sample (according to the AOAC procedure) to release the free form from the bioavailable form that is bound to NAD and NADP. Results. The most plentiful sources of niacin were found in smoked fish with the highest amounts in butterfish, after warm temperature smoking, and in mackerel; respectively 9.03 and 8.90 mg/100 g. Such 100 g portions of smoked fish are a good dietary source of niacin, in that for men and women above 19 years of age, they constitute respectively 22% - 56% and 25% - 64% of the RDA (Recommended Daily Allowance). The highest levels of niacin in frozen fish were found in butterfish and Norwegian salmon; respectively 8.05 and 5.75 mg/100 g which in turn represent respectively 10% - 50% and 11% - 56% of the RDA in men and women aged above 19 years. Conclusions. Niacin concentrations varied according to fish species. The richest dietary sources were smoked fish consisting of butterfish, after warm temperature smoking, and mackerel. In frozen fish, butterfish and Norwegian salmon had the highest niacin amounts. A 100 g serving of such sea fish can, to quite a large extent, satisfy the adult RDA.Wprowadzenie. Ryby zwłaszcza morskie stanowią naturalne źródło łatwo przyswajalnego białka oraz wielu cennych witamin i minerałów. Witamina B3 to grupa związków w skład których wchodzą kwas nikotynowy (niacyna) oraz amid kwasu nikotynowego (nikotynamid). Stosunkowo łatwo uzupełniać niedobory niacyny spożywając regularnie produkty bogate w tą witaminę, jak i białko lub szeroko dostępne na rynku suplementy diety. Cel badań. Celem pracy było oznaczenie zawartości niacyny w łatwo dostępnych na rynku rybach morskich, a także ocena analizowanych ryb jako potencjalnego dobrego źródła niacyny w diecie człowieka (RDA) w różnych grupach wiekowych. Materiał i metody. Oznaczono zawartość niacyny w piętnastu rodzajach ryb słonowodnych. w rybach wędzonych (sieja, ryba maślana, szprot, pstrąg, śledź oraz makrela) i mrożonych (ryba maślana, łosoś norweski, sola, grenadier, panga). Łącznie przebadano 150 produktów rybnych. Niacynę oznaczono metodą mikrobiologiczną według AOAC stosując hydrolizę enzymatyczną za pomocą papainy i diastazy w celu wyodrębnienia witaminy z analizowanych próbek. Metoda enzymatyczna pozwala na wyodrębnienie tylko biologicznie dostępnych form niacyny związanych w NAD i NADP. Wyniki. Najlepszym źródłem niacyny były ryby wędzone, a najwięcej witaminy stwierdzono w wędzonych na ciepło rybie maślanej (9,03 mg/100 g) i makreli (8,90 mg/100 g). Porcja ryby wędzonej (100 g) może być bardzo dobrym źródłem niacyny realizując normy dziennego zapotrzebowania dla kobiet i mężczyzn w wieku powyżej 19 lat, odpowiednio w zakresie wartości od 24% do 64% i od 21% do 56%. W grupie badanych ryb mrożonych najwyższą zawartość niacyny zawierała ryba maślana (7,89 mg/100 g) i łosoś norweski (5,75 mg/100 g). Porcja ryby mrożonej (100 g) pokrywała dzienne zapotrzebowanie na niacynę normy dla kobiet i mężczyzn w wieku powyżej 19 lat, odpowiednio w zakresach od 11% do 56% i od 10% do 49%. Wnioski. Przeprowadzone analizy zawartości niacyny wykazały zróżnicowanie pomiędzy poszczególnymi gatunkami ryb. Wykazano, iż najlepszym źródłem niacyny są ryby wędzone, spośród których najwięcej analizowanej witaminy posiadają ryba maślana wędzona na ciepło oraz makrela. W grupie ryb mrożonych najwyższą zawartość niacyny oznaczono w rybie maślanej oraz w łososiu norweskim. Porcja ryby morskiej (100 g) może być bardzo dobrym źródłem niacyny