RESUMEN

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation.

Asunto(s)

RESUMEN

The correct inheritance of chromatin structure is key for maintaining genome function and cell identity and preventing cellular transformation. DEK, a conserved non-histone chromatin protein, has recognized tumor-promoting properties, its overexpression being associated with poor prognosis in various cancer types. At the cellular level, DEK displays pleiotropic functions, influencing differentiation, apoptosis and stemness, but a characteristic oncogenic mechanism has remained elusive. Here, we report the identification of DEK bodies, focal assemblies of DEK that regularly occur at specific, yet unidentified, sites of heterochromatin replication exclusively in late S-phase. In these bodies, DEK localizes in direct proximity to active replisomes in agreement with a function in the early maturation of heterochromatin. A high-throughput siRNA screen, supported by mutational and biochemical analyses, identifies SUMO as one regulator of DEK body formation, linking DEK to the complex SUMO protein network that controls chromatin states and cell fate. This work combines and refines our previous data on DEK as a factor essential for heterochromatin integrity and facilitating replication under stress, and delineates an avenue of further study for unraveling the contribution of DEK to cancer development.

Asunto(s)

RESUMEN

Drug resistance limits the effectiveness of oesophageal adenocarcinoma (OAC) chemotherapies, leading to a poor prognosis for this disease. Elucidation of the underlying resistance mechanisms is key to enabling the identification of more effective treatments. This study, therefore, aims to identify novel therapeutic and/or chemotherapy sensitising drug targets in OAC. Transcriptional data from a cohort of 273 pre-treatment OAC biopsies, from patients who received neoadjuvant chemotherapy followed by surgical resection, were analysed using gene set enrichment analysis (GSEA) to determine differential gene expression between responding and non-responding OAC tumours. From this, 80 genes were selected for high-throughput siRNA screening in OAC cell lines with or without standard chemotherapy treatment. In parallel, cell viability assays were performed using a panel of FDA-approved drugs and combination index (CI) values were calculated to evaluate drug synergy with standard chemotherapy. Mechanisms of synergy were investigated using western blot, propidium iodide flow cytometry, and proliferation assays. Taken together, the screens identified that targeting Src, using either siRNA or the small molecule inhibitor dasatinib, enhanced the efficacy of chemotherapy in OAC cells. Further in vitro functional analysis confirmed Src inhibition to be synergistic with standard OAC chemotherapies, 5-fluorouracil (5-FU), and cisplatin (CDDP). In conclusion, a compound screen together with a functional genomic approach identified Src as a potential chemosensitising target in OAC, which could be assessed in a clinical study for poor prognosis OAC patients.

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic, which has led to a devastating global health crisis. The emergence of variants that escape neutralizing responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2 biology. Using a comprehensive identification of RNA-binding proteins (RBPs) by mass spectrometry (ChIRP-MS) approach, we identify 107 high-confidence cellular factors that interact with the SARS-CoV-2 genome during infection. By systematically knocking down their expression in human lung epithelial cells, we find that the majority of the identified RBPs are SARS-CoV-2 proviral factors. In particular, we show that HNRNPA2B1, ILF3, QKI, and SFPQ interact with the SARS-CoV-2 genome and promote viral RNA amplification. Our study provides valuable resources for future investigations into the mechanisms of SARS-CoV-2 replication and the identification of host-centered antiviral therapies.

Asunto(s)

RESUMEN

In cells undergoing cell-intrinsic apoptosis, mitochondrial outer membrane permeabilization (MOMP) typically marks an irreversible step in the cell death process. However, in some cases, a subpopulation of treated cells can exhibit a sublethal response, termed "minority MOMP." In this phenomenon, the affected cells survive, despite a low level of caspase activation and subsequent limited activation of the endonuclease caspase-activated DNase (DNA fragmentation factor subunit beta). Consequently, these cells can experience DNA damage, increasing the probability of oncogenesis. However, little is known about the minority MOMP response. To discover genes that affect the MOMP response in individual cells, we conducted an imaging-based phenotypic siRNA screen. We identified multiple candidate genes whose downregulation increased the heterogeneity of MOMP within single cells, among which were genes related to mitochondrial dynamics and mitophagy that participate in the mitochondrial quality control (MQC) system. Furthermore, to test the hypothesis that functional MQC is important for reducing the frequency of minority MOMP, we developed an assay to measure the clonogenic survival of caspase-engaged cells. We found that cells deficient in various MQC genes were indeed prone to aberrant post-MOMP survival. Our data highlight the important role of proteins involved in mitochondrial dynamics and mitophagy in preventing apoptotic dysregulation and oncogenesis.

Asunto(s)

RESUMEN

The functional status of the tumor suppressor p53 is a critical component in determining the sensitivity of cancer cells to many chemotherapeutic agents. DNA topoisomerase II (Top2) plays essential roles in DNA metabolism and is the target of FDA approved chemotherapeutic agents. Topoisomerase targeting drugs convert the enzyme into a DNA damaging agent and p53 influences cellular responses to these agents. We assessed the impact of the loss of p53 function on the formation of DNA damage induced by the Top2 poison etoposide. Using human HCT116 cells, we found resistance to etoposide in cell growth assays upon the functional loss of p53. Nonetheless, cells lacking fully functional p53 were etoposide hypersensitive in clonogenic survival assays. This complex role of p53 led us to directly examine the effects of p53 status on topoisomerase-induced DNA damage. A deficiency in functional p53 resulted in elevated levels of the Top2 covalent complexes (Top2cc) in multiple cell lines. Employing genome-wide siRNA screens, we identified a set of genes for which reduced expression resulted in enhanced synthetic lethality upon etoposide treatment of p53 defective cells. We focused on one hit from this screen, ATR, and showed that decreased expression sensitized the p53-defective cells to etoposide in all assays and generated elevated levels of Top2cc in both p53 proficient and deficient cells. Our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53 defective cells could be used to enhance the therapeutic efficacy of Top2 targeting agents.

Asunto(s)

RESUMEN

Type 2 diabetes (T2D) is a chronic metabolic disorder affecting almost half a billion people worldwide. Impaired function of pancreatic ß-cells is both a hallmark of T2D and an underlying factor in the pathophysiology of the disease. Understanding the cellular mechanisms regulating appropriate insulin secretion has been of long-standing interest in the scientific and clinical communities. To identify novel genes regulating insulin secretion we developed a robust arrayed siRNA screen measuring basal, glucose-stimulated, and augmented insulin secretion by EndoC-ßH1 cells, a human ß-cell line, in a 384-well plate format. We screened 521 candidate genes selected by text mining for relevance to T2D biology and identified 23 positive and 68 negative regulators of insulin secretion. Among these, we validated ghrelin receptor (GHSR), and two genes implicated in endoplasmic reticulum stress, ATF4 and HSPA5. Thus, we have demonstrated the feasibility of using EndoC-ßH1 cells for large-scale siRNA screening to identify candidate genes regulating ß-cell insulin secretion as potential novel drug targets. Furthermore, this screening format can be adapted to other disease-relevant functional endpoints to enable large-scale screening for targets regulating cellular mechanisms contributing to the progressive loss of functional ß-cell mass occurring in T2D.

RESUMEN

The bacterial pathogen Listeria monocytogenes invades host cells, ruptures the internalization vacuole, and reaches the cytosol for replication. A high-content small interfering RNA (siRNA) microscopy screen allowed us to identify epithelial cell factors involved in L. monocytogenes vacuolar rupture, including the serine/threonine kinase Taok2. Kinase activity inhibition using a specific drug validated a role for Taok2 in favoring L. monocytogenes cytoplasmic access. Furthermore, we showed that Taok2 recruitment to L. monocytogenes vacuoles requires the presence of pore-forming toxin listeriolysin O. Overall, our study identified the first set of host factors modulating L. monocytogenes vacuolar rupture and cytoplasmic access in epithelial cells.

Asunto(s)

RESUMEN

Synthetic lethal interactions can assist in characterizing protein functions and cellular processes, but they can also be used to identify novel drug targets for the development of innovative cancer therapeutic strategies. Despite recent technological advancements including CRISPR/Cas9 approaches, the systematic assessment of all pairwise gene interactions in humans (~ 200 million pairs) remains an unmet goal. Thus, hypothesis-driven approaches, which prioritize subsets of promising candidate SL interactions for experimental assessment, are critical to expedite the identification of novel SL interactions. Here, we provide a guide to screen and validate focused libraries of promising candidate SL interactions, typically consisting of 50-500 targets. First, we describe two siRNA and image-based screening protocols to rapidly assess candidate SL interactions. Subsequently, we provide methods to validate a subset of the most promising interactions uncovered in the screens. These approaches employ commercially available reagents and standard laboratory equipment to facilitate and expedite the identification of bona fide human SL interactions.

Asunto(s)

RESUMEN

Inflammatory bowel disease (IBD) is thought to be caused by an aberrant host response to the commensal enteric flora in genetically susceptible individuals. Dendritic cells (DCs) play a key role in the regulation of this response as they sample gut commensals. In healthy individuals DCs actively contribute to tolerance upon recognition of these resident bacteria, whereas in individuals with IBD, DCs will initiate an inflammatory response. To mimic the disease response in vitro, human monocyte-derived DCs were matured with E. coli causing the cells to produce high levels of the pro-inflammatory cytokine IL-12/IL-23p40 (p40) and low levels of the anti-inflammatory cytokine IL-10. A siRNA-based screening assay was developed and screened to identify potential therapeutic targets that shift this balance towards an immunosuppressive state with lower levels of p40 and higher levels of IL-10. The screening assay was optimized and quality controlled using non-targeting controls and positive control siRNAs targeting IL12B and TLR4 transcripts. In the primary screen, smartpool siRNAs were screened for reduction in p40 expression, induction of IL-10 levels, or increase in IL-10:p40 ratios without affecting cell viability. All potential targets were taken forward into a confirmation screen in a different DC donor in which four individual siRNAs per target were screened. At least two siRNAs per target should have an effect to be considered a valid target. This screen resulted in a concise list of ten genes, of which their role in DC maturation is currently being investigated.

Asunto(s)

RESUMEN

The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics. As cell productivity directly affects the cost of production, methods are developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity. However, there are no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins. Here, a large-scale, CHO cell-specific small interfering RNA screen is performed to identify genes that consistently enhance antibody production when silenced in a panel of seven CHO cell lines. Four genes, namely, Cyp1a2, Atp5s, Dgki, and P3h2, are identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines. Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, results in a more than 90% increase in specific antibody productivity. Overall, the knockout of Cyp1a2 demonstrates the most significant improvement of antibody production, with a minimal impact on cell growth.

Asunto(s)

RESUMEN

Human Cytomegalovirus (HCMV) is a highly prevalent herpesvirus, persistently infecting between 30 and 100% of the population, depending on socio-economic status (Fields et al., 2013). HCMV remains an important clinical pathogen accounting for more than 60% of complications associated with solid organ transplant patients (Kotton, 2013; Kowalsky et al., 2013; Bruminhent and Razonable, 2014). It is also the leading cause of infectious congenital birth defects and has been linked to chronic inflammation and immune aging (Ballard et al., 1979; Griffith et al., 2016; Jergovic et al., 2019). There is currently no effective vaccine and HCMV antivirals have significant side effects. As current antivirals target viral genes, the virus can develop resistance, reducing drug efficacy. There is therefore an urgent need for new antiviral agents that are effective against HCMV, have better toxicity profiles and are less vulnerable to the emergence of resistant strains. Targeting of host factors that are critical to virus replication is a potential strategy for the development of novel antivirals that circumvent the development of viral resistance. Systematic high throughput approaches provide powerful methods for the identification of novel host-virus interactions. As well as contributing to our basic understanding of virus and cell biology, such studies provide potential targets for the development of novel antiviral agents. High-throughput studies, such as RNA sequencing, proteomics, and RNA interference screens, are useful tools to identify HCMV-induced global changes in host mRNA and protein expression levels and host factors important for virus replication. Here, we summarize new findings on HCMV lytic infection from high-throughput studies since 2014 and how screening approaches have evolved.

Asunto(s)

RESUMEN

Neurofibromatosis type 1 (NF1) and Neurofibromatosis type 2 (NF2) are two dominantly inherited disorders that cause tumors in Schwann cells. NF1 patients have a high risk for malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. NF2 patients have a high risk for schwannomas. To identify potential therapeutic targets in these two tumors, we screened the NF1 MPNST cell line, ST88-14, and the NF2 schwannoma cell line, HEI-193, against ~2000 drugs of known mechanisms of action (including ~600 cancer relevant drugs), and also screened the cell lines against an siRNA library targeting most protein kinases. Both the drug screen and the siRNA screen identified Polo-like kinase 1 (PLK1) among the most potent hits in both cell lines. Since PLK1 acts on the cell cycle primarily at the G2/M transition, the same stage where aurora kinase (AURKA) acts, we explored PLK1 and its relationship to aurora kinase in MPNST. Quantitative profiling of PLK1 inhibitors against a panel of 10 neurofibromatosis cell lines found that they were potent inhibitors and, unlike AURKA inhibitors, were not more selective for NF1 over NF2 tumor cells. Furthermore, one PLK1 inhibitor, BI6727 stabilized tumor volume in MPNST xenografts. We conclude that PLK1 is a therapeutic target for MPNSTs and schwannomas, but inhibitors may have a narrow therapeutic index that limits their use as a single agent.

RESUMEN

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.

Asunto(s)

RESUMEN

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-like vacuole through activation of a Dot/Icm-type IVB secretion system and subsequent translocation of effectors that remodel the host cell. Here a genome-wide small interfering RNA screen and reporter assay were used to identify host proteins required for Dot/Icm effector translocation. Significant, and independently validated, hits demonstrated the importance of multiple protein families required for endocytic trafficking of the C. burnetii-containing vacuole to the lysosome. Further analysis demonstrated that the degradative activity of the lysosome created by proteases, such as TPP1, which are transported to the lysosome by receptors, such as M6PR and LRP1, are critical for C. burnetii virulence. Indeed, the C. burnetii PmrA/B regulon, responsible for transcriptional up-regulation of genes encoding the Dot/Icm apparatus and a subset of effectors, induced expression of a virulence-associated transcriptome in response to degradative products of the lysosome. Luciferase reporter strains, and subsequent RNA-sequencing analysis, demonstrated that particular amino acids activate the C. burnetii PmrA/B two-component system. This study has further enhanced our understanding of C. burnetii pathogenesis, the host-pathogen interactions that contribute to bacterial virulence, and the different environmental triggers pathogens can sense to facilitate virulence.

Asunto(s)

RESUMEN

Cu, Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Although the precise mechanisms of SOD1 mutant (SOD1mut)-induced motoneuron toxicity are still unclear, defects in SOD1 proteostasis are known to have a critical role in ALS pathogenesis. We previously reported that the SOD1mut adopts a conformation that exposes a Derlin-1-binding region (DBR) and that DBR-exposed SOD1 interacts with Derlin-1, leading to motoneuron death. We also found that an environmental change, i.e. zinc depletion, induces a conformational change in WT SOD1 (SOD1WT) to the DBR-exposed conformation, suggesting the presence of an equilibrium state between the DBR-masked and DBR-exposed states even with SOD1WT Here, we conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change, and we used a genome-wide siRNA screen to search for regulators of SOD1 proteostasis. This screen yielded 30 candidate genes that maintained an absence of the DBR-exposed SOD1WT conformation. Among these genes was one encoding DDB1- and CUL4-associated factor 4 (DCAF4), a substrate receptor of the E3 ubiquitin-protein ligase complex. Of note, we found that DCAF4 mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of DBR-exposed SOD1. In summary, our screen identifies DCAF4 as being required for proper proteostasis of DBR-exposed SOD1, which may have potential relevance for the development of therapies for managing ALS.

Asunto(s)

RESUMEN

The cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following DNA damage using an RNAi library of chromatin regulators. We identified genes involved in chromatin remodeling, sister chromatid cohesion, and histone acetylation not previously associated with checkpoint recovery. Among these is the PHD finger protein 6 (PHF6), a gene mutated in Börjeson-Forssman-Lehmann syndrome and leukemic cancers. We find that loss of PHF6 dramatically compromises checkpoint recovery in G2 phase cells. Moreover, PHF6 is rapidly recruited to sites of DNA lesions in a PARP-dependent manner and required for efficient DNA repair through classical non-homologous end joining. These results indicate that PHF6 is a novel DNA damage response regulator that promotes end joining-mediated repair, thereby stimulating timely recovery from the G2 checkpoint.

Asunto(s)

RESUMEN

As obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High-throughput small interfering RNA (siRNA) screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes, including the mediator complex, proteasome function, and mRNA splicing. Focused characterization of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus 1 or influenza A virus replication, suggesting that the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued 7 days postinfection with the addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the nonessential amino acid asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients.IMPORTANCE HCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem, and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and that knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients.

Asunto(s)

RESUMEN

The understanding of muscle tissue formation and regeneration is essential for the development of therapeutic approaches to treat muscle diseases or loss of muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres. To identify new genes controlling myoblast fusion, we performed a siRNA screen in c2c12 myoblasts. The genes identified during this screen were then studied in vivo by knockdown in zebrafish using morpholino. We found that N-alpha-acetyltransferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion, suggesting that Naa15 negatively regulates myogenic cell fusion. We identified two Naa15 orthologous genes in the zebrafish genome: Naa15a and Naa15b. These two orthologs were expressed in the myogenic domain of the somite. Knockdown of zebrafish Naa15a and Naa15b genes induced a "U"-shaped segmentation of the myotome and alteration of myotome boundaries, resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together, these results show that Naa15 regulates myotome formation and myogenesis in fish.

Asunto(s)

RESUMEN

The proteasome is the proteolytic machinery at the center of regulated intracellular protein degradation and participates in various cellular processes. Maintaining the quality of the proteasome is therefore important for proper cell function. It is unclear, however, how proteasomes change over time and how aged proteasomes are disposed. Here, we show that the proteasome undergoes specific biochemical alterations as it ages. We generated Rpn11-Flag/enhanced green fluorescent protein (EGFP) tag-exchangeable knock-in mice and established a method for selective purification of old proteasomes in terms of their molecular age at the time after synthesis. The half-life of proteasomes in mouse embryonic fibroblasts isolated from these knock-in mice was about 16 h. Using this tool, we found increased association of Txnl1, Usp14, and actin with the proteasome and specific phosphorylation of Rpn3 at Ser 6 in 3-day-old proteasomes. We also identified CSNK2A2 encoding the catalytic α' subunit of casein kinase II (CK2α') as a responsible gene that regulates the phosphorylation and turnover of old proteasomes. These findings will provide a basis for understanding the mechanism of molecular aging of the proteasome.

Asunto(s)