Biology subjects

Proud, C. G.

Publications and source records attributed to Proud, C. G..

6 recordsLinked to original sources

bioRxiv2024

PEBP1 amplifies mitochondrial dysfunction induced integrated stress response

Mitochondrial dysfunction is involved in numerous diseases and the aging process. The integrated stress response (ISR) serves as a critical adaptation mechanism to a variety of stresses, including those originating from mitochondria. By utilizing thermal proteome profiling (TPP), we uncovered that phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitory protein (RKIP), is thermally stabilized by stresses which induce mitochondrial ISR. Depletion of PEBP1 impaired mitochondrial ISR activation by reducing eIF2 phosphorylation and subsequent ISR gene expression, which was independent of PEBP1s role in inhibiting the RAF/MEK/ERK pathway. Consistently, overexpression of PEBP1 potentiated ISR activation by heme-regulated inhibitor kinase (HRI), the principal eIF2 kinase in the mitochondrial ISR pathway. Real-time interaction analysis using luminescence complementation in live cells revealed an interaction between PEBP1 and eIF2, which was disrupted by eIF2 S51 phosphorylation. These findings suggest a role for PEBP1 in amplifying mitochondrial stress signals, thereby facilitating an effective cellular response to mitochondrial dysfunction. Therefore, PEBP1 may be a potential therapeutic target for diseases associated with mitochondrial dysfunction.

cell biology↗

bioRxiv2021

Glutamine deficiency in solid tumors confers resistance to ribosomal RNA synthesis inhibitors

Ribosome biogenesis involves the processing of precursor ribosomal RNAs (pre-rRNAs) and sequential assembly with ribosomal proteins. Here we report that nutrient deprivation severely impairs pre-rRNA processing and leads to the accumulation of unprocessed rRNAs. Upon nutrient restoration, the accumulated pre-rRNAs are processed into mature rRNAs that are utilized for ribosome biogenesis. Failure to accumulate pre-rRNAs under nutrient deprivation leads to perturbed ribosome assembly during nutrient restoration and subsequent apoptosis via uL5/uL18-mediated activation of p53. Restoration of glutamine alone activates p53 by triggering uL5/uL18 translation. Induction of uL5/uL18 protein synthesis by glutamine was dependent on the translation factor eukaryotic elongation factor 2 (eEF2), which was in turn dependent on Raf/MEK/ERK signalling. Depriving cells of glutamine prevents the activation of p53 by rRNA synthesis inhibitors. Our data reveals a mechanism that cancer cells can exploit to suppress p53-mediated apoptosis during fluctuations in environmental nutrient availability.

cancer biology↗

bioRxiv2020

TSC-Insensitive Rheb Mutations Induce Oncogenic Transformation Through a Combination of Hyperactive mTORC1 Signalling and Metabolic Reprogramming

The mechanistic target of rapamycin complex 1 (mTORC1) is an important regulator of cellular metabolism that is commonly hyperactivated in cancer. Recent cancer genome screens have identified multiple mutations in Ras-homolog enriched in brain (Rheb), the primary activator of mTORC1, that might act as driver oncogenes by causing hyperactivation of mTORC1. Here, we show that a number of recurrently occurring Rheb mutants drive hyperactive mTORC1 signalling through differing levels of insensitivity to the primary inactivator of Rheb, Tuberous Sclerosis Complex. We show that two activated mutants, Rheb-T23M and E40K, strongly drive increased cell growth, proliferation and anchorage-independent growth resulting in enhanced tumour growth in vivo. Proteomic analysis of cells expressing the mutations revealed, surprisingly, that these two mutants promote distinct oncogenic pathways with Rheb-T23M driving metabolic reprogramming and an increased rate of glycolysis, while Rheb-E40K regulates the translation factor eEF2 and autophagy, likely through a differential interaction with AMPK. Our findings suggest that unique bespoke combination therapies may be utilised to treat cancers according to which Rheb mutant they harbour.

cell biology↗

bioRxiv2020

eEF2 kinase enhances the expression of PD-L1 by promoting the translation of its mRNA

Emerging advances in cancer therapy have transformed the landscape from conventional therapies towards cancer immunotherapy regimens. Recent discoveries have resulted in the development of clinical immune checkpoint inhibitors that are game-changers for cancer immunotherapy. Here we show that eEF2K, an atypical protein kinase that inhibits the elongation stage of protein synthesis, actually promotes the synthesis of PD-L1, an immune checkpoint protein which helps cancer cells to escape from immunosurveillance. Ablation of eEF2K in prostate and lung cancer cells markedly reduced the expression levels of the PD-L1 protein. We show that eEF2K promotes the association of PD-L1 mRNAs with translationally active polyribosomes and that translation of the PD-L1 mRNA is regulated by a uORF (upstream open reading-frame) within its 5-UTR (5-untranslated region) which starts with a non-canonical CUG codon. This inhibitory effect is attenuated by eEF2K thereby allowing higher levels of translation of the PD-L1 coding region and enhanced expression of the PD-L1 protein. Moreover, eEF2K-depleted cancer cells are more vulnerable to immune attack by natural killer cells. Therefore, control of translation elongation can modulate the translation of this specific mRNA, one which contains an uORF that starts with CUG, and perhaps others that contain a similar feature. Taken together, our data reveal that eEF2K regulates PD-L1 expression at the level of the translation of its mRNA by virtue of a uORF in its 5-region. This, and other roles of eEF2K in cancer cell biology (e.g., in cell survival and migration), may be exploited for the design of future therapeutic strategies.

biochemistry↗

bioRxiv2020

Identification of DNA response elements regulating expression of CCAAT/enhancer-binding protein (C/EBP) β and δ during early adipogenesis

Given the high and increasing prevalence of obesity and associated disorders, such as type-2 diabetes, it is important to understand the mechanisms that regulate lipid storage and the differentiation of fat cells, a process termed adipogenesis. Using the well-established mouse 3T3-L1 in vitro model of adipogenesis, we refine how the induction of two key adipogenic transcription factors, CCAAT/enhancer-binding proteins (C/EBPs) {beta} and {delta} are regulated during early adipogenesis. We identify, in the gene promoters of Cebpb and Cebpd, the DNA response elements responsible for binding transcription factors that are activated by cAMP or glucocorticoids. We also show that mitogen-activated protein kinase (MAPK)-interacting kinase 2 (MNK2; Mknk2), which plays a distinct role in diet-induced obesity, is induced during early adipogenesis and identify the functional DNA response elements responsible for regulating its expression. Mknk2 expression is maintained in differentiated 3T3-L1 adipocytes and is expressed at high levels across a range of mouse adipose tissue depots. Together, these new insights help to clarify the transcriptional program of early adipogenesis and identify Mknk2 as one of potentially many genes up-regulated during adipogenesis.

biochemistry↗

bioRxiv2020

capCLIP: a new tool to probe protein synthesis in human cells through capture and identification of the eIF4E-mRNA interactome.

Translation of eukaryotic mRNAs starts with binding of the m7G cap to the protein eIF4E followed by recruitment of other translation initiation factors. eIF4Es essential role in translation suggests the cellular eIF4E-mRNA interactome (or eIF4E cap-ome) may serve as a faithful proxy of cellular translational activity. Here we describe capCLIP, a novel method to systematically capture and quantify the eIF4E cap-ome. To validate capCLIP, we identified the cap-omes in human cells {+/-} the partial mTORC1 inhibitor rapamycin. As expected, TOP (terminal oligopyrimidine) mRNA representation is systematically reduced in rapamycin-treated cells. capCLIP tag data permits refinement of a 7-nucleotide TOP motif (5'-CUYUYYC-3'). We also apply capCLIP to probe the consequences of phosphorylation of eIF4E, whose function had remained unclear. eIF4E phosphorylation drives an overall reduction in eIF4E-mRNA association; strikingly, mRNAs most sensitive to phosphorylation possess short 5'-UTRs. capCLIP provides a sensitive and comprehensive measure of cellular translational activity. We foresee its application as a high-throughput way to assess translation in contexts not amenable to existing methodologies.

biochemistry↗