Distinct types of intramitochondrial protein aggregates protect mitochondria against proteotoxic stress.

Mitochondria consist of hundreds of proteins, most of which are inaccessible to the proteasomal quality control system of the cytosol. How cells stabilize the mitochondrial proteome during challenging conditions remains poorly understood. Here, we show that mitochondria form spatially defined protein aggregates as a stress-protecting mechanism. Two different types of intramitochondrial protein aggregates can be distinguished. The mitoribosomal protein Var1 (uS3m) undergoes a stress-induced transition from a soluble, chaperone-stabilized protein that is prevalent under benign conditions to an insoluble, aggregated form upon acute stress. The formation of Var1 bodies stabilizes mitochondrial proteostasis, presumably by sequestration of aggregation-prone proteins. The AAA chaperone Hsp78 is part of a second type of intramitochondrial aggregate that transiently sequesters proteins and promotes their folding or Pim1-mediated degradation. Thus, mitochondrial proteins actively control the formation of distinct types of intramitochondrial protein aggregates, which cooperate to stabilize the mitochondrial proteome during proteotoxic stress conditions.

The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

The adaptive strategies of cells in the face of CIN.

An increased frequency of chromosome segregation errors, known as chromosomal instability (CIN), leads to accumulation of aneuploid cells with abnormal chromosomal numbers, which impairs viability through negative effects on survival and proliferation under most conditions. Two recent papers find by independent approaches that the key to surviving high levels of CIN is reducing the instability itself, showcasing the remarkable adaptability of the chromosome segregation machinery, in particular the microtubule-kinetochore interface, and highlighting the crucial role that maintaining chromosomal stability plays in cell proliferation.

MitoStores: chaperone-controlled protein granules store mitochondrial precursors in the cytosol.

Hundreds of nucleus-encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post-translational manner. However, the early processes associated with mitochondrial protein targeting remain poorly understood. Here, we show that in Saccharomyces cerevisiae, the cytosol has the capacity to transiently store mitochondrial matrix-destined precursors in dedicated deposits that we termed MitoStores. Competitive inhibition of mitochondrial protein import via clogging of import sites greatly enhances the formation of MitoStores, but they also form during physiological cell growth on nonfermentable carbon sources. MitoStores are enriched for a specific subset of nucleus-encoded mitochondrial proteins, in particular those containing N-terminal mitochondrial targeting sequences. Our results suggest that MitoStore formation suppresses the toxic potential of aberrantly accumulating mitochondrial precursor proteins and is controlled by the heat shock proteins Hsp42 and Hsp104. Thus, the cytosolic protein quality control system plays an active role during the early stages of mitochondrial protein targeting through the coordinated and localized sequestration of mitochondrial precursor proteins.

Experimental Approaches to Generate and Isolate Human Tetraploid Cells.

Cancer cells are frequently affected by large-scale chromosome copy number changes, such as polyploidy or whole chromosome aneuploidy, and thus understanding the consequences of these changes is important for cancer research. In the past, it has been difficult to study the consequences of large-scale genomic changes, especially in pure isogenic populations. Here, we describe two methods to generate tetraploid cells induced either by cytokinesis failure or mitotic slippage. These treatments result in mixed population of diploids and tetraploids that can be analyzed directly. Alternatively, tetraploid populations can be established by single cell clone selection or by fluorescence activated cell sorting. These methods enable to analyze and compare the consequences of whole-genome doubling between the parental cell line, freshly arising tetraploid cells, and post-tetraploid aneuploid clones.

Consequences of trisomy syndromes - 21 and beyond.

The mechanisms underlying pathologies in Down syndrome remain poorly understood. In this forum article we compare the cellular phenotypes of chromosome 21 trisomy with other trisomic cells. We argue that both effects of the extra chromosome 21 and the global consequences of chromosome gain must be considered to understand complex pathologies of Down syndrome.

Consequences of chromosome gain: A new view on trisomy syndromes.

Chromosome gains are detrimental for the development of the human embryo. As such, autosomal trisomies almost always result in spontaneous abortion, and the rare embryos surviving until live birth suffer from a plethora of pathological defects. There is no treatment currently available to ameliorate the consequences of trisomies, such as Down syndrome (trisomy of chromosome 21). Identifying the source of the phenotypes observed in cells with extra chromosomes is crucial for understanding the underlying molecular causes of trisomy syndromes. Although increased expression of the genes localized on the extra chromosome triggers several pathological phenotypes, an alternative model suggests that global, aneuploidy-associated changes in cellular physiology also contribute to the pathology. Here, we compare the molecular consequences of trisomy syndromes in vivo against engineered cell lines carrying various chromosome gains in vitro. We point out several phenotypes that are shared by variable trisomies and, therefore, might be caused by the presence of an extra chromosome per se, independent of its identity. This alternative view may provide useful insights for understanding Down syndrome pathology and open additional opportunities for diagnostics and treatments.

Whole-Genome Duplication Shapes the Aneuploidy Landscape of Human Cancers.

Aneuploidy is a hallmark of cancer with tissue-specific prevalence patterns that suggest it plays a driving role in cancer initiation and progression. However, the contribution of aneuploidy to tumorigenesis depends on both cellular and genomic contexts. Whole-genome duplication (WGD) is a common macroevolutionary event that occurs in more than 30% of human tumors early in tumorigenesis. Although tumors that have undergone WGD are reported to be more permissive to aneuploidy, it remains unknown whether WGD also affects aneuploidy prevalence patterns. Here we analyzed clinical tumor samples from 5,586 WGD- tumors and 3,435 WGD+ tumors across 22 tumor types and found distinct patterns of aneuploidy in WGD- and WGD+ tumors. WGD+ tumors were characterized by more promiscuous aneuploidy patterns, in line with increased aneuploidy tolerance. Moreover, the genetic interactions between chromosome arms differed between WGD- and WGD+ tumors, giving rise to distinct cooccurrence and mutual exclusivity aneuploidy patterns. The proportion of whole-chromosome aneuploidy compared with arm-level aneuploidy was significantly higher in WGD+ tumors, indicating distinct dominant mechanisms for aneuploidy formation. Human cancer cell lines successfully reproduced these WGD/aneuploidy interactions, confirming the relevance of studying this phenomenon in culture. Finally, induction of WGD and assessment of aneuploidy in isogenic WGD-/WGD+ human colon cancer cell lines under standard or selective conditions validated key findings from the clinical tumor analysis, supporting a causal link between WGD and altered aneuploidy landscapes. We conclude that WGD shapes the aneuploidy landscape of human tumors and propose that this interaction contributes to tumor evolution. SIGNIFICANCE: These findings suggest that the interactions between whole-genome duplication and aneuploidy are important for tumor evolution, highlighting the need to consider genome status in the analysis and modeling of cancer aneuploidy.

Consequences of Chromosome Loss: Why Do Cells Need Each Chromosome Twice?

Aneuploidy is a cellular state with an unbalanced chromosome number that deviates from the usual euploid status. During evolution, elaborate cellular mechanisms have evolved to maintain the correct chromosome content over generations. The rare errors often lead to cell death, cell cycle arrest, or impaired proliferation. At the same time, aneuploidy can provide a growth advantage under selective conditions in a stressful, frequently changing environment. This is likely why aneuploidy is commonly found in cancer cells, where it correlates with malignancy, drug resistance, and poor prognosis. To understand this "aneuploidy paradox", model systems have been established and analyzed to investigate the consequences of aneuploidy. Most of the evidence to date has been based on models with chromosomes gains, but chromosome losses and recurrent monosomies can also be found in cancer. We summarize the current models of chromosome loss and our understanding of its consequences, particularly in comparison to chromosome gains.

Genetic instability from a single S phase after whole-genome duplication.

Diploid and stable karyotypes are associated with health and fitness in animals. By contrast, whole-genome duplications-doublings of the entire complement of chromosomes-are linked to genetic instability and frequently found in human cancers1-3. It has been established that whole-genome duplications fuel chromosome instability through abnormal mitosis4-8; however, the immediate consequences of tetraploidy in the first interphase are not known. This is a key question because single whole-genome duplication events such as cytokinesis failure can promote tumorigenesis9. Here we find that human cells undergo high rates of DNA damage during DNA replication in the first S phase following induction of tetraploidy. Using DNA combing and single-cell sequencing, we show that DNA replication dynamics is perturbed, generating under- and over-replicated regions. Mechanistically, we find that these defects result from a shortage of proteins during the G1/S transition, which impairs the fidelity of DNA replication. This work shows that within a single interphase, unscheduled tetraploid cells can acquire highly abnormal karyotypes. These findings provide an explanation for the genetic instability landscape that favours tumorigenesis after tetraploidization.

Loss of USP28 and SPINT2 expression promotes cancer cell survival after whole genome doubling.

BACKGROUND: Whole genome doubling is a frequent event during cancer evolution and shapes the cancer genome due to the occurrence of chromosomal instability. Yet, erroneously arising human tetraploid cells usually do not proliferate due to p53 activation that leads to CDKN1A expression, cell cycle arrest, senescence and/or apoptosis. METHODS: To uncover the barriers that block the proliferation of tetraploids, we performed a RNAi mediated genome-wide screen in a human colorectal cancer cell line (HCT116). RESULTS: We identified 140 genes whose depletion improved the survival of tetraploid cells and characterized in depth two of them: SPINT2 and USP28. We found that SPINT2 is a general regulator of CDKN1A transcription via histone acetylation. Using mass spectrometry and immunoprecipitation, we found that USP28 interacts with NuMA1 and affects centrosome clustering. Tetraploid cells accumulate DNA damage and loss of USP28 reduces checkpoint activation, thus facilitating their proliferation. CONCLUSIONS: Our results indicate three aspects that contribute to the survival of tetraploid cells: (i) increased mitogenic signaling and reduced expression of cell cycle inhibitors, (ii) the ability to establish functional bipolar spindles and (iii) reduced DNA damage signaling.

Systems approaches identify the consequences of monosomy in somatic human cells.

Chromosome loss that results in monosomy is detrimental to viability, yet it is frequently observed in cancers. How cancers survive with monosomy is unknown. Using p53-deficient monosomic cell lines, we find that chromosome loss impairs proliferation and genomic stability. Transcriptome and proteome analysis demonstrates reduced expression of genes encoded on the monosomes, which is partially compensated in some cases. Monosomy also induces global changes in gene expression. Pathway enrichment analysis reveals that genes involved in ribosome biogenesis and translation are downregulated in all monosomic cells analyzed. Consistently, monosomies display defects in protein synthesis and ribosome assembly. We further show that monosomies are incompatible with p53 expression, likely due to defects in ribosome biogenesis. Accordingly, impaired ribosome biogenesis and p53 inactivation are associated with monosomy in cancer. Our systematic study of monosomy in human cells explains why monosomy is so detrimental and reveals the importance of p53 for monosomy occurrence in cancer.

Processes shaping cancer genomes - From mitotic defects to chromosomal rearrangements.

Sequencing of cancer genomes revealed a rich landscape of somatic single nucleotide variants, structural changes of chromosomes, as well as chromosomal copy number alterations. These chromosome changes are highly variable, and simple translocations, deletions or duplications have been identified, as well as complex events that likely arise through activity of several interconnected processes. Comparison of the cancer genome sequencing data with our knowledge about processes important for maintenance of genome stability, namely DNA replication, repair and chromosome segregation, provides insights into the mechanisms that may give rise to complex chromosomal patterns, such as chromothripsis, a complex form of multiple focal chromosome rearrangements. In addition, observations gained from model systems that recapitulate the rearrangements patterns under defined experimental conditions suggest that mitotic errors and defective DNA replication and repair contribute to their formation. Here, we review the molecular mechanisms that contribute to formation of chromosomal aberrations observed in cancer genomes.

Chromosomal instability accelerates the evolution of resistance to anti-cancer therapies.

Aneuploidy is a ubiquitous feature of human tumors, but the acquisition of aneuploidy typically antagonizes cellular fitness. To investigate how aneuploidy could contribute to tumor growth, we triggered periods of chromosomal instability (CIN) in human cells and then exposed them to different culture environments. We discovered that transient CIN reproducibly accelerates the acquisition of resistance to anti-cancer therapies. Single-cell sequencing revealed that these resistant populations develop recurrent aneuploidies, and independently deriving one chromosome-loss event that was frequently observed in paclitaxel-resistant cells was sufficient to decrease paclitaxel sensitivity. Finally, we demonstrated that intrinsic levels of CIN correlate with poor responses to numerous therapies in human tumors. Our results show that, although CIN generally decreases cancer cell fitness, it also provides phenotypic plasticity to cancer cells that can allow them to adapt to diverse stressful environments. Moreover, our findings suggest that aneuploidy may function as an under-explored cause of therapy failure.

Profiling the physiological pitfalls of anti-hepatitis C direct-acting agents in budding yeast.

Sofosbuvir and Daclatasvir are among the direct-acting antiviral (DAA) medications prescribed for the treatment of chronic hepatitis C (CHC) virus infection as combination therapy with other antiviral medications. DAA-based therapy achieves high cure rates, reaching up to 97% depending on the genotype of the causative hepatitis C virus (HCV). While DAAs have been approved as an efficient and well-tolerated therapy for CHC, emerging concerns about adverse cardiac side effects, higher risk of recurrence and occurrence of hepatocellular carcinoma (HCC) and doubts of genotoxicity have been reported. In our study, we investigated in detail physiological off-targets of DAAs and dissected the effects of these drugs on cellular organelles using budding yeast, a unicellular eukaryotic organism. DAAs were found to disturb the architecture of the endoplasmic reticulum (ER) and the mitochondria, while showing no apparent genotoxicity or DNA damaging effect. Our study provides evidence that DAAs are not associated with genotoxicity and highlights the necessity for adjunctive antioxidant therapy to mitigate the adverse effects of DAAs on ER and mitochondria.

Genotoxic stress in constitutive trisomies induces autophagy and the innate immune response via the cGAS-STING pathway.

Gain of even a single chromosome leads to changes in human cell physiology and uniform perturbations of specific cellular processes, including downregulation of DNA replication pathway, upregulation of autophagy and lysosomal degradation, and constitutive activation of the type I interferon response. Little is known about the molecular mechanisms underlying these changes. We show that the constitutive nuclear localization of TFEB, a transcription factor that activates the expression of autophagy and lysosomal genes, is characteristic of human trisomic cells. Constitutive nuclear localization of TFEB in trisomic cells is independent of mTORC1 signaling, but depends on the cGAS-STING activation. Trisomic cells accumulate cytoplasmic dsDNA, which activates the cGAS-STING signaling cascade, thereby triggering nuclear accumulation of the transcription factor IRF3 and, consequently, upregulation of interferon-stimulated genes. cGAS depletion interferes with TFEB-dependent upregulation of autophagy in model trisomic cells. Importantly, activation of both the innate immune response and autophagy occurs also in primary trisomic embryonic fibroblasts, independent of the identity of the additional chromosome. Our research identifies the cGAS-STING pathway as an upstream regulator responsible for activation of autophagy and inflammatory response in human cells with extra chromosomes, such as in Down syndrome or other aneuploidy-associated pathologies.

SUMOylation stabilizes sister kinetochore biorientation to allow timely anaphase.

During mitosis, sister chromatids attach to microtubules from opposite poles, called biorientation. Sister chromatid cohesion resists microtubule forces, generating tension, which provides the signal that biorientation has occurred. How tension silences the surveillance pathways that prevent cell cycle progression and correct erroneous kinetochore-microtubule attachments remains unclear. Here we show that SUMOylation dampens error correction to allow stable sister kinetochore biorientation and timely anaphase onset. The Siz1/Siz2 SUMO ligases modify the pericentromere-localized shugoshin (Sgo1) protein before its tension-dependent release from chromatin. Sgo1 SUMOylation reduces its binding to protein phosphatase 2A (PP2A), and weakening of this interaction is important for stable biorientation. Unstable biorientation in SUMO-deficient cells is associated with persistence of the chromosome passenger complex (CPC) at centromeres, and SUMOylation of CPC subunit Bir1 also contributes to timely anaphase onset. We propose that SUMOylation acts in a combinatorial manner to facilitate dismantling of the error correction machinery within pericentromeres and thereby sharpen the metaphase-anaphase transition.