Puromycin

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.

Puromycin is an amino-acyl transfer RNA analog widely employed in studies of protein synthesis. Since puromycin is covalently incorporated into nascent polypeptide chains, anti-puromycin immunofluorescence enables visualization of nascent protein synthesis. A common assumption in studies of local messenger RNA translation is that the anti-puromycin staining of puromycylated nascent polypeptides in fixed cells accurately reports on their original site of translation, particularly when ribosomes are stalled with elongation inhibitors prior to puromycin treatment. However, when we attempted to implement a proximity ligation assay to detect ribosome-puromycin complexes, we found no evidence to support this assumption. We further demonstrated, using biochemical assays and live cell imaging of nascent polypeptides in mammalian cells, that puromycylated nascent polypeptides rapidly dissociate from ribosomes even in the presence of elongation inhibitors. Our results suggest that attempts to define precise subcellular translation sites using anti-puromycin immunostaining may be confounded by release of puromycylated nascent polypeptide chains prior to fixation.

Glomerular diseases are commonly characterized by podocyte injury including apoptosis, actin cytoskeleton rearrangement and detachment. However, the strategies for preventing podocyte damage remain insufficient. Recently autophagy has been regarded as a vital cytoprotective mechanism for keeping podocyte homeostasis. Thus, it is reasonable to utilize this mechanism to attenuate podocyte injury. Trehalose, a natural disaccharide, is an mTOR independent autophagy inducer. It is unclear whether trehalose alleviates podocyte injury. Therefore, we investigated the efficacy of trehalose in puromycin aminonucleoside (PAN)-treated podocytes which mimic cell damage in minimal change nephrotic syndrome in vitro. Human conditional immortalized podocytes were treated with trehalose with or without PAN. Autophagy was investigated by immunofluorescence staining for LC3 puncta and Western blotting for LC3, Atg5, p-AMPK, p-mTOR and its substrates. Podocyte apoptosis and necrosis were evaluated by flow cytometry and by measuring lactate dehydrogenase activity respectively. We also performed migration assay to examine podocyte recovery. It was shown that trehalose induced podocyte autophagy in an mTOR independent manner and without reactive oxygen species involvement. Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect. Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility. These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.

BackgroundThe gut-kidney axis has been implicated in chronic kidney disease, however its role in minimal change nephrotic syndrome (MCNS) is poorly understood. We investigated the impact of gut microbiota on proteinuria in MCNS.MethodsA puromycin aminonucleoside (PAN)-induced rat model of MCNS was used. Rats received a cocktail of antibiotics, PBS (control), or antibiotics plus indoxyl sulfate (IS). To assess causality, fecal microbiota transplantation (FMT) was performed in additional PAN rats. Urinary protein, kidney histology, urinary IS, 8-hydroxy-2'-deoxyguanosine (8-OHdG), and gut microbiota composition were evaluated.ResultsOn day 8 after PAN injection, antibiotic-treated rats exhibited markedly reduced proteinuria (1.4 g/gCre) compared with controls (16.8 g/gCre, p = 0.014), whereas IS-treated rats developed severe proteinuria (117.3 g/gCre). Electron microscopy revealed podocyte foot process effacement in control and IS-treated rats but not in antibiotic-treated rats. Antibiotic-treatment decreased indole-producing bacteria, lowered urinary IS, and reduced 8-OHdG levels, indicating attenuation of oxidative stress. Importantly, FMT abolished the protective effect of antibiotics, re-emerging proteinuria.ConclusionDepletion of the gut microbiota by antibiotic treatment in a rat MCNS model alleviated proteinuria, which was reversed by FMT. This causally implicates gut microbiota, particularly indole-producing bacteria that generate toxins including IS, as a key therapeutic target for MCNS.ImpactThis study demonstrated that depleting the gut microbiota with antibiotics reduced proteinuria in a rat model of minimal change nephrotic syndrome, suggesting that harmful gut bacteria play a critical role in this disease. This research also identified indoxyl sulfate as a key uremic toxin produced by gut bacteria that worsens proteinuria and kidney damage, highlighting its role in disease progression. These findings could lead to novel treatments that target gut microbiota, including antibiotics or activated charcoal adsorbents that reduce proteinuria in minimal change nephrotic syndrome, and potentially minimize steroid use.