The major clinical associations with the progression of diabetic kidney disease (DKD) are glycemic control and systemic hypertension. Recent studies have continued to emphasize vasoactive hormone ...pathways including aldosterone and endothelin which suggest a key role for vasoconstrictor pathways in promoting renal damage in diabetes. The role of glucose per se remains difficult to define in DKD but appears to involve key intermediates including reactive oxygen species (ROS) and dicarbonyls such as methylglyoxal which activate intracellular pathways to promote fibrosis and inflammation in the kidney. Recent studies have identified a novel molecular interaction between hemodynamic and metabolic pathways which could lead to new treatments for DKD. This should lead to a further improvement in the outlook of DKD building on positive results from RAAS blockade and more recently newer classes of glucose-lowering agents such as SGLT2 inhibitors and GLP1 receptor agonists.
Mesenchymal stem cells (MSCs) are stromal cells that have the ability to self-renew and also exhibit multilineage differentiation into both mesenchymal and nonmesenchymal lineages. The intrinsic ...properties of these cells make them an attractive candidate for clinical applications. MSCs are of keen interest because they can be isolated from a small aspirate of bone marrow or adipose tissues and can be easily expanded in vitro. Moreover, their ability to modulate immune responses makes them an even more attractive candidate for regenerative medicine as allogeneic transplant of these cells is feasible without a substantial risk of immune rejection. MSCs secrete various immunomodulatory molecules which provide a regenerative microenvironment for a variety of injured tissues or organ to limit the damage and to increase self-regulated tissue regeneration. Autologous/allogeneic MSCs delivered via the bloodstream augment the titers of MSCs that are drawn to sites of tissue injury and can accelerate the tissue repair process. MSCs are currently being tested for their potential use in cell and gene therapy for a number of human debilitating diseases and genetic disorders. This paper summarizes the current clinical and nonclinical data for the use of MSCs in tissue repair and potential therapeutic role in various diseases.
Answer to August 2019 Photo Quiz Manski, Scott A; O'Brien, Jennifer J; Patel, Devang M
Journal of clinical microbiology,
08/2019, Letnik:
57, Številka:
8
Journal Article
Reporter methods to quantitatively measure the efficiency and specificity of genome editing tools are important for the development of novel editing techniques and successful applications of ...available ones. However, the existing methods have major limitations in sensitivity, accuracy, and/or readiness for in vivo applications. Here, we aim to develop a straight-forward method by using nucleotide insertion/deletion resulted from genome editing. In this system, a target sequence with frame-shifting length is inserted after the start codon of a cerulean fluorescence protein (CFP) to inactivate its fluorescence. As such, only a new insertion/deletion event in the target sequence will reactivate the fluorescence. This reporter is therefore termed as "Insertion/deletion-activated frame-shift fluorescence protein". To increase its traceability, an internal ribosome entry site and a red fluorescence protein mCherryFP are placed downstream of the reporter. The percentage of CFP-positive cells can be quantified by fluorescence measuring devices such as flow cytometer as the readout for genome editing frequency.
To test the background noise level, sensitivity, and quantitative capacity of this new reporter, we applied this approach to examine the efficiency of genome editing of CRISPR/Cas9 on two different targeting sequences and in three different cell lines, in the presence or absence of guide-RNAs with or without efficiency-compromising mutations. We found that the insertion/deletion-activated frame-shift fluorescence protein has very low background signal, can detect low-efficiency genome editing events driven by mutated guideRNAs, and can quantitatively distinguish genome editing by normal or mutated guideRNA. To further test whether the positive editing event detected by this reporter indeed correspond to genuine insertion/deletion on the genome, we enriched the CFP-positive cells to examine their fluorescence under confocal microscope and to analyze the DNA sequence of the reporter in the genome by Sanger sequencing. We found that the positive events captured by this reporter indeed correlates with genuine DNA insertion/deletion in the expected genome location.
The insertion/deletion-activated frame-shift fluorescence protein reporter has very low background, high sensitivity, and is quantitative in nature. It will be able to facilitate the development of new genome editing tools as well as the application of existing tools.
The response to oxygen availability is a fundamental process concerning metabolism and survival/death in all mitochondria-containing eukaryotes. However, the known oxygen-sensing mechanism in ...mammalian cells depends on pVHL, which is only found among metazoans but not in other species. Here, we present an alternative oxygen-sensing pathway regulated by ATE1, an enzyme ubiquitously conserved in eukaryotes that influences protein degradation by posttranslational arginylation. We report that ATE1 centrally controls the hypoxic response and glycolysis in mammalian cells by preferentially arginylating HIF1α that is hydroxylated by PHD in the presence of oxygen. Furthermore, the degradation of arginylated HIF1α is independent of pVHL E3 ubiquitin ligase but dependent on the UBR family proteins. Bioinformatic analysis of human tumor data reveals that the ATE1/UBR and pVHL pathways jointly regulate oxygen sensing in a transcription-independent manner with different tissue specificities. Phylogenetic analysis suggests that eukaryotic ATE1 likely evolved during mitochondrial domestication, much earlier than pVHL.
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•ATE1 controls glycolysis and hypoxic response in mammalian cells by affecting HIF1α•HIF1α is arginylated by ATE1 and degraded by UBRs independently of pVHL•ATE1-dependent oxygen-sensing pathway evolved much earlier than the pVHL pathway
Moorthy et al. find an alternative pathway for oxygen-dependent degradation of HIF1α, mediated by ATE1 rather than pVHL. Unlike pVHL, which is not found outside metazoans, ATE1 is conserved throughout eukaryotes, and its evolution likely accompanied the domestication of mitochondria.
Most prostate cancer cases remain indolent for long periods of time, but metastatic progression quickly worsens the prognosis and leads to mortality. However, little is known about what promotes the ...metastasis of prostate cancer and there is a lack of effective prognostic indicators, making it immensely difficult to manage options for treatment or surveillance. Arginyltransferase 1 (Ate1) is the enzyme mediating post-translational protein arginylation, which has recently been identified as a master regulator affecting many cancer-relevant pathways including stress response, cell cycle checkpoints, and cell migration/adhesion. However, the precise role of Ate1 in cancer remains unknown. In this study, we found the occurrence of metastasis of prostate cancer is inversely correlated with the levels of Ate1 protein and mRNA in the primary tumor. We also found that metastatic prostate cancer cell lines have a reduced level of Ate1 protein compared to non-metastatic cell lines, and that a depletion of Ate1 drives prostate cancer cells towards more aggressive pro-metastatic phenotypes without affecting proliferation rates. Furthermore, we demonstrated that a reduction of Ate1 can result from chronic stress, and that shRNA-reduced Ate1 increases cellular resistance to stress, and drives spontaneous and stress-induced genomic mutations. Finally, by using a prostate orthotropic xenograft mouse model, we found that a reduction of Ate1 was sufficient to enhance the metastatic phenotypes of prostate cancer cell line PC-3 in vivo. Our study revealed a novel role of Ate1 in suppressing prostate cancer metastasis, which has a profound significance for establishing metastatic indicators for prostate cancer, and for finding potential treatments to prevent its metastasis.
Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that is activated by collagens that is involved in the pathogenesis of fibrotic disorders. Interestingly, de novo production of the ...collagen type I (Col I) has been observed in Col4a3 knockout mice, a mouse model of Alport Syndrome (AS mice). Deletion of the DDR1 in AS mice was shown to improve survival and renal function. However, the mechanisms driving DDR1-dependent fibrosis remain largely unknown.
Podocyte pDDR1 levels, Collagen and cluster of differentiation 36 (CD36) expression was analyzed by Real-time PCR and Western blot. Lipid droplet accumulation and content was determined using Bodipy staining and enzymatic analysis. CD36 and DDR1 interaction was determined by co-immunoprecipitation. Creatinine, BUN, albuminuria, lipid content, and histological and morphological assessment of kidneys harvested from AS mice treated with Ezetimibe and/or Ramipril or vehicle was performed.
We demonstrate that Col I-mediated DDR1 activation induces CD36-mediated podocyte lipotoxic injury. We show that Ezetimibe interferes with the CD36/DDR1 interaction in vitro and prevents lipotoxicity in AS mice thus preserving renal function similarly to ramipril.
Our study suggests that Col I/DDR1-mediated lipotoxicity contributes to renal failure in AS and that targeting this pathway may represent a new therapeutic strategy for patients with AS and with chronic kidney diseases (CKD) associated with Col4 mutations.
This study is supported by the NIH grants R01DK117599, R01DK104753, R01CA227493, U54DK083912, UM1DK100846, U01DK116101, UL1TR000460 (Miami Clinical Translational Science Institute, National Center for Advancing Translational Sciences and the National Institute on Minority Health and Health Disparities), F32DK115109, Hoffmann-La Roche and Alport Syndrome Foundation.
Arginyltransferase 1 (Ate1) mediates protein arginylation, a poorly understood protein posttranslational modification (PTM) in eukaryotic cells. Previous evidence suggest a potential involvement of ...arginylation in stress response and this PTM was traditionally considered anti-apoptotic based on the studies of individual substrates. However, here we found that arginylation promotes cell death and/or growth arrest, depending on the nature and intensity of the stressing factor. Specifically, in yeast, mouse and human cells, deletion or downregulation of the ATE1 gene disrupts typical stress responses by bypassing growth arrest and suppressing cell death events in the presence of disease-related stressing factors, including oxidative, heat, and osmotic stresses, as well as the exposure to heavy metals or radiation. Conversely, in wild-type cells responding to stress, there is an increase of cellular Ate1 protein level and arginylation activity. Furthermore, the increase of Ate1 protein directly promotes cell death in a manner dependent on its arginylation activity. Finally, we found Ate1 to be required to suppress mutation frequency in yeast and mammalian cells during DNA-damaging conditions such as ultraviolet irradiation. Our study clarifies the role of Ate1/arginylation in stress response and provides a new mechanism to explain the link between Ate1 and a variety of diseases including cancer. This is also the first example that the modulation of the global level of a PTM is capable of affecting DNA mutagenesis.
Protein arginylation is an emerging post-translational modification that targets a number of metabolic enzymes; however, the mechanisms and downstream effects of this modification are unknown. Here ...we show that lack of arginylation renders cells vulnerable to purine nucleotide synthesis inhibitors and affects the related glycine and serine biosynthesis pathways. We show that the purine nucleotide biosynthesis enzyme PRPS2 is selectively arginylated, unlike its close homologue PRPS1, and that arginylation of PRPS2 directly facilitates its biological activity. Moreover, selective arginylation of PRPS2 but not PRPS1 is regulated through a coding sequence-dependent mechanism that combines elements of mRNA secondary structure with lysine residues encoded near the N-terminus of PRPS1. This mechanism promotes arginylation-specific degradation of PRPS1 and selective retention of arginylated PRPS2 in vivo. We therefore demonstrate that arginylation affects both the activity and stability of a major metabolic enzyme.
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