Protein synthesis is a key regulated cellular process that links nutrient availability and organismal growth. It has long been known that some cellular proteins continue to be synthesized under ...conditions where global translation is severely compromised. One prominent example is the selective translation of heat shock proteins (Hsps) under stress conditions. Although the transcriptional regulation of Hsp genes has been well established, neither the specific translation-promoting features nor the regulatory mechanism of the translation machinery have been clearly defined. Here we show that the stress-induced preferential translation of Hsp70 mRNA is negatively regulated by PI3K-mTORC1 signaling. Despite the transcriptional up-regulation, the translation of Hsp70 mRNA is deficient in cells lacking tuberous sclerosis complex 2. Conversely, Hsp70 synthesis is enhanced under the reduced PI3K-mTORC1 signaling. We found that the 5′ UTR of Hsp70 mRNA contributes to cap-independent translation without exhibiting typical features of internal ribosome entry site. Our findings imply a plausible mechanism for how persistent PI3K-mTORC1 signaling favors the development of age-related pathologies by attenuating stress resistance.
The cytoplasm is protected against the perils of protein misfolding by two mechanisms: molecular chaperones (which facilitate proper folding) and the ubiquitin-proteasome system, which regulates ...degradation of misfolded proteins. CHIP (carboxyl terminus of Hsp70-interacting protein) is an Hsp70-associated ubiquitin ligase that participates in this process by ubiquitylating misfolded proteins associated with cytoplasmic chaperones. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. Using a proteomics approach, we have identified BAG2, a previously uncharacterized BAG domain-containing protein, as a common component of CHIP holocomplexes in vivo. Binding assays indicate that BAG2 associates with CHIP as part of a ternary complex with Hsc70, and BAG2 colocalizes with CHIP under both quiescent conditions and after heat shock. In vitro and in vivo ubiquitylation assays indicate that BAG2 is an efficient and specific inhibitor of CHIP-dependent ubiquitin ligase activity. This activity is due, in part, to inhibition of interactions between CHIP and its cognate ubiquitin-conjugating enzyme, UbcH5a, which may in turn be facilitated by ATP-dependent remodeling of the BAG2-Hsc70-CHIP heterocomplex. The association of BAG2 with CHIP provides a cochaperone-dependent regulatory mechanism for preventing unregulated ubiquitylation of misfolded proteins by CHIP.
Translational Regulation in Nutrigenomics Liu, Botao; Qian, Shu-Bing
Advances in nutrition (Bethesda, Md.),
November 2011, 2011-Nov, 2011-11-00, 20111101, Letnik:
2, Številka:
6
Journal Article
Recenzirano
Odprti dostop
The emergence of genome-wide analysis to interrogate cellular DNA, RNA, and protein content has revolutionized the study of the control network that mediates cellular homeostasis. Nutrigenomics ...addresses the effect of nutrients on gene expression, which provides a basis for understanding the biological activity of dietary components. Translation of mRNAs represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular, under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are influenced by nutrient signaling. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during pathophysiological conditions by translation of selective mRNAs. Here we describe recent advances in our understanding of translational control, nutrient signaling, and their dysregulation in aging and cancer. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health.
Approximately 30% of polypeptides synthesized by mammalian cells are degraded with a half-life of <10 min by proteasomes. These rapidly degraded polypeptides (RDPs) constitute the bulk of proteasome ...substrates and are the principal source of viral and self-peptide ligands for major histocompatibility complex class I molecules. Here we provide evidence that ∼75% of RDPs are degraded by the standard ubiquitin 26 S proteasome system and that their degradation is regulated by modulating Hsc70 activity in cells. Surprisingly, the remaining ∼25% of RDPs are degraded without ubiquitylation by 20 S proteasomes independently of 19 S regulators and in a manner that is largely unaffected by modulating Hsc70 activity. This latter pathway is utilized for generating an antigenic peptide from viral-defective ribosomal products. The dichotomy in the behavior of RDPs points to a novel quality control level for nascent proteins that is independent of the well established Hsc70-ubiquitin 26 S proteasome pathway.
Rhenium tricarbonyl complexes have been recently investigated as novel anticancer agents. However, little is understood about their mechanisms of action, as well as the means by which cancer cells ...respond to chronic exposure to these compounds. To gain a deeper mechanistic insight into these rhenium anticancer agents, we developed and characterized an ovarian cancer cell line that is resistant to a previously studied compound Re(CO)3(dmphen)(ptolICN)+, where dmphen=2,9‐dimethyl‐1,10‐phenanthroline and ptolICN=para‐tolyl isonitrile, called TRIP. This TRIP‐resistant ovarian cancer cell line, A2780TR, was found to be 9 times less sensitive to TRIP compared to the wild‐type A2780 ovarian cancer cell line. Furthermore, the cytotoxicities of established drugs and other rhenium anticancer agents in the TRIP‐resistant cell line were determined. Notably, the drug taxol was found to exhibit a 184‐fold decrease in activity in the A2780TR cell line, suggesting that mechanisms of resistance towards TRIP and this drug are similar. Accordingly, expression levels of the ATP‐binding cassette transporter P‐glycoprotein, an efflux transporter known to detoxify taxol, were found to be elevated in the A2780TR cell line. Additionally, a gene expression analysis using the National Cancer Institute 60 cell line panel identified the MT1E gene to be overexpressed in cells that are less sensitive to TRIP. Because this gene encodes for metallothioneins, this result suggests that detoxification by this class of proteins is another mechanism for resistance to TRIP. The importance of this gene in the A2780TR cell line was assessed, confirming that its expression is elevated in this cell line as well. As the first study to investigate and identify the cancer cell resistance pathways in response to a rhenium complex, this report highlights important similarities and differences in the resistance responses of ovarian cancer cells to TRIP and conventional drugs.
TRIPping up resistance! A rhenium‐resistant ovarian cancer cell line was developed by prolonged treatment with a rhenium(I) tricarbonyl isonitrile complex. The cell line was characterized and found to overexpress the ACB transporters P‐glycoprotein and ABCC1, the metallothionein MT1E, and exhibit cross‐resistance with the known anticancer agents taxol and doxorubicin.
Protein ubiquitination regulates numerous cellular functions in eukaryotes. The prevailing view about the role of RING or U-box ubiquitin ligases (E3) is to provide precise positioning between the ...attached substrate and the ubiquitin-conjugating enzyme (E2). However, the mechanism of ubiquitin transfer remains obscure. Using the carboxyl terminus of Hsc70-interacting protein as a model E3, we show herein that although U-box binding is required, it is not sufficient to trigger the transfer of ubiquitin onto target substrates. Furthermore, additional regions of the E3 protein that have no direct contact with E2 play critical roles in mediating ubiquitin transfer from E2 to attached substrates. By combining computational structure modeling and protein engineering approaches, we uncovered a conformational flexibility of E3 that is required for substrate ubiquitination. Using an engineered version of the carboxyl terminus of Hsc70-interacting protein ubiquitin ligase as a research tool, we demonstrate a striking flexibility of ubiquitin conjugation that does not affect substrate specificity. Our results not only reveal conformational changes of E3 during ubiquitin transfer but also provide a promising approach to custom-made E3 for targeted proteolysis.
The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving ...reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.
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•SNAT2 activity mediates biphasic translational control in osmoadaptation•Early-phase stress inhibits translation and induces distinct RBP condensates•Late-phase SNAT2-mediated uptake of amino acids reverses translation inhibition•SNAT2 activity disassembles early-phase RBP condensates
Krokowski et al. report that osmotolerance, in response to mild hypertonic stress, involves a biphasic response of translational control that includes the amino acid transporter SNAT2 and mTOR, independently of ISR. SNAT2-mediated amino acid uptake functions as a critical molecular switch of the biphasic response and thus establishment of osmoadaptation.
Due to a rapidly aging global population, osteoporosis and the associated risk of bone fractures have become a wide-spread public health problem. However, osteoporosis is very heterogeneous, and the ...existing standard diagnostic measure is not sufficient to accurately identify all patients at risk of osteoporotic fractures and to guide therapy. Here, we constructed the first prospective multi-omics atlas of the largest osteoporosis cohort to date (longitudinal data from 366 participants at three time points), and also implemented an explainable data-intensive analysis framework (DLSF: Deep Latent Space Fusion) for an omnigenic model based on a multi-modal approach that can capture the multi-modal molecular signatures (M3S) as explicit functional representations of hidden genotypes. Accordingly, through DLSF, we identified two subtypes of the osteoporosis population in Chinese individuals with corresponding molecular phenotypes, i.e., clinical intervention relevant subtypes (CISs), in which bone mineral density benefits response to calcium supplements in 2-year follow-up samples. Many snpGenes associated with these molecular phenotypes reveal diverse candidate biological mechanisms underlying osteoporosis, with xQTL preferences of osteoporosis and its subtypes indicating an omnigenic effect on different biological domains. Finally, these two subtypes were found to have different relevance to prior fracture and different fracture risk according to 4-year follow-up data. Thus, in clinical application, M3S could help us further develop improved diagnostic and treatment strategies for osteoporosis and identify a new composite index for fracture prediction, which were remarkably validated in an independent cohort (166 participants).
Translation is a crucial process in cancer development and progression. Many oncogenic signaling pathways target the translation initiation stage to satisfy the increased anabolic demands of cancer ...cells. Using quantitative profiling of initiating ribosomes, we found that ribosomal pausing at the start codon serves as a "brake" to restrain the translational output. In response to oncogenic RAS signaling, the initiation pausing relaxes and contributes to the increased translational flux. Intriguingly, messenger RNA (mRNA) m
A modification in the vicinity of start codons influences the behavior of initiating ribosomes. Under oncogenic RAS signaling, the reduced mRNA methylation leads to relaxed initiation pausing, thereby promoting malignant transformation and tumor growth. Restored initiation pausing by inhibiting m
A demethylases suppresses RAS-mediated oncogenic translation and subsequent tumorigenesis. Our findings unveil a paradigm of translational control that is co-opted by RAS mutant cancer cells to drive malignant phenotypes.
The most abundant mRNA post-transcriptional modification is
N
6
-methyladenosine (m
6
A) that has broad roles in RNA biology
1
-
5
. In mammalian cells, the asymmetric distribution of m
6
A along ...mRNAs leaves relatively less methylation in the 5′ untranslated region (5′UTR) compared to other regions
6
,
7
. However, whether and how 5′UTR methylation is regulated is poorly understood. Despite the crucial role of the 5′UTR in translation initiation, very little is known whether m
6
A modification influences mRNA translation. Here we show that in response to heat shock stress, m
6
A is preferentially deposited to the 5′UTR of newly transcribed mRNAs. We found that the dynamic 5′UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well characterized m
6
A “reader”. Upon heat shock stress, the nuclear YTHDF2 preserves 5′UTR methylation of stress-induced transcripts by limiting the m
6
A “eraser” FTO from demethylation. Remarkably, the increased 5′UTR methylation in the form of m
6
A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single site m
6
A modification in the 5′UTR enables translation initiation independent of the 5′ end m
7
G cap. The elucidation of the dynamic feature of 5′UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m
6
A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.
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