scholarly journals A mutant p53/Hif1α/miR-30d axis reprograms the secretory pathway promoting the release of a prometastatic secretome

Cell Stress ◽  
2020 ◽  
Vol 4 (11) ◽  
pp. 261-264
Author(s):  
Valeria Capaci ◽  
Fiamma Mantovani ◽  
Giannino Del Sal
Keyword(s):  
Secretory Pathway ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Wild Type ◽  
Metastatic Colonization ◽  
Non Invasive ◽  
Xenograft Models ◽  
Non Coding Rnas ◽  
Cellular Pathways ◽  
And Function

TP53 missense mutations are frequent driver events during tumorigenesis. The majority of TP53 mutations are missense and occur within the DNA binding domain of p53, leading to expression of mutant p53 (mut-p53) proteins that not only lose the tumor suppressive functions of the wild-type (wt-p53) form, but can also acquire novel oncogenic features fostering tumor growth, metastasis and chemoresistance. Mut-p53 affects fundamental cellular pathways and functions through different mechanisms, a major one being the alteration of gene expression. In our recent work (Capaci et al., 2020, Nat Commun) we found that mut-p53, via miR-30d, modifies structure and function of the Golgi apparatus (GA) and induces increased rate of trafficking. This culminates in the release of a pro-malignant secretome, which is capable of remodeling the tumor microenvironment (TME), to increase stiffness of the extracellular matrix (ECM), favouring metastatic colonization, as shown by cell-based assays and experiments of metastatic niche preconditioning in mouse xenograft models. This study provides new insights into the mechanisms by which mut-p53, through induction of non-coding RNAs, can exert pro-tumorigenic functions in a non-cell-autonomous fashion, and highlights potential non-invasive biomarkers and therapeutic targets to treat tumors harboring mut-p53 (Figure 1).

Differential expression of microRNAs in porcine placentas on Days 30 and 90 of gestation

2010 ◽  
Vol 22 (8) ◽  
pp. 1175 ◽  
Author(s):  
Lijie Su ◽  
Shuhong Zhao ◽  
Mengjin Zhu ◽  
Mei Yu
Keyword(s):  
Differential Expression ◽  
Differentially Expressed ◽  
Locked Nucleic Acid ◽  
Chain Reaction ◽  
Stem Loop ◽  
Non Invasive ◽  
Differentially Expressed Mirnas ◽  
Non Coding Rnas ◽  
Polymerase Chain ◽  
And Function

The porcine placenta is classified as a non-invasive epitheliochorial type. To meet the increasing demands for nutrients by the rapidly growing conceptus and/or fetus, the placental microscopic folds undergo significant morphological and biochemical changes during two periods critical for conceptus and/or fetus, namely Days 30–40 and after Day 90 of gestation. MicroRNAs (miRNAs) are a class of small non-coding RNAs that can modulate gene activity by inhibiting the translation or regulation of mRNA degradation. In the present study, we identified 17 differentially expressed miRNAs in porcine placenta on Days 30 and 90 of gestation using a locked nucleic acid (LNA) microRNA array. Stem–loop real-time reverse transcription–polymerase chain reaction confirmed the differential expression of eight selected miRNAs (miR-24, miR-125b, miR-92b, miR-106a, miR-17, let-7i, miR-27a and miR-20). Analysis of targets and the pathways in which these miRNAs are involved revealed that the differentially expressed miRNAs target many genes that are important in various processes, including cell growth, trophoblast differentiation, angiogenesis and formation and maintenance of adherens junctions. The results of the present study suggest potential roles for these differentially expressed miRNAs in porcine placental growth and function.

A functional analysis of the putative polymorphisms A91V and N252S and 22 missense perforin mutations associated with familial hemophagocytic lymphohistiocytosis

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4700-4706 ◽  
Author(s):  
Ilia Voskoboinik ◽  
Marie-Claude Thia ◽  
Joseph A. Trapani
Keyword(s):  
Functional Analysis ◽  
Hemophagocytic Lymphohistiocytosis ◽  
Autosomal Recessive ◽  
Missense Mutations ◽  
Wild Type ◽  
Lytic Function ◽  
Functional Defect ◽  
Target Cell Membrane ◽  
And Function ◽  
Prf1 Gene

Abstract Up to 60% of cases of the autosomal recessive immunodeficiency hemophagocytic lymphohistiocytosis (HLH) are associated with mutations in the perforin (PRF1) gene. In this study, we expressed wild-type and mutated perforin in rat basophil leukemia cells to study the effect on lytic function of the substitutions A91V and N252S (commonly considered to be neutral polymorphisms) and 22 perforin missense substitutions first identified in HLH patients. Surprisingly, we found that A91V perforin was expressed at reduced levels compared with wild-type perforin, resulting in partial loss of lytic capacity. In contrast, expression and function of N252S-substituted perforin were normal. Most HLH-associated mutations resulted in protein degradation (probably due to misfolding) and complete loss of perforin activity, the exception being R232H, which retained approximately 30% wild-type activity. Several other mutated proteins (H222Q, C73R, F157V, and D313V) had no detectable lytic activity but were expressed at normal levels, suggesting that their functional defect might map downstream at the level of the target cell membrane. One further perforin substitution identified in an HLH patient (V183G) was normally expressed and displayed normal lysis. This report represents the first systematic functional analysis of HLH-associated missense mutations and the 2 most common perforin polymorphisms. (Blood. 2005;105:4700-4706)

scholarly journals Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning

2017 ◽  
Vol 114 (9) ◽  
pp. 2265-2270 ◽  
Author(s):  
Justin R. Klesmith ◽  
John-Paul Bacik ◽  
Emily E. Wrenbeck ◽  
Ryszard Michalczyk ◽  
Timothy A. Whitehead
Keyword(s):  
Surface Display ◽  
Protein Solubility ◽  
Fitness Landscapes ◽  
Yeast Surface Display ◽  
Crystallographic Structure ◽  
Missense Mutations ◽  
Classification Models ◽  
Wild Type ◽  
Trade Offs ◽  
And Function

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.

Naturally occurring mutations in the thrombomodulin gene leading to impaired expression and function

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3646-3653 ◽  
Author(s):  
Gabriella Kunz ◽  
Ann-Kristin Öhlin ◽  
Antonella Adami ◽  
Bengt Zöller ◽  
Peter Svensson ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein C ◽  
Thromboembolic Disease ◽  
Missense Mutations ◽  
Wild Type ◽  
Michaelis Constant ◽  
Complementary Dna ◽  
Naturally Occurring ◽  
Cofactor Activity ◽  
And Function

Sporadic mutations in the thrombomodulin (TM) gene occur in patients with both arterial and venous thrombosis, but the effects of these mutations on expression and function are largely unexplored. Full-length wild-type TM complementary DNA (cDNA) was incorporated into vector pcDNA6 for transfection into COS-7 cells for transient expression. Mutagenesis was performed to create 7 TM mutants with natural mutations either previously identified (Ala25Thr, Gly61Ala, Asp468Tyr, Pro477Ser, Pro483Leu) or reported here (an 11-base pair [bp] deletion, del791-801, leading to STOP306, and a missense mutation, Arg385Ser). Four mutations were found to detrimentally affect the level of expression of the TM protein. Of the missense mutations, 3 had reduced expression compared to wild-type TM (100%), Arg385Ser (50.2% ± 5%, P < .001), Pro477Ser (76.8% ± 1%, P < .001), Pro483Leu (82.1% ± 8%, P < .007). No TM protein expression could be detected on the cell surface for mutation del791-801. The cofactor activity of TM in protein C activation was also evaluated. The Michaelis constant (Km) for wild-type thrombin-TM complex was 634 ± 6 nmol/L. Two mutants, with Arg385Ser and Pro477Ser, had increased (P < .0001) Km, 2967 ±  283 nM, and 2342 ± 219 nM, respectively, demonstrating impaired function of the thrombin-TM complex. This work presents biochemical evidence that certain (but not all) natural mutations in the TM gene reduce expression and impair function of the protein on the cell surface, and helps clarify the suggested contribution that these mutations might make to the risk of thromboembolic disease.

scholarly journals Schizophrenia and autism associated mutations and disrupted m6A signal by YTHDF1 cause defects in microtubule function and neurodevelopment

2020 ◽  
Author(s):  
Rohini Roy ◽  
Xiangru Li ◽  
Shengqun Hou ◽  
Yoshie Fujiwara ◽  
Momoe Sukegawa ◽  
...  
Keyword(s):  
Axon Growth ◽  
Mrna Localization ◽  
Microtubule Assembly ◽  
Rna Modification ◽  
Missense Mutations ◽  
Risk Gene ◽  
Cellular Pathways ◽  
And Function ◽  
Neuronal Functions ◽  
Microtubule Function

AbstractBuilding and maintaining neuronal networks and cognitive functions require mRNA localization and regulated protein synthesis in neurons. RNA modification N6-methyl-adenosine (m6A) has recently been shown in axonal and synaptically localized mRNAs whose local activity is required for axon growth, synaptogenesis, and neuronal plasticity. However, no cellular pathways engaging local epitranscriptomic modulation are known to underlie these post-mitotic neuronal functions. Now we report that cytoplasmic m6A reader YTHDF1 is enriched in neurons and required for axonal, dendritic, and spine development. We show that m6A and YTHDF1 are part of a microtubule plus-end associated RNA granule that contains extensive networks of mRNAs organized by autism risk gene adenomatous polyposis coli (APC). Disrupting m6A signals by knocking down methyltransferase METTL14 or YTHDF1, or overexpressing autism or schizophrenia-associated missense mutations I311V or S399L in human METTL14, reduce expression of APC granule and tubulin, disrupt microtubule assembly and function. These results reveal a novel neuronal subcellular locus for epitranscriptomic regulation to promote post-mitotic neurodevelopment.

scholarly journals Rerouting of a Follicle-Stimulating Hormone Analog to the Regulated Secretory Pathway

Endocrinology ◽  
2010 ◽  
Vol 151 (1) ◽  
pp. 388-393 ◽  
Author(s):  
Christopher A. Pearl ◽  
Albina Jablonka-Shariff ◽  
Irving Boime
Keyword(s):  
Secretory Pathway ◽  
Follicular Development ◽  
Biological Action ◽  
Gh3 Cells ◽  
Wild Type ◽  
Hormone Analog ◽  
Intracellular Sorting ◽  
Regulated Secretory Pathway ◽  
Secretion Pathways ◽  
And Function

Abstract LH and FSH are produced by the same gonadotrope cells of the anterior pituitary but differ in their mode of secretion. This coordinated secretion of LH and FSH is essential for normal follicular development and ovulation in females and for spermatogenesis in males. The structural signals encoded in the LH and FSH subunits that govern the intracellular sorting of LH through the regulated secretory pathway and FSH through the constitutive pathway are largely unknown. Our laboratory recently identified the seven amino acid carboxy tail of LHβ as a sorting signal for LH in GH3 cells. Here we compared the morphological features of GH3 cells expressing an FSH analog containing the heptapeptide (FL7AA) with wild-type FSH using confocal microscopy. These experiments were performed to develop a rerouting model for examining structure-function links between secretion pathways of FSH/LH and their biological action. Both FSH- and LH-expressing cells exhibit a fluorescence pattern of randomly dispersed cytoplasmic puncta. FL7AA expressing cells have more intracellular accumulation compared with wild-type FSH and display a unique halo pattern of fluorescence near the plasma membrane. Such a pattern was not observed in cells expressing FSH or LH. Our results demonstrate that this FSH analog containing the carboxy heptapeptide of LHβ is rerouted to the regulated secretory pathway in GH3 cells. This rerouted gonadotropin provides a unique model to study the trafficking, regulation, and function of LH and FSH.

scholarly journals A posttranslational modification code for CFTR maturation is altered in cystic fibrosis

2019 ◽  
Vol 12 (562) ◽  
pp. eaan7984 ◽  
Author(s):  
Sandra Pankow ◽  
Casimir Bamberger ◽  
John R. Yates
Keyword(s):  
Cystic Fibrosis ◽  
Posttranslational Modifications ◽  
Protein Misfolding ◽  
Secretory Pathway ◽  
Point Mutations ◽  
Wild Type ◽  
Er Quality Control ◽  
Multiple Organs ◽  
Δf508 Cftr ◽  
And Function

The multistep process regulating the maturation of membrane proteins in the endoplasmic reticulum (ER) and the secretory pathway is disrupted in many protein misfolding disorders. Mutations in the ion channel CFTR that impair its folding and subsequent localization to the plasma membrane cause cystic fibrosis (CF), an inherited and eventually lethal disease that impairs the function of multiple organs, mostly the lungs. Here, we found that proper maturation of CFTR is dependent on cross-talk between phosphorylation and methylation events in the regulatory insertion (RI) element of the protein. Manipulating these posttranslational modifications (PTMs) prevented the maturation of wild-type CFTR and instead induced its degradation by ER quality control systems. Deletion of Phe508(ΔF508), the most prevalent mutation in CF, and other mutations in CFTR that impair its trafficking, such as N1303K, also led to quantitative and qualitative PTM changes that prevented the maturation of misfolded CFTR. Further analysis revealed that a wild-type CFTR–like PTM pattern and function was restored in ΔF508 CFTR when cells were cultured at 28°C but only in the presence of the kinase CK2α. Furthermore, the ability to replicate this PTM pattern predicted the efficacy of treatments in restoring ΔF508 CFTR activity. Accordingly, evaluation of patient information revealed that point mutations of several of the modification sites are associated with clinical CF. These findings identify a minimal quantitative and qualitative PTM code for CFTR maturation that distinguishes correctly folded from misfolded CFTR.

scholarly journals The Function of the Mutant p53-R175H in Cancer

Cancers ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 4088
Author(s):  
Yen-Ting Chiang ◽  
Yi-Chung Chien ◽  
Yu-Heng Lin ◽  
Hui-Hsuan Wu ◽  
Dung-Fang Lee ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Gene Mutations ◽  
Metabolic Reprogramming ◽  
Cancer Development ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Wild Type ◽  
Downstream Signaling ◽  
Hotspot Mutations ◽  
Wild Type P53

Wild-type p53 is known as “the guardian of the genome” because of its function of inducing DNA repair, cell-cycle arrest, and apoptosis, preventing the accumulation of gene mutations. TP53 is highly mutated in cancer cells and most TP53 hotspot mutations are missense mutations. Mutant p53 proteins, encoded by these hotspot mutations, lose canonical wild-type p53 functions and gain functions that promote cancer development, including promoting cancer cell proliferation, migration, invasion, initiation, metabolic reprogramming, angiogenesis, and conferring drug resistance to cancer cells. Among these hotspot mutations, p53-R175H has the highest occurrence. Although losing the transactivating function of the wild-type p53 and prone to aggregation, p53-R175H gains oncogenic functions by interacting with many proteins. In this review, we summarize the gain of functions of p53-R175H in different cancer types, the interacting proteins of p53-R175H, and the downstream signaling pathways affected by p53-R175H to depict a comprehensive role of p53-R175H in cancer development. We also summarize treatments that target p53-R175H, including reactivating p53-R175H with small molecules that can bind to p53-R175H and alter it into a wild-type-like structure, promoting the degradation of p53-R175H by targeting heat-shock proteins that maintain the stability of p53-R175H, and developing immunotherapies that target the p53-R175H–HLA complex presented by tumor cells.

scholarly journals Selective functional inhibition of a tumor-derived p53 mutant by cytosolic chaperones identified using split-YFP in budding yeast

2021 ◽  
Author(s):  
Ashley S Denney ◽  
Andrew D Weems ◽  
Michael A McMurray
Keyword(s):  
Three Dimensional ◽  
Temperature Sensitive ◽  
Mutant P53 ◽  
Missense Mutations ◽  
Tumor Suppressor P53 ◽  
Wild Type ◽  
Hsp90 Inhibition ◽  
New Approach ◽  
Type Function ◽  
Chaperone Interactions

Abstract Life requires the oligomerization of individual proteins into higher-order assemblies. In order to form functional oligomers, monomers must adopt appropriate three-dimensional structures. Molecular chaperones transiently bind nascent or misfolded proteins to promote proper folding. Single missense mutations frequently cause disease by perturbing folding despite chaperone engagement. A misfolded mutant capable of oligomerizing with wild-type proteins can dominantly poison oligomer function. We previously found evidence that human-disease-linked mutations in Saccharomyces cerevisiae septin proteins slow folding and attract chaperones, resulting in a kinetic delay in oligomerization that prevents the mutant from interfering with wild-type function. Here we build upon our septin studies to develop a new approach for identifying chaperone interactions in living cells, and use it to expand our understanding of chaperone involvement, kinetic folding delays, and oligomerization in the recessive behavior of tumor-derived mutants of the tumor suppressor p53. We find evidence of increased binding of several cytosolic chaperones to a recessive, misfolding-prone mutant, p53(V272M). Similar to our septin results, chaperone overexpression inhibits the function of p53(V272M) with minimal effect on the wild type. Unlike mutant septins, p53(V272M) is not kinetically delayed under conditions in which it is functional. Instead, it interacts with wild-type p53 but this interaction is temperature sensitive. At high temperatures or upon chaperone overexpression, p53(V272M) is excluded from the nucleus and cannot function or perturb wild-type function. Hsp90 inhibition liberates mutant p53 to enter the nucleus. These findings provide new insights into the effects of missense mutations.

scholarly journals GENE-59. NOT ALL p53 MUTATIONS ARE CREATED EQUAL: A MURINE ASTROCYTE MODEL FOR HIGH-THROUGHPUT FUNCTIONAL ASSESSMENT OF p53 MISSENSE MUTATIONS

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi110-vi110
Author(s):  
Nathan Rockwell ◽  
Max Staller ◽  
Maria Cannella ◽  
Barak Cohen ◽  
Joshua Rubin
Keyword(s):  
Tumor Suppressor ◽  
P53 Protein ◽  
Single Copy ◽  
Wide Distribution ◽  
Mutant P53 ◽  
Missense Mutations ◽  
P53 Mutations ◽  
Wild Type ◽  
Binding Partners ◽  
Cancer Phenotypes

Abstract The tumor suppressor TP53 (p53) is the most commonly mutated gene in cancer and among the most frequently mutated genes in glioblastoma (GBM). The majority of p53 mutations in GBM are missense mutations in the DNA binding domain that lead to the production of full length mutant p53 protein. In addition to the complete loss of tumor suppressor function, these mutations have gain-of-function (GOF) properties either through attenuation of wild-type function or neomorphic functions. The variability in GOF mutations results in heterogeneity in cancer phenotypes between mutants that remain poorly understood. Here, we developed a murine astrocyte model to functionally assess a library of p53 mutants in parallel. Primary astrocytes were isolated from postnatal day one pups possessing a single copy of wild-type p53 flanked by loxP sites (TRP53f/-). We then built a library of 17 individual alleles of recurring mutations in GBM with flanking loxP sites. When co-transfected into the mouse astrocytes with a plasmid expressing Cre recombinase, the endogenous WT p53 was excised and replaced with a single copy of the mutant allele. In this way, all astrocytes expressed a single copy of mutant p53 from the endogenous p53 locus. As the mutant p53 cells expanded, aliquots of cells were extracted for targeted genomic sequencing of the p53 allele. Comparing the allelic frequencies of each mutant overtime revealed a wide distribution of growth rates between mutants. To validate the screen results, wildtype astrocytes were transduced with mutant p53-IRES-eGFP retrovirus to overexpress one of three mutations with divergent growth phenotypes. As observed in the initial screen, equivalent overexpression of the different p53 mutants was sufficient to induce significant differences in growth phenotype, with astrocytes expressing the Y217C growing the fastest, R172H second, and Y202C growing the slowest. Ongoing studies are evaluating mutation-specific p53 binding partners and transcriptional outputs.

Sign in / Sign up

Export Citation Format

Share Document