A single synonymous mutation determines the phosphorylation and stability of the nascent protein

Abstract p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

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Inherited and Acquired RUNX1 Mutations in Hematologic Disorders

Blood ◽

10.1182/blood.v122.21.sci-5.sci-5 ◽

2013 ◽

Vol 122 (21) ◽

pp. SCI-5-SCI-5

Author(s):

Nancy A. Speck ◽

Xiongwei Cai ◽

Jingping Ge ◽

Philip J. Mason

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Conflicts Of Interest ◽

P53 Protein ◽

Lymphoblastic Leukemia ◽

Genotoxic Stress ◽

Myelogenous Leukemia ◽

Phenotypic Properties ◽

Hematopoietic Stem ◽

Protein Levels

Abstract RUNX1, a DNA binding subunit of core binding factors, is frequently mutated or rearranged in hematopoietic malignancies, including acute myelogenous leukemia (AML), chronic myelomonocytic leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome (MDS). Mutations in RUNX1 can be early events in leukemia, and generate a long-lived pre-leukemic stem cell (pre-LSC). Additionally, it has been reported that loss of function RUNX1 mutations are particularly frequent in radiation-associated MDS and AML, suggesting that pre-existing RUNX1 mutations in a pre-LSC may predispose patients to MDS/AML following DNA damage. Discussion will focus on the phenotypic properties of Runx1-deficient pre-LSCs, and the mechanisms by which Runx1 deficiency contributes to these phenotypes. Pan-hematopoietic Runx1 loss in mice causes a G1 to S-cell cycle delay and decreases apoptosis of pre-LSCs. Runx1-deficient pre-LSCs are radiation- and chemotherapy-resistant, and this correlates with decreased p53 protein levels and an attenuated p53 pathway response. Both p53 protein levels and apoptosis are increased following treatment with Nutlin-3. Runx1-deficient pre-LSCs are smaller, consume less glucose, and produce less ATP than normal hematopoietic stem cells (HSCs). Runx1-deficient stem and progenitor cells have lower overall ribosomal content and skewing in the relative amounts of rRNA and mRNA encoding ribosomal proteins. Analysis of AKT pathway components suggests that the decreased ribosome biogenesis is unlikely to be primarily caused by lower AKT signaling. We hypothesize that one or more of the above-mentioned properties (low p53 levels, decreased metabolism) render Runx1-deficient pre-LSCs less sensitive to genotoxic stress than normal HSCs, allowing a Runx1-deficient pre-LSC population to both perdure and expand in the bone marrow. Disclosures: No relevant conflicts of interest to declare.

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An intrinsically disordered nascent protein interacts with specific regions of the ribosomal surface near the exit tunnel

Communications Biology ◽

10.1038/s42003-021-02752-4 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Valeria Guzman-Luna ◽

Andrew M. Fuchs ◽

Anna J. Allen ◽

Alexios Staikos ◽

Silvia Cavagnero

Keyword(s):

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Intrinsically Disordered Protein ◽

Protein Chain ◽

Net Charge ◽

Weakly Interact ◽

Intrinsically Disordered ◽

Disordered Protein ◽

Exit Tunnel

AbstractThe influence of the ribosome on nascent chains is poorly understood, especially in the case of proteins devoid of signal or arrest sequences. Here, we provide explicit evidence for the interaction of specific ribosomal proteins with ribosome-bound nascent chains (RNCs). We target RNCs pertaining to the intrinsically disordered protein PIR and a number of mutants bearing a variable net charge. All the constructs analyzed in this work lack N-terminal signal sequences. By a combination chemical crosslinking and Western-blotting, we find that all RNCs interact with ribosomal protein L23 and that longer nascent chains also weakly interact with L29. The interacting proteins are spatially clustered on a specific region of the large ribosomal subunit, close to the exit tunnel. Based on chain-length-dependence and mutational studies, we find that the interactions with L23 persist despite drastic variations in RNC sequence. Importantly, we also find that the interactions are highly Mg+2-concentration-dependent. This work is significant because it unravels a novel role of the ribosome, which is shown to engage with the nascent protein chain even in the absence of signal or arrest sequences.

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Ribosomal protein S19 deficiency in zebrafish leads to developmental abnormalities and defective erythropoiesis through activation of p53 protein family

Blood ◽

10.1182/blood-2008-01-132290 ◽

2008 ◽

Vol 112 (13) ◽

pp. 5228-5237 ◽

Cited By ~ 191

Author(s):

Nadia Danilova ◽

Kathleen M. Sakamoto ◽

Shuo Lin

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

P53 Protein ◽

Genotoxic Stress ◽

Erythroid Progenitors ◽

P53 Family ◽

Congenital Diseases ◽

Developmental Abnormalities ◽

Ribosomal Protein S19 ◽

Craniofacial Defects

Abstract Mutations in several ribosomal proteins (RPs) lead to Diamond-Blackfan anemia (DBA), a syndrome characterized by defective erythropoiesis, congenital anomalies, and increased frequency of cancer. RPS19 is the most frequently mutated RP in DBA. RPS19 deficiency impairs ribosomal biogenesis, but how this leads to DBA or cancer remains unknown. We have found that rps19 deficiency in ze-brafish results in hematopoietic and developmental abnormalities resembling DBA. Our data suggest that the rps19-deficient phenotype is mediated by dysregulation of deltaNp63 and p53. During gastrulation, deltaNp63 is required for specification of nonneural ectoderm and its up-regulation suppresses neural differentiation, thus contributing to brain/craniofacial defects. In rps19-deficient embryos, deltaNp63 is induced in erythroid progenitors and may contribute to blood defects. We have shown that suppression of p53 and deltaNp63 alleviates the rps19-deficient phenotypes. Mutations in other ribosomal proteins, such as S8, S11, and S18, also lead to up-regulation of p53 pathway, suggesting it is a common response to ribosomal protein deficiency. Our finding provides new insights into pathogenesis of DBA. Ribosomal stress syndromes represent a broader spectrum of human congenital diseases caused by genotoxic stress; therefore, imbalance of p53 family members may become a new target for therapeutics.

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Polyamine depletion stabilizes p53 resulting in inhibition of normal intestinal epithelial cell proliferation

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.3.c941 ◽

2001 ◽

Vol 281 (3) ◽

pp. C941-C953 ◽

Cited By ~ 61

Author(s):

Li Li ◽

Jaladanki N. Rao ◽

Xin Guo ◽

Lan Liu ◽

Rachel Santora ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Growth Inhibition ◽

Intestinal Mucosa ◽

P53 Protein ◽

P53 Gene ◽

Small Intestinal Mucosa ◽

Small Intestinal ◽

The Stability ◽

P53 Mrna

The p53 nuclear phosphoprotein plays a critical role in transcriptional regulation of target genes involved in growth arrest and apoptosis. The natural polyamines, including spermidine, spermine, and their precursor putrescine, are required for cell proliferation, and decreasing cellular polyamines inhibits growth of the small intestinal mucosa. In the current study, we investigated the mechanisms of regulation of p53 gene expression by cellular polyamines and further determined the role of the gene product in the process of growth inhibition after polyamine depletion. Studies were conducted both in vivo and in vitro using rats and the IEC-6 cell line, derived from rat small intestinal crypt cells. Levels for p53 mRNA and protein, transcription and posttranscription of the p53 gene, and cell growth were examined. Depletion of cellular polyamines by treatment with α-difluoromethylornithine (DFMO) increased p53 gene expression and caused growth inhibition in the intact small intestinal mucosa and the cultured cells. Polyamine depletion dramatically increased the stability of p53 mRNA as measured by the mRNA half-life but had no effect on p53 gene transcription in IEC-6 cells. Induction of p53 mRNA levels in DFMO-treated cells was paralleled by an increase in the rate of newly synthesized p53 protein. The stability of p53 protein was also increased after polyamine depletion, which was associated with a decrease in Mdm2 expression. When polyamine-deficient cells were exposed to exogenous spermidine, a decrease in p53 gene expression preceded an increase in cellular DNA synthesis. Inhibition of the p53 gene expression by using p53 antisense oligodeoxyribonucleotides significantly promoted cell growth in the presence of DFMO. These findings indicate that polyamines downregulate p53 gene expression posttranscriptionally and that growth inhibition of small intestinal mucosa after polyamine depletion is mediated, at least partially, through the activation of p53 gene.

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Poly(C)-binding Protein 2 Regulates the p53 Expression via Interactions with the 5′-Terminal Region of p53 mRNA

International Journal of Molecular Sciences ◽

10.3390/ijms222413306 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13306

Author(s):

Damian M. Janecki ◽

Agata Swiatkowska ◽

Joanna Szpotkowska ◽

Anna Urbanowicz ◽

Martyna Kabacińska ◽

...

Keyword(s):

Binding Protein ◽

P53 Protein ◽

Mrna Level ◽

Genotoxic Stress ◽

Stress Conditions ◽

Mobility Shift ◽

P53 Expression ◽

Double Function ◽

Mobility Shift Assay ◽

P53 Mrna

The p53 protein is one of the major transcriptional factors which guards cell homeostasis. Here, we showed that poly(C)-binding protein 2 (PCBP2) can bind directly to the 5′ terminus of p53 mRNA by means of electrophoretic mobility shift assay. Binding sites of PCBP2 within this region of p53 mRNA were mapped using Pb2+-induced cleavage and SAXS methods. Strikingly, the downregulation of PCBP2 in HCT116 cells resulted in a lower level of p53 protein under normal and stress conditions. Quantitative analysis of p53 mRNA in PCBP2-downregulated cells revealed a lower level of p53 mRNA under normal conditions suggesting the involvement of PCBP2 in p53 mRNA stabilisation. However, no significant change in p53 mRNA level was observed upon PCBP2 depletion under genotoxic stress. Moreover, a higher level of p53 protein in the presence of rapamycin or doxorubicin and the combination of both antibiotics was noticed in PCBP2-overexpressed cells compared to control cells. These observations indicate the potential involvement of PCBP2 in cap-independent translation of p53 mRNA especially occurring under stress conditions. It has been postulated that the PCBP2 protein is engaged in the enhancement of p53 mRNA stability, probably via interacting with its 3′ end. Our data show that under stress conditions PCBP2 also modulates p53 translation through binding to the 5′ terminus of p53 mRNA. Thus PCBP2 emerges as a double-function factor in the p53 expression.

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Reading the phosphorylation code: binding of the 14-3-3 protein to multivalent client phosphoproteins

Biochemical Journal ◽

10.1042/bcj20200084 ◽

2020 ◽

Vol 477 (7) ◽

pp. 1219-1225 ◽

Cited By ~ 4

Author(s):

Nikolai N. Sluchanko

Keyword(s):

Protein Function ◽

Intrinsically Disordered Protein ◽

Structural Basis ◽

Major Protein ◽

Post Translational Modifications ◽

Protein Protein Interaction ◽

Intrinsically Disordered ◽

Biochemical Journal ◽

Multiple Binding ◽

And Control

Many major protein–protein interaction networks are maintained by ‘hub’ proteins with multiple binding partners, where interactions are often facilitated by intrinsically disordered protein regions that undergo post-translational modifications, such as phosphorylation. Phosphorylation can directly affect protein function and control recognition by proteins that ‘read’ the phosphorylation code, re-wiring the interactome. The eukaryotic 14-3-3 proteins recognizing multiple phosphoproteins nicely exemplify these concepts. Although recent studies established the biochemical and structural basis for the interaction of the 14-3-3 dimers with several phosphorylated clients, understanding their assembly with partners phosphorylated at multiple sites represents a challenge. Suboptimal sequence context around the phosphorylated residue may reduce binding affinity, resulting in quantitative differences for distinct phosphorylation sites, making hierarchy and priority in their binding rather uncertain. Recently, Stevers et al. [Biochemical Journal (2017) 474: 1273–1287] undertook a remarkable attempt to untangle the mechanism of 14-3-3 dimer binding to leucine-rich repeat kinase 2 (LRRK2) that contains multiple candidate 14-3-3-binding sites and is mutated in Parkinson's disease. By using the protein-peptide binding approach, the authors systematically analyzed affinities for a set of LRRK2 phosphopeptides, alone or in combination, to a 14-3-3 protein and determined crystal structures for 14-3-3 complexes with selected phosphopeptides. This study addresses a long-standing question in the 14-3-3 biology, unearthing a range of important details that are relevant for understanding binding mechanisms of other polyvalent proteins.

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Self-Assembling Micelles Based on an Intrinsically Disordered Protein Domain

10.26434/chemrxiv.7157507 ◽

2018 ◽

Author(s):

Sarah Klass ◽

Matthew J. Smith ◽

Tahoe Fiala ◽

Jessica Lee ◽

Anthony Omole ◽

...

Keyword(s):

Drug Delivery ◽

Fusion Proteins ◽

Organic Molecules ◽

Intrinsically Disordered Protein ◽

Protein Domain ◽

Enzymatic Cleavage ◽

Intrinsically Disordered ◽

Disordered Protein ◽

Self Assembling ◽

Protein Tag

Herein, we describe a new series of fusion proteins that have been developed to self-assemble spontaneously into stable micelles that are 27 nm in diameter after enzymatic cleavage of a solubilizing protein tag. The sequences of the proteins are based on a human intrinsically disordered protein, which has been appended with a hydrophobic segment. The micelles were found to form across a broad range of pH, ionic strength, and temperature conditions, with critical micelle concentration (CMC) values below 1 µM being observed in some cases. The reported micelles were found to solubilize hydrophobic metal complexes and organic molecules, suggesting their potential suitability for catalysis and drug delivery applications.

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