Mitosis is commonly thought to be associated with reduced cap-dependent protein translation. Here we show an alternative control mechanism for maintaining cap-dependent translation during mitosis ...revealed by a viral oncoprotein, Merkel cell polyomavirus small T (MCV sT). We find MCV sT to be a promiscuous E3 ligase inhibitor targeting the anaphase-promoting complex, which increases cell mitogenesis. MCV sT binds through its Large T stabilization domain region to cell division cycle protein 20 (Cdc20) and, possibly, cdc20 homolog 1 (Cdh1) E3 ligase adapters. This activates cyclin-dependent kinase 1/cyclin B1 (CDK1/CYCB1) to directly hyperphosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP1) at authentic sites, generating a mitosis-specific, mechanistic target of rapamycin (mTOR) inhibitor-resistant δ phospho-isoform not present in G1-arrested cells. Recombinant 4E-BP1 inhibits capped mRNA reticulocyte translation, which is partially reversed by CDK1/CYCB1 phosphorylation of 4E-BP1. eIF4G binding to the eIF4E–m ⁷GTP cap complex is resistant to mTOR inhibition during mitosis but sensitive during interphase. Flow cytometry, with and without sT, reveals an orthogonal pH3 S¹⁰⁺ mitotic cell population having higher inactive p4E-BP1 ᵀ³⁷/ᵀ⁴⁶⁺ saturation levels than pH3 S¹⁰– interphase cells. Using a Click-iT flow cytometric assay to directly measure mitotic protein synthesis, we find that most new protein synthesis during mitosis is cap-dependent, a result confirmed using the eIF4E/4G inhibitor drug 4E1RCat. For most cell lines tested, cap-dependent translation levels were generally similar between mitotic and interphase cells, and the majority of new mitotic protein synthesis was cap-dependent. These findings suggest that mitotic cap-dependent translation is generally sustained during mitosis by CDK1 phosphorylation of 4E-BP1 even under conditions of reduced mTOR signaling.
Significance Cancer cell proliferation is highly dependent on cap-dependent protein synthesis, which is generally assumed to be inhibited during mitosis. Using a viral oncoprotein that enforces mitosis, we show that CDK1 substitutes for mTOR interphase functions to phosphorylate eukaryotic initiation factor 4E-binding protein (4E-BP1) to a mitosis-specific δ isoform. Flow cytometric assays reveal that mitotic cells have high levels of inactivated 4E-BP1 and do not generally show specific loss of cap-dependent translation compared with interphase cells. This appears to be due to cyclin-dependent kinase 1 (CDK1) activity during mitosis. Mitotic cells typically represent less than 1% of all cells in bulk culture, and mitosis-arresting drugs, such as nocodazole, can directly inhibit mitotic protein translation, potentially explaining differences between our findings and previous studies showing reduced cap-dependent translation during mitosis.
4E-BP1 is a tumor suppressor regulating cap-dependent translation that is in turn controlled by mechanistic target of rapamycin (mTOR) or cyclin-dependent kinase 1 (CDK1) phosphorylation. 4E-BP1 ...serine 82 (S82) is phosphorylated by CDK1, but not mTOR, and the consequences of this mitosis-specific phosphorylation are unknown. Knock-in mice were generated with a single 4E-BP1 S82 alanine (S82A) substitution leaving other phosphorylation sites intact. S82A mice were fertile and exhibited no gross developmental or behavioral abnormalities, but the homozygotes developed diffuse and severe polycystic liver and kidney disease with aging, and lymphoid malignancies after irradiation. Sublethal irradiation caused immature T-cell lymphoma only in S82A mice while S82A homozygous mice have normal T-cell hematopoiesis before irradiation. Whole genome sequencing identified PTEN mutations in S82A lymphoma and impaired PTEN expression was verified in S82A lymphomas derived cell lines. Our study suggests that the absence of 4E-BP1S82 phosphorylation, a subtle change in 4E-BP1 phosphorylation, might predispose to polycystic proliferative disease and lymphoma under certain stressful circumstances, such as aging and irradiation.
In this article, we consider the role of heterogeneous nucleation in β‐amyloid aggregation. Heterogeneous nucleation is more common and occurs at lower levels of supersaturation than homogeneous ...nucleation. The nucleation period is also the stage at which most of the polymorphism of amyloids arises, this being one of the defining features of amyloids. We focus on several well‐known heterogeneous nucleators of β‐amyloid, including lipid surfaces, especially those enriched in gangliosides and cholesterol, and divalent metal ions. These two broad classes of nucleators affect β‐amyloid particularly in light of the amphiphilicity of these peptides: the N‐terminal region, which is largely polar and charged, contains the metal binding site, whereas the C‐terminal region is aliphatic and is important in lipid binding. Notably, these two classes of nucleators can interact cooperatively, aggregation begetting greater aggregation.
► MCV was discovered in 2008 by digital transcriptome subtraction and is one of seven new human polyomaviruses described in the past five years. ► Merkel cell polyomavirus (MCV), a new human ...polyomavirus, is clonally integrated in 70–80% of Merkel cell carcinoma (MCC) tumors. ► MCV is part of the normal, healthy skin flora but causes cancer after viral genome mutations eliminate its replication capacity. ► While similar to known polyomaviruses, MCV oncogenes act in new ways, such as activation of the survivin oncoprotein and PP2A-independent targeting of cap-dependent translation. ► In four years, the diagnosis and treatment potential for an intractable and enigmatic cancer has dramatically changed through discovery of the viral cause of MCC.
Merkel cell polyomavirus (MCV), discovered in 2008, is clonally integrated in ∼80% Merkel cell carcinoma (MCC). MCV is a common skin flora and initiates cancer in susceptible hosts only after it acquires a precise set of mutations that render it replication incompetent. Both MCV large and small T proteins promote cancer cell survival and proliferation. Large T targets pocket proteins regulating cell cycle transit while small T activates cap-dependent translation critical for cancer cell growth. These findings already have led to new diagnostics and clinical trials to target MCV-induced survivin and to promote antitumor immunity. In four years, the cause, diagnosis and therapy for an intractable cancer has been changed due to the molecular discovery of MCV.
ABSTRACT
Chronic obstructive pulmonary disease (COPD) is a growing cause of morbidity and mortality worldwide. Cigarette smoke (CS) exposure, a major cause of COPD, dysregulates airway epithelial ion ...transport and diminishes airway surface liquid (ASL) volume. Short palate lung and nasal epithelial clone 1 (SPLUNC1) is secreted into the airway lumen where it maintains airway hydration via interactions with the epithelial Na+ channel (ENaC). Although ASL hydration is dysregulated in CS‐exposed/COPD airways, effects of CS on SPLUNC1 have not been elucidated. We hypothesized that CS alters SPLUNC1 activity, therefore contributing to ASL dehydration. CS exposure caused irreversible SPLUNC1 aggregation and prevented SPLUNC1 from internalizing ENaC and maintaining ASL hydration. Proteomic analysis revealed αβ‐unsaturated aldehyde modifications to SPLUNCL's cysteine residues. Removal of these cysteines prevented SPLUNC1 from regulating ENaC/ASL volume. In contrast, SPX‐101, a peptide mimetic of natural SPLUNC1, that internalizes ENaC, but does not contain cysteines was unaffected by CS. SPX‐101 increased ASL hydration and attenuated ENaC activity in airway cultures after CS exposure and prolonged survival in a chronic airway disease model. These findings suggest that the CS‐induced defects in SPLUNC1 can be circumvented, thus making SPX‐101 a novel candidate for the treatment of mucus dehydration in COPD.—Moore, P. J., Reidel, B., Ghosh, A., Sesma, J., Kesimer, M., Tarran, R. Cigarette smoke modifies and inactivates SPLUNC1, leading to airway dehydration. FASEB J. 32, 6559–6574 (2018). www.fasebj.org
Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been ...identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses.
Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.
The continuing discoveries of potentially active small RNAs at an unprecedented rate using high-throughput sequencing have raised the need for methods that can reliably detect and quantitate the ...expression levels of small RNAs. Currently, northern blot is the most widely used method for validating small RNAs that are identified by methods such as high-throughput sequencing. We describe a new northern blot-based protocol (LED) for small RNA (~15-40 bases) detection using digoxigenin (DIG)-labeled oligonucleotide probes containing locked nucleic acids (LNA) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide for cross-linking the RNA to the membrane. LED generates clearly visible signals for RNA amounts as low as 0.05 fmol. This method requires as little as a few seconds of membrane exposure to outperform the signal intensity using overnight exposure of isotope-based methods, corresponding to ~1000-fold improvement in exposure-time. In contrast to commonly used radioisotope-based methods, which require freshly prepared and hazardous probes, LED probes can be stored for at least 6 months, facilitate faster and more cost-effective experiments, and are more environmentally friendly. A detailed protocol of LED is provided in the Supplementary Data.
Context:
Insulin resistance can be compensated by increased functional pancreatic β-cell mass; otherwise, diabetes ensues. Such compensation depends not only on environmental and genetic factors but ...also on the baseline β-cell mass from which the expansion originates.
Objective:
Little is known about assembly of a baseline β-cell mass in humans. Here, we examined formation of β-cell populations relative to other pancreatic islet cell types and associated neurons throughout the normal human lifespan.
Design and Methods:
Human pancreatic sections derived from normal cadavers aged 24 wk premature to 72 yr were examined by immunofluorescence. Insulin, glucagon, and somatostatin were used as markers for β-, α-, and δ-cells, respectively. Cytokeratin-19 marked ductal cells, Ki67 cell proliferation, and Tuj1 (neuronal class III β-tubulin) marked neurons.
Results:
Most β-cell neogenesis was observed preterm with a burst of β-cell proliferation peaking within the first 2 yr of life. Thereafter, little indication of β-cell growth was observed. Postnatal proliferation of α- and δ-cells was rarely seen, but a wave of ductal cell proliferation was found mostly associated with exocrine cell expansion. The β-cell to α-cell ratio doubled neonatally, reflecting increased growth of β-cells, but during childhood, there was a 7-fold change in the β-cell to δ-cell ratio, reflecting an additional loss of δ-cells. A close association of neurons to pancreatic islets was noted developmentally and retained throughout adulthood. Negligible neuronal association to exocrine pancreas was observed.
Conclusion:
Human baseline β-cell population and appropriate association with other islet cell types is established before 5 yr of age.
The use of continuous glucose monitoring has been suggested as a method of determining glucose control in prediabetes, including understanding glucose variability, which may be a risk factor in the ...progression to type 2 diabetes. This pilot research study aimed to determine glycemic variability (%CV) in people diagnosed with impaired glucose tolerance (IGT)/prediabetes. The study included people aged 18 or over, with their most recent HbA1c 5.7-6.4% (39-47 mmol/mol) recorded in medical notes in the last 12 months. Pregnant patients were excluded, as were patients with diabetes. A total of 43 participants (60.5% female) from 5 primary care sites in the UK enrolled in the 2-week single arm study, wearing a FreeStyle Libre Pro Flash Glucose Monitoring SystemTM (glucose data was not available to participants). On average, HbA1c was 6.06±0.25% (42.7±2.6 mmol/mol), age was 62.5±8.3 years, BMI was 32.1±6.6 kg/m2, average time since diagnosis was 18±19 months, average Q diabetes score was 28.4±23.7% (mean±SD). Glucose variability (%CV) was 17.3±3.8%, %CV was greater during daytime hours (06:00 to 23:00) than at night (23:00 to 06:00), 17.7±4.1% and 12.9±3.5% respectively (mean±SD). %CV was greater in those with BMI<30 kg/m2 compared to those ≥30 kg/m2; 18.9±3.3% and 15.8±3.5% respectively (mean±SD). Mean glucose was 101.2±10.0 mg/dL, time in hyperglycemia (>180 mg/dL, 10.0 mmol/L) was 0.09±0.23 hours per day (mean±SD). Ten anticipated sensor insertion site symptoms were experienced by five participants: erythema (n=2, well-defined redness), pain (n=1), bruising (n=1), itching (n=2), rash (n=2), bleeding (n=1) and other (n=1, ‘skin irritation’), all were mild in severity and resolved. This population, with prediabetes experienced glucose variability of 17.3% (%CV). This was greater during daytime hours and in those with lower BMI (<30 kg/m2).
Disclosure
K. Douglas: None. N. Annamalai: None. P.J. Moore: None. S. Thomson: Research Support; Self; Abbott. Research Support; Spouse/Partner; Abbott.
Merkel cell carcinoma (MCC) is a neuroendocrine skin cancer associated with high mortality. Merkel cell polyomavirus (MCV), discovered in 2008, is associated with ~80% of MCC. The MCV large tumor ...(LT) oncoprotein upregulates the cellular oncoprotein survivin through its conserved retinoblastoma protein-binding motif. We confirm here that YM155, a survivin suppressor, is cytotoxic to MCV-positive MCC cells in vitro at nanomolar levels. Mouse survival was significantly improved for NOD-Scid-Gamma mice treated with YM155 in a dose and duration dependent manner for 3 of 4 MCV-positive MCC xenografts. One MCV-positive MCC xenograft (MS-1) failed to significantly respond to YM155, which corresponds with in vitro dose-response activity. Combination treatment of YM155 with other chemotherapeutics resulted in additive but not synergistic cell killing of MCC cell lines in vitro. These results suggest that survivin targeting is a promising therapeutic approach for most but not all MCV-positive MCCs.
PDF
