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18 pages, 2660 KiB

Open AccessArticle

Structural Integrity of Nucleolin Is Required to Suppress TDP-43-Mediated Cytotoxicity in Yeast and Human Cell Models

by Caterina Peggion, Maria Lina Massimino, Daniel Pereira, Sara Granuzzo, Francesca Righetto, Raissa Bortolotto, Jessica Agostini, Geppo Sartori, Alessandro Bertoli and Raffaele Lopreiato

Int. J. Mol. Sci. 2023, 24(24), 17466; https://doi.org/10.3390/ijms242417466 - 14 Dec 2023

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Abstract

The Transactivating response (TAR) element DNA-binding of 43 kDa (TDP-43) is mainly implicated in the regulation of gene expression, playing multiple roles in RNA metabolism. Pathologically, it is implicated in amyotrophic lateral sclerosis and in a class of neurodegenerative diseases broadly going under [...] Read more.

The Transactivating response (TAR) element DNA-binding of 43 kDa (TDP-43) is mainly implicated in the regulation of gene expression, playing multiple roles in RNA metabolism. Pathologically, it is implicated in amyotrophic lateral sclerosis and in a class of neurodegenerative diseases broadly going under the name of frontotemporal lobar degeneration (FTLD). A common hallmark of most forms of such diseases is the presence of TDP-43 insoluble inclusions in the cell cytosol. The molecular mechanisms of TDP-43-related cell toxicity are still unclear, and the contribution to cell damage from either loss of normal TDP-43 function or acquired toxic properties of protein aggregates is yet to be established. Here, we investigate the effects on cell viability of FTLD-related TDP-43 mutations in both yeast and mammalian cell models. Moreover, we focus on nucleolin (NCL) gene, recently identified as a genetic suppressor of TDP-43 toxicity, through a thorough structure/function characterization aimed at understanding the role of NCL domains in rescuing TDP-43-induced cytotoxicity. Using functional and biochemical assays, our data demonstrate that the N-terminus of NCL is necessary, but not sufficient, to exert its antagonizing effects on TDP-43, and further support the relevance of the DNA/RNA binding central region of the protein. Concurrently, data suggest the importance of the NCL nuclear localization for TDP-43 trafficking, possibly related to both TDP-43 physiology and toxicity. Full article

(This article belongs to the Special Issue Yeast as a Model System to Study Human Diseases)

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18 pages, 2479 KiB

Open AccessArticle

Overexpression of Hsp104 by Causing Dissolution of the Prion Seeds Cures the Yeast [PSI⁺] Prion

by Katherine E. Stanford, Xiaohong Zhao, Nathan Kim, Daniel C. Masison and Lois E. Greene

Int. J. Mol. Sci. 2023, 24(13), 10833; https://doi.org/10.3390/ijms241310833 - 29 Jun 2023

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Abstract

The yeast Sup35 protein misfolds into the infectious [PSI⁺] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression [...] Read more.

The yeast Sup35 protein misfolds into the infectious [PSI⁺] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [PSI⁺] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [PSI⁺] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104. Full article

(This article belongs to the Special Issue Yeast as a Model System to Study Human Diseases)

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17 pages, 3712 KiB

Open AccessArticle

Resistance to Chemotherapeutic 5-Fluorouracil Conferred by Modulation of Heterochromatic Integrity through Ino80 Function in Fission Yeast

by Kim Kiat Lim, Nathaniel Zhi Hao Koh, Yi Bing Zeng, Jun Kai Chuan, Raechell Raechell and Ee Sin Chen

Int. J. Mol. Sci. 2023, 24(13), 10687; https://doi.org/10.3390/ijms241310687 - 26 Jun 2023

Cited by 1 | Viewed by 1156

Abstract

5-Fluorouracil (5-FU) is a conventional chemotherapeutic drug widely used in clinics worldwide, but development of resistance that compromises responsiveness remains a major hurdle to its efficacy. The mechanism underlying 5-FU resistance is conventionally attributed to the disruption of nucleotide synthesis, even though research [...] Read more.

5-Fluorouracil (5-FU) is a conventional chemotherapeutic drug widely used in clinics worldwide, but development of resistance that compromises responsiveness remains a major hurdle to its efficacy. The mechanism underlying 5-FU resistance is conventionally attributed to the disruption of nucleotide synthesis, even though research has implicated other pathways such as RNA processing and chromatin dysregulation. Aiming to clarify resistance mechanisms of 5-FU, we tested the response of a collection of fission yeast (Schizosaccharomyces pombe) null mutants, which confer multiple environmental factor responsiveness (MER). Our screen identified disruption of membrane transport, chromosome segregation and mitochondrial oxidative phosphorylation to increase cellular susceptibility towards 5-FU. Conversely, we revealed several null mutants of Ino80 complex factors exhibited resistance to 5-FU. Furthermore, attenuation of Ino80 function via deleting several subunit genes reversed loss of chromosome-segregation fidelity in 5-FU in the loss-of-function mutant of the Argonaute protein, which regulates RNA interference (RNAi)-dependent maintenance of pericentromeric heterochromatin. Our study thus uncovered a critical role played by chromatin remodeling Ino80 complex factors in 5-FU resistance, which may constitute a possible target to modulate in reversing 5-FU resistance. Full article

(This article belongs to the Special Issue Yeast as a Model System to Study Human Diseases)

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20 pages, 2090 KiB

Open AccessArticle

The Histone Deacetylases Hst1 and Rpd3 Integrate De Novo NAD⁺ Metabolism with Phosphate Sensing in Saccharomyces cerevisiae

by Benjamin Groth, Yi-Ching Lee, Chi-Chun Huang, Matilda McDaniel, Katie Huang, Lan-Hsuan Lee and Su-Ju Lin

Int. J. Mol. Sci. 2023, 24(9), 8047; https://doi.org/10.3390/ijms24098047 - 28 Apr 2023

Viewed by 1335

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) is a critical cofactor essential for various cellular processes. Abnormalities in NAD⁺ metabolism have also been associated with a number of metabolic disorders. The regulation and interconnection of NAD⁺ metabolic pathways are not yet completely [...] Read more.

Nicotinamide adenine dinucleotide (NAD⁺) is a critical cofactor essential for various cellular processes. Abnormalities in NAD⁺ metabolism have also been associated with a number of metabolic disorders. The regulation and interconnection of NAD⁺ metabolic pathways are not yet completely understood. By employing an NAD⁺ intermediate-specific genetic system established in the model organism S. cerevisiae, we show that histone deacetylases (HDACs) Hst1 and Rpd3 link the regulation of the de novo NAD⁺ metabolism-mediating BNA genes with certain aspects of the phosphate (Pi)-sensing PHO pathway. Our genetic and gene expression studies suggest that the Bas1–Pho2 and Pho2–Pho4 transcription activator complexes play a role in this co-regulation. Our results suggest a model in which competition for Pho2 usage between the BNA-activating Bas1–Pho2 complex and the PHO-activating Pho2–Pho4 complex helps balance de novo activity with PHO activity in response to NAD⁺ or phosphate depletion. Interestingly, both the Bas1–Pho2 and Pho2–Pho4 complexes appear to also regulate the expression of the salvage-mediating PNC1 gene negatively. These results suggest a mechanism for the inverse regulation between the NAD⁺ salvage pathways and the de novo pathway observed in our genetic models. Our findings help provide a molecular basis for the complex interplay of two different aspects of cellular metabolism. Full article

(This article belongs to the Special Issue Yeast as a Model System to Study Human Diseases)

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19 pages, 4679 KiB

Open AccessArticle

A Functional Yeast-Based Screen Identifies the Host Microtubule Cytoskeleton as a Target of Numerous Chlamydia pneumoniae Proteins

by Carolin Wevers, Mona Höhler, Abel R. Alcázar-Román, Johannes H. Hegemann and Ursula Fleig

Int. J. Mol. Sci. 2023, 24(8), 7618; https://doi.org/10.3390/ijms24087618 - 20 Apr 2023

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Abstract

Bacterial pathogens have evolved intricate ways to manipulate the host to support infection. Here, we systematically assessed the importance of the microtubule cytoskeleton for infection by Chlamydiae, which are obligate intracellular bacteria that are of great importance for human health. The elimination [...] Read more.

Bacterial pathogens have evolved intricate ways to manipulate the host to support infection. Here, we systematically assessed the importance of the microtubule cytoskeleton for infection by Chlamydiae, which are obligate intracellular bacteria that are of great importance for human health. The elimination of microtubules in human HEp-2 cells prior to C. pneumoniae infection profoundly attenuated the infection efficiency, demonstrating the need for microtubules for the early infection processes. To identify microtubule-modulating C. pneumoniae proteins, a screen in the model yeast Schizosaccharomyces pombe was performed. Unexpectedly, among 116 selected chlamydial proteins, more than 10%, namely, 13 proteins, massively altered the yeast interphase microtubule cytoskeleton. With two exceptions, these proteins were predicted to be inclusion membrane proteins. As proof of principle, we selected the conserved CPn0443 protein, which caused massive microtubule instability in yeast, for further analysis. CPn0443 bound and bundled microtubules in vitro and co-localized partially with microtubules in vivo in yeast and human cells. Furthermore, CPn0443-transfected U2OS cells had a significantly reduced infection rate by C. pneumoniae EBs. Thus, our yeast screen identified numerous proteins encoded using the highly reduced C. pneumoniae genome that modulated microtubule dynamics. Hijacking of the host microtubule cytoskeleton must be a vital part of chlamydial infection. Full article

(This article belongs to the Special Issue Yeast as a Model System to Study Human Diseases)

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Review

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26 pages, 991 KiB

Open AccessReview

More than Just Bread and Wine: Using Yeast to Understand Inherited Cytochrome Oxidase Deficiencies in Humans

by Chenelle A. Caron-Godon, Emma Collington, Jessica L. Wolf, Genna Coletta and D. Moira Glerum

Int. J. Mol. Sci. 2024, 25(7), 3814; https://doi.org/10.3390/ijms25073814 - 29 Mar 2024

Viewed by 675

Abstract

Inherited defects in cytochrome c oxidase (COX) are associated with a substantial subset of diseases adversely affecting the structure and function of the mitochondrial respiratory chain. This multi-subunit enzyme consists of 14 subunits and numerous cofactors, and it requires the function of some [...] Read more.

Inherited defects in cytochrome c oxidase (COX) are associated with a substantial subset of diseases adversely affecting the structure and function of the mitochondrial respiratory chain. This multi-subunit enzyme consists of 14 subunits and numerous cofactors, and it requires the function of some 30 proteins to assemble. COX assembly was first shown to be the primary defect in the majority of COX deficiencies 36 years ago. Over the last three decades, most COX assembly genes have been identified in the yeast Saccharomyces cerevisiae, and studies in yeast have proven instrumental in testing the impact of mutations identified in patients with a specific COX deficiency. The advent of accessible genome-wide sequencing capabilities has led to more patient mutations being identified, with the subsequent identification of several new COX assembly factors. However, the lack of genotype–phenotype correlations and the large number of genes involved in generating a functional COX mean that functional studies must be undertaken to assign a genetic variant as being causal. In this review, we provide a brief overview of the use of yeast as a model system and briefly compare the COX assembly process in yeast and humans. We focus primarily on the studies in yeast that have allowed us to both identify new COX assembly factors and to demonstrate the pathogenicity of a subset of the mutations that have been identified in patients with inherited defects in COX. We conclude with an overview of the areas in which studies in yeast are likely to continue to contribute to progress in understanding disease arising from inherited COX deficiencies. Full article

(This article belongs to the Special Issue Yeast as a Model System to Study Human Diseases)

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20 pages, 1106 KiB

Open AccessReview

Homo cerevisiae—Leveraging Yeast for Investigating Protein–Protein Interactions and Their Role in Human Disease

by Florent Laval, Georges Coppin, Jean-Claude Twizere and Marc Vidal

Int. J. Mol. Sci. 2023, 24(11), 9179; https://doi.org/10.3390/ijms24119179 - 24 May 2023

Cited by 1 | Viewed by 1608

Abstract

Understanding how genetic variation affects phenotypes represents a major challenge, particularly in the context of human disease. Although numerous disease-associated genes have been identified, the clinical significance of most human variants remains unknown. Despite unparalleled advances in genomics, functional assays often lack sufficient [...] Read more.

Understanding how genetic variation affects phenotypes represents a major challenge, particularly in the context of human disease. Although numerous disease-associated genes have been identified, the clinical significance of most human variants remains unknown. Despite unparalleled advances in genomics, functional assays often lack sufficient throughput, hindering efficient variant functionalization. There is a critical need for the development of more potent, high-throughput methods for characterizing human genetic variants. Here, we review how yeast helps tackle this challenge, both as a valuable model organism and as an experimental tool for investigating the molecular basis of phenotypic perturbation upon genetic variation. In systems biology, yeast has played a pivotal role as a highly scalable platform which has allowed us to gain extensive genetic and molecular knowledge, including the construction of comprehensive interactome maps at the proteome scale for various organisms. By leveraging interactome networks, one can view biology from a systems perspective, unravel the molecular mechanisms underlying genetic diseases, and identify therapeutic targets. The use of yeast to assess the molecular impacts of genetic variants, including those associated with viral interactions, cancer, and rare and complex diseases, has the potential to bridge the gap between genotype and phenotype, opening the door for precision medicine approaches and therapeutic development. Full article

(This article belongs to the Special Issue Yeast as a Model System to Study Human Diseases)

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18 pages, 1447 KiB

Open AccessReview

TTT (Tel2-Tti1-Tti2) Complex, the Co-Chaperone of PIKKs and a Potential Target for Cancer Chemotherapy

by Sankhadip Bhadra and Yong-jie Xu

Int. J. Mol. Sci. 2023, 24(9), 8268; https://doi.org/10.3390/ijms24098268 - 05 May 2023

Cited by 2 | Viewed by 2967

Abstract

The heterotrimeric Tel2-Tti1-Tti2 or TTT complex is essential for cell viability and highly conserved in eukaryotes. As the co-chaperone of ATR, ATM, DNA-PKcs, mTOR, SMG1, and TRRAP, the phosphatidylinositol 3-kinase-related kinases (PIKKs) and a group of large proteins of 300–500 kDa, the TTT [...] Read more.

The heterotrimeric Tel2-Tti1-Tti2 or TTT complex is essential for cell viability and highly conserved in eukaryotes. As the co-chaperone of ATR, ATM, DNA-PKcs, mTOR, SMG1, and TRRAP, the phosphatidylinositol 3-kinase-related kinases (PIKKs) and a group of large proteins of 300–500 kDa, the TTT plays crucial roles in genome stability, cell proliferation, telomere maintenance, and aging. Most of the protein kinases in the kinome are targeted by co-chaperone Cdc37 for proper folding and stability. Like Cdc37, accumulating evidence has established the mechanism by which the TTT interacts with chaperone Hsp90 via R2TP (Rvb1-Rvb2-Tah1-Pih1) complex or other proteins for co-translational maturation of the PIKKs. Recent structural studies have revealed the α-solenoid structure of the TTT and its interactions with the R2TP complex, which shed new light on the co-chaperone mechanism and provide new research opportunities. A series of mutations of the TTT have been identified that cause disease syndrome with neurodevelopmental defects, and misregulation of the TTT has been shown to contribute to myeloma, colorectal, and non-small-cell lung cancers. Surprisingly, Tel2 in the TTT complex has recently been found to be a target of ivermectin, an antiparasitic drug that has been used by millions of patients. This discovery provides mechanistic insight into the anti-cancer effect of ivermectin and thus promotes the repurposing of this Nobel-prize-winning medicine for cancer chemotherapy. Here, we briefly review the discovery of the TTT complex, discuss the recent studies, and describe the perspectives for future investigation. Full article

(This article belongs to the Special Issue Yeast as a Model System to Study Human Diseases)

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