Ribosome biogenesis and p53: Who is regulating whom?

Comment on: Krastev DB, et al. Nat Cell Biol 2011; 13:809-18.     

The PGC-1α-dependent pathway of mitochondrial biogenesis is upregulated in type I endometrial cancer

PGC-1α-dependent pathway of mitochondrial biogenesis was investigated for the first time in type I endometrial cancer and in normal endometrium. In cancer endometrial tissue the citrate synthase activity, the mitochondrial DNA content and the TFAM level were found doubled compared to control endometrial tissue. Moreover, a 1.6- and 1.8-fold increase, respectively, of NRF-1 and PGG-1α expression was found. This study demonstrates, for the first time, that the increased mitochondrial biogenesis in type I endometrial cancer is associated to the upregulation of PGC-1α signalling pathway.     

??(?)lentiginosine-induced apoptosis involves the intrinsic pathway and is p53-independent

We have recently found that 𝒟(−)lentiginosine, a synthetic iminosugar exerting glucosidase inhibitory activity, but not its natural enantiomer lentiginosine, is endowed with an unexpected, pro-apoptotic activity. Here, we investigated mechanisms involved in apoptosis induced by 𝒟−)lentiginosine in MOLT-3, HT-29 and SH-SY5Y tumour cell lines. The results showed that 𝒟−)lentiginosine increased caspase 9 expression at 18 h in all the cell lines from 1.5–3.1 folds. Cytochrome c in the cytoplasm was found to be increased from 2.3–2.6 folds in treated cells with respect to control cells. These effects were accompanied by a remarkable collapse of the mitochondrial membrane potential and by the downregulation of anti-apoptotic genes, as well as the upregulation of pro-apoptotic genes of the Bcl-2 family. U937Bcl-2 transfectants, highly expressing Bcl-2, were reluctant to undergo apoptosis even following treatment with 500 μM 𝒟−)lentiginosine, whereas apoptosis by 𝒟−)lentiginosine was induced also in U937 cells, naturally deficient in P53. Thus, our study establishes that the enantiomer of a natural iminosugar is endowed with a possible anti-tumorigenic effect that might be ascribed not only to their capacity to inhibit glycosidases but also to other unknown mechanisms. These data encourage further investigation on similar compounds to make them an interesting platform for the generation of new anticancer drugs.     

A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase—extensive moonlighting in mitochondrial tRNA biogenesis

Transfer RNAs (tRNAs) reach their mature functional form through several steps of processing and modification. Some nucleotide modifications affect the proper folding of tRNAs, and they are crucial in case of the non-canonically structured animal mitochondrial tRNAs, as exemplified by the apparently ubiquitous methylation of purines at position 9. Here, we show that a subcomplex of human mitochondrial RNase P, the endonuclease removing tRNA 5′ extensions, is the methyltransferase responsible for m¹G9 and m¹A9 formation. The ability of the mitochondrial tRNA:m¹R9 methyltransferase to modify both purines is uncommon among nucleic acid modification enzymes. In contrast to all the related methyltransferases, the human mitochondrial enzyme, moreover, requires a short-chain dehydrogenase as a partner protein. Human mitochondrial RNase P, thus, constitutes a multifunctional complex, whose subunits moonlight in cascade: a fatty and amino acid degradation enzyme in tRNA methylation and the methyltransferase, in turn, in tRNA 5′ end processing.     

Human rpL3 induces G₁/S arrest or apoptosis by modulating p21waf1/cip1 levels in a p53-independent manner

It is now largely accepted that ribosomal proteins may be implicated in a variety of biological functions besides that of components of the translation machinery. Many evidences show that a subset of ribosomal proteins are involved in the regulation of the cell cycle and apoptosis through modulation of p53 activity. In addition, p53-independent mechanisms of cell cycle arrest in response to alterations of ribosomal proteins availability have been described. Here, we identify human rpL3 as a new regulator of cell cycle and apoptosis through positive regulation of p21 expression in a p53-independent system. We demonstrate that the rpL3-mediated p21 upregulation requires the specific interaction between rpL3 and Sp1. Furthermore, in our experimental system, p21 overexpression leads to a dual outcome, activating the G₁/S arrest of the cell cycle or the apoptotic pathway through mitochondria, depending on its intracellular levels. It is noteworthy that depletion of p21 abrogates both effects. Taken together, our findings unravel a novel extraribosomal function of rpL3 and reinforce the proapoptotic role of p21 in addition to its widely reported ability as an inhibitor of cell proliferation.     

Metazoan stress granule assembly is mediated by P-eIF2α-dependent and -independent mechanisms

Stress granules (SGs) are cytoplasmic bodies wherein translationally silenced mRNAs are recruited for triage in response to environmental stress. We report that Drosophila cells form SGs in response to arsenite and heat shock. Drosophila SGs, like mammalian SGs, are distinct from but adjacent to processing bodies (PBs, sites of mRNA silencing and decay), require polysome disassembly, and are in dynamic equilibrium with polysomes. We further examine the role of the two Drosophila eIF2α kinases, PEK and GCN2, in regulating SG formation in response to heat and arsenite stress. While arsenite-induced SGs are dependent upon eIF2α phosphorylation, primarily via PEK, heat-induced SGs are phospho-eIF2α-independent. In contrast, heat-induced SGs require eIF2α phosphorylation in mammalian cells, as non-phosphorylatable eIF2α Ser51Ala mutant murine embryonic fibroblasts do not form SGs even after severe heat shock. These results suggest that mammals evolved alternative mechanisms for dealing with thermal stress.     

Resveratrol recruits rat muscle microvasculature via a nitric oxide-dependent mechanism that is blocked by TNFα

Resveratrol, a polyphenol found in many plants, has antioxidant and anti-inflammatory actions. It also improves endothelial function and may be cardioprotective. Tumor necrosis factor-α (TNFα) causes oxidative stress and microvascular endothelial dysfunction. Whether resveratrol affects microvascular function in vivo and, if so, whether inflammatory cytokines antagonize its microvascular action are not clear. In cultured bovine aortic endothelial cells (BAECs), resveratrol (100 nM) increased the phosphorylation of protein kinase B (Akt), endothelial nitric oxide (NO) synthase (eNOS), and ERK1/2 within 15 min by more than twofold, and this effect lasted for at least 2 h. Treatment of BAECs with TNFα (10 ng/ml) significantly increased the NADPH oxidase activity and the production of hydrogen peroxide and superoxide. Pretreatment of cells with resveratrol (100 nM) prevented each of these. Injection (ip) of resveratrol in rats potently increased muscle microvascular blood volume (MBV; P = 0.007) and flow (MBF; P < 0.02) within 30 min, and this was sustained for at least 2 h. The phosphorylation of Akt in liver or muscle was unchanged. Superimposed systemic infusion of L-NAME (NOS inhibitor) completely abolished resveratrol-induced increases in MBV and MBF. Similarly, systemic infusion of TNFα prevented resveratrol-induced muscle microvascular recruitment. In conclusion, resveratrol activates eNOS and increases muscle microvascular recruitment via an NO-dependent mechanism. Despite the potent antioxidant effect of resveratrol, TNFα at concentrations that block insulin-mediated muscle microvascular recruitment completely neutralized resveratrol's microvascular action. Thus, chronic inflammation, as seen in type 2 diabetes, may limit resveratrol's vasodilatory actions on muscle microvasculature.     

Promotion of mitochondrial biogenesis via the regulation of PARIS and PGC-1α by parkin as a mechanism of neuroprotection by carnosic acid

BackgroundImpairment of mitochondrial biogenesis is associated with the pathological progression of Parkinson's disease (PD). Parkin-interacting substrate (PARIS) can be ubiquitinated by parkin and prevents the repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α).PurposeThis study investigated whether the neuroprotective mechanism of carnosic acid (CA) from rosemary is mediated via the regulation of PARIS and PGC-1α by parkin.MethodsThe Western blotting and RT-PCR were used to determine protein and mRNA, respectively. To investigate the protein-protein interaction of between PARIS and ubiquitin, the immunoprecipitation assay (IP assay) was utilized. Silencing of endogenous parkin or PGC-1α was performed by using transient transfection of small interfering RNA (siRNA).ResultsSH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA) increased PARIS protein, decreased PGC-1α protein, and reduced protein and mRNA of mitochondrial biogenesis-related genes. CA pretreatment reversed the effects of 6-OHDA. By IP assay, the interaction of PARIS with ubiquitin protein caused by CA was stronger than that caused by 6-OHDA. Moreover, knockdown of parkin attenuated the ability of CA to reverse the 6-OHDA-induced increase in PARIS and decrease in PGC-1α expression. PGC-1α siRNA was used to investigate how CA influenced the effect of 6-OHDA on the modulation of mitochondrial biogenesis and apoptosis. In the presence of PGC-1α siRNA, CA could no longer significantly reverse the reduction of mitochondrial biogenesis or the induction of cleavage of apoptotic-related proteins by 6-OHDA.ConclusionThe cytoprotective of CA is related to the enhancement of mitochondrial biogenesis by inhibiting PARIS and inducing PGC-1α by parkin. The activation of PGC-1α-mediated mitochondrial biogenesis by CA prevents the degeneration of dopaminergic neurons, CA may have therapeutic application in PD.     

Evidence that expression of SpαI/65 hereditary elliptocytosis is compounded by a genetic factor that is linked to the homologous α-spectrin allele

Summary Many cases of hereditary elliptocytosis (HE) result from mutated spectrin -chains. It has repeatedly been observed that the amount of a mutant -chain is different in various affected individuals, resulting in clinical pictures of variable severity. The different levels are thought to result from different percentages of the spectrin allele in trans. Such percentages, in turn, could be under genetic control. We tested this hypothesis in a large Algerian family with Sp^I/65 HE. In an informative sibship, we found three persons with a distinctly high level of expression of the Sp^I/65 variant, suggesting the existence, in trans, of a low percentage -allele. The -spectrin gene haplotype associated with the latter was constantly – +–, based on the XbaI, PvuII and MspI polymorphic sites. In contrast, a basal level of expression of the Sp^I/65 variant in the same sibship indicated, in trans, the existence of a normal percentage -allele. The haplotype corresponding to this other -allele was + – +. Study of another generation of the family showed, however, that the – + – haplotype could also be linked to a normal percentage -allele. These results are consistent with the view that the expression level of ^I/65 spectrin (and of other types of -variants) is compounded by a genetic factor that is linked to the normal -allele in trans. The low percentage allele itself remains silent in the simple heterozygous state.     

Is G1 arrest in plant seeds induced by a p53-related pathway?

In mammals, p53 is crucial for inducing the genes that lead to G1 arrest following DNA damage, enabling DNA repair. However, the possibility that such a system exists in plants has attracted little attention. Even though some plant cDNA sequences with partial homology to p53 have been reported recently, there has been little analysis of how these molecules might relate to DNA damage. The lack of investigation into whether a DNA-damage-induced, p53-mediated G1-arrest pathway might exist in plants is remarkable given that plant DNA, like that of all organisms, is continually under the threat of attack.     

Mycoplasma capricolum membranes induce tumor necrosis factor α by a mechanism different from that of lipopolysaccharide

Summary Heat-inactivated (60°C, 45 min)Mycoplasma capricolum strain JR cells activate murine macrophages to secrete high levels of tumór necrosis factor (TNF) and to lyse tumor target cells efficiently. Fractionation of the intactM. capricolum cells, obtained from cells harvested at the exponential phase of growth, shows that their capacity to induce TNF secretion by macrophage resides exclusively in the membrane fraction. The macrophage-mediated cytolysis following activation byM. capricolum membranes was significantly inhibited by specific anti-recombinant murine TNF antibodies.M. capricolum membranes are a potent inducer of TNF as the commonly used bacterial lipopolysaccharide, indicated by their doseresponse curve for macrophage activation. Our study further showed thatM. capricolum membranes and lipopolysaccharide synergize to augment TNF secretion by C57BL/6-derived macrophages markedly. Moreover, lipopolysaccharide-unresponsive C3H/HeJ-derived macrophages, were pronouncedly activated byM. capricolum membranes, which do not contain lipopolysaccharide. These findings suggest that the mechanism by whichM. capricolum membranes activate macrophages differs from that of lipopolysaccharide. Results of preliminary experiments show that human monocytes as well secrete TNF following activation byM. capricolum membranes. Thus, in contrast with the prohibitive toxicity of lipopolysaccharide to animals and humans,M. capricolum membranes, which contain no lipopolysaccharide and are nontoxic in nature, may be of therapeutic value in the treatment of cancer.This study was supported by the Ernst David Bergmann Fund of the Hebrew University, Jerusalem, the Concern II Foundation for Cancer Research, Los Angeles, and the Society of Research Associates of the Lautenberg Center