Search Results (11)

Background: Obesity, a chronic metabolic disorder, is related to cardiovascular diseases, diabetes, cancer, and reproductive disorders. The relationship between obesity and male infertility is now well recognized, but the mechanisms involved are unclear. We aimed to observe the effect of obesity on spermatogenesis and to investigate the role of histone ubiquitination and acetylation modifications in obesity-induced spermatogenesis disorders. Methods: Thirty male C57BL/6J mice were randomly divided into two groups. The control group was fed with a general maintenance diet (12% fat), while a high-fat diet (HFD) group was fed with 40% fat for 10 weeks; then, they were mated with normal females. The fertility of male mice was calculated, testicular and sperm morphology were observed, and the expression levels of key genes and the levels of histone acetylation and ubiquitination modification during spermatogenesis were detected. Results: The number of sperm was decreased, as well as the sperm motility, while the number of sperm with malformations was increased. In the testes, the mRNA and protein expression levels of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), chromosome region maintenance-1 protein (CRM1), high-mobility group B2 (HMGB2), phosphoglycerate kinase 2 (PGK2), and testicular angiotensin-converting enzyme (tACE) were decreased. Furthermore, obesity led to a decrease in ubiquitinated H2A (ubH2A) and reduced levels of histone H3 acetylation K18 (H3AcK18) and histone H4 acetylation K5, K8, K12, and K16 (H4tetraAck), which disrupted protamine 1 (Prm1) deposition in testis tissue. Conclusion: These results suggest that low levels of histone ubiquitination and acetylation are linked with obesity-induced disorders during spermatogenesis, contributing to a better understanding of obesity-induced damage to male reproduction. Full article

The interactions between Acute Myeloid Leukaemia (AML) leukemic stem cells and the bone marrow (BM) microenvironment play a critical role during AML progression and resistance to drug treatments. Therefore, the identification of novel therapies requires drug-screening methods using in vitro co-culture models that closely recreate the cytoprotective BM setting. We have developed a new fluorescence-based in vitro co-culture system scalable to high throughput for measuring the concomitant effect of drugs on AML cells and the cytoprotective BM microenvironment. eGFP-expressing AML cells are co-cultured in direct contact with mCherry-expressing BM stromal cells for the accurate assessment of proliferation, viability, and signaling in both cell types. This model identified several efficacious compounds that overcome BM stroma-mediated drug resistance against daunorubicin, including the chromosome region maintenance 1 (CRM1/XPO1) inhibitor KPT-330. In silico analysis of genes co-expressed with CRM1, combined with in vitro experiments using our new methodology, also indicates that the combination of KPT-330 with the AURKA pharmacological inhibitor alisertib circumvents the cytoprotection of AML cells mediated by the BM stroma. This new experimental model and analysis provide a more precise screening method for developing improved therapeutics targeting AML cells within the cytoprotective BM microenvironment. Full article

Single-cell imaging has emerged as a powerful means to study viral replication dynamics and identify sites of virus–host interactions. Multivariate aspects of viral replication cycles yield challenges inherent to handling large, complex imaging datasets. Herein, we describe the design and implementation of an automated, imaging-based strategy, “Human Immunodeficiency Virus Red-Green-Blue” (HIV RGB), for deriving comprehensive single-cell measurements of HIV-1 unspliced (US) RNA nuclear export, translation, and bulk changes to viral RNA and protein (HIV-1 Rev and Gag) subcellular distribution over time. Differentially tagged fluorescent viral RNA and protein species are recorded using multicolor long-term (>24 h) time-lapse video microscopy, followed by image processing using a new open-source computational imaging workflow dubbed “Nuclear Ring Segmentation Analysis and Tracking” (NR-SAT) based on ImageJ plugins that have been integrated into the Konstanz Information Miner (KNIME) analytics platform. We describe a typical HIV RGB experimental setup, detail the image acquisition and NR-SAT workflow accompanied by a step-by-step tutorial, and demonstrate a use case wherein we test the effects of perturbing subcellular localization of the Rev protein, which is essential for viral US RNA nuclear export, on the kinetics of HIV-1 late-stage gene regulation. Collectively, HIV RGB represents a powerful platform for single-cell studies of HIV-1 post-transcriptional RNA regulation. Moreover, we discuss how similar NR-SAT-based design principles and open-source tools might be readily adapted to study a broad range of dynamic viral or cellular processes. Full article

Gastric cancer remains an unmet clinical problem in urgent need of newer and effective treatments. Here we show that the nuclear export protein, Exportin 1 (XPO1, chromosome region maintenance 1 or CRM1), is a promising molecular target in gastric cancer. We demonstrate significant overexpression of XPO1 in a cohort of histologically diverse gastric cancer patients with primary and metastatic disease. XPO1 RNA interference suppressed gastric cancer cell growth. Anti-tumor activity was observed with specific inhibitor of nuclear export (SINE) compounds (selinexor/XPOVIO), second-generation compound KPT-8602/eltanexor, KPT-185 and +ve control Leptomycin B in three distinct gastric cancer cell lines. SINE compounds inhibited gastric cancer cell proliferation, disrupted spheroid formation, induced apoptosis and halted cell cycle progression at the G1/S phase. Anti-tumor activity was concurrent with nuclear retention of tumor suppressor proteins and inhibition of colony formation. In combination studies, SINE compounds enhanced the efficacy of nab-paclitaxel in vitro and in vivo. More significantly, using non-coding RNA sequencing studies, we demonstrate for the first time that SINE compounds can alter the expression of non-coding RNAs (microRNAs and piwiRNAs). SINE treatment caused statistically significant downregulation of oncogenic miR-33b-3p in two distinct cell lines. These studies demonstrate the therapeutic significance of XPO1 in gastric cancer that warrants further clinical investigation. Full article

Nuclear transport receptors of the karyopherin superfamily of proteins transport macromolecules from one compartment to the other and are critical for both cell physiology and pathophysiology. The nuclear transport machinery is tightly regulated and essential to a number of key cellular processes since the spatiotemporally expression of many proteins and the nuclear transporters themselves is crucial for cellular activities. Dysregulation of the nuclear transport machinery results in localization shifts of specific cargo proteins and associates with the pathogenesis of disease states such as cancer, inflammation, viral illness and neurodegenerative diseases. Therefore, inhibition of the nuclear transport system has future potential for therapeutic intervention and could contribute to the elucidation of disease mechanisms. In this review, we recapitulate clue findings in the pathophysiological significance of nuclear transport processes and describe the development of nuclear transport inhibitors. Finally, clinical implications and results of the first clinical trials are discussed for the most promising nuclear transport inhibitors. Full article

Pancreatic cancer is a deadly disease that is resistant to most available therapeutics. Pancreatic cancer to date has no effective drugs that could enhance the survival of patients once their disease has metastasized. There is a need for the identification of novel actionable drug targets in this unusually recalcitrant cancer. Nuclear protein transport is an important mechanism that regulates the function of several tumor suppressor proteins (TSPs) in a compartmentalization-dependent manner. High expression of the nuclear exporter chromosome maintenance region 1 (CRM1) or exportin 1 (XPO1), a common feature of several cancers including pancreatic cancer, results in excessive export of critical TSPs to the incorrect cellular compartment, leading to their functional inactivation. Small molecule inhibitors of XPO1 can block this export, retaining very important and functional TSPs in the nucleus and leading to the effective killing of the cancer cells. This review highlights the current knowledge on the role of XPO1 in pancreatic cancer and how this serves as a unique and clinically viable target in this devastating and by far incurable cancer. Full article

Two primary causes of respiratory tract infections are respiratory syncytial virus (RSV) and influenza viruses, both of which remain major public health concerns. There are a limited number of antiviral drugs available for the treatment of RSV and influenza, each having limited effectiveness and each driving selective pressure for the emergence of drug-resistant viruses. Novel broad-spectrum antivirals are needed to circumvent problems with current disease intervention strategies, while improving the cytokine-induced immunopathology associated with RSV and influenza infections. In this review, we examine the use of Verdinexor (KPT-335, a novel orally bioavailable drug that functions as a selective inhibitor of nuclear export, SINE), as an antiviral with multifaceted therapeutic potential. KPT-335 works to (1) block CRM1 (i.e., Chromosome Region Maintenance 1; exportin 1 or XPO1) mediated export of viral proteins critical for RSV and influenza pathogenesis; and (2) repress nuclear factor κB (NF-κB) activation, thus reducing cytokine production and eliminating virus-associated immunopathology. The repurposing of SINE compounds as antivirals shows promise not only against RSV and influenza virus but also against other viruses that exploit the nucleus as part of their viral life cycle. Full article

Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation. Full article

Influenza remains a serious worldwide public health problem. After infection, viral genomic RNA is replicated in the nucleus and packed into viral ribonucleoprotein, which will then be exported to the cytoplasm via a cellular chromosome region maintenance 1 (CRM1)-dependent pathway for further assembly and budding. However, the nuclear export mechanism of influenza virus remains controversial. Here, we identify cellular nuclear transport factor 2 (NTF2)-like export protein 1 (NXT1) as a novel binding partner of nucleoprotein (NP) that stimulates NP-mediated nuclear export via the CRM1-dependent pathway. NXT1-knockdown cells exhibit decreased viral replication kinetics and nuclear accumulated viral RNA and NP. By contrast, NXT1 overexpression promotes nuclear export of NP in a CRM1-dependent manner. Pull-down assays suggest the formation of an NXT1, NP, and CRM1 complex, and demonstrate that NXT1 binds to the C-terminal region of NP. These findings reveal a distinct mechanism for nuclear export of the influenza virus and identify the NXT1/NP interaction as a potential target for antiviral drug development. Full article

Tumour suppressor proteins, such as p53, BRCA1, and ABC, play key roles in preventing the development of a malignant phenotype, but those that function as transcriptional regulators need to enter the nucleus in order to function. The export of proteins between the nucleus and cytoplasm is complex. It occurs through nuclear pores and exported proteins need a nuclear export signal (NES) to bind to nuclear exportin proteins, including CRM1 (Chromosomal Region Maintenance protein 1), and the energy for this process is provided by the RanGTP/RanGDP gradient. Due to the loss of DNA repair and cell cycle checkpoints, drug resistance is a major problem in cancer treatment, and often an initially successful treatment will fail due to the development of resistance. An important mechanism underlying resistance is nuclear export, and a number of strategies that can prevent nuclear export may reverse resistance. Examples include inhibitors of CRM1, antibodies to the nuclear export signal, and alteration of nuclear pore structure. Each of these are considered in this review. Full article

Recent studies have demonstrated the interference of nucleocytoplasmic trafficking with the establishment and maintenance of various cancers. Nucleocytoplasmic transport is highly regulated and coordinated, involving different nuclear transport factors or receptors, importins and exportins, that mediate cargo transport from the cytoplasm into the nucleus or the other way round, respectively. The exportin CRM1 (Chromosome region maintenance 1) exports a plethora of different protein cargoes and ribonucleoprotein complexes. Structural and biochemical analyses have enabled the deduction of individual steps of the CRM1 transport cycle. In addition, CRM1 turned out to be a valid target for anticancer drugs as it exports numerous proto-oncoproteins and tumor suppressors. Clearly, detailed understanding of the flexibility, regulatory features and cooperative binding properties of CRM1 for Ran and cargo is a prerequisite for the design of highly effective drugs. The first compound found to inhibit CRM1-dependent nuclear export was the natural drug Leptomycin B (LMB), which blocks export by competitively interacting with a highly conserved cleft on CRM1 required for nuclear export signal recognition. Clinical studies revealed serious side effects of LMB, leading to a search for alternative natural and synthetic drugs and hence a multitude of novel therapeutics. The present review examines recent progress in understanding the binding mode of natural and synthetic compounds and their inhibitory effects. Full article