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1.
Mol Cell ; 82(1): 159-176.e12, 2022 01 06.
Article in English | MEDLINE | ID: mdl-34847357

ABSTRACT

The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.


Subject(s)
Cell Nucleus/enzymology , Cell Proliferation , DNA Replication , Exosomes/enzymology , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/enzymology , RNA Polymerase II/metabolism , Transcription, Genetic , Animals , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Cell Line, Tumor , Cell Nucleus/genetics , DNA Breaks, Double-Stranded , Exoribonucleases/genetics , Exoribonucleases/metabolism , Exosomes/genetics , Female , Gene Expression Regulation, Neoplastic , HEK293 Cells , Humans , Male , Mice , N-Myc Proto-Oncogene Protein/genetics , NIH 3T3 Cells , Neuroblastoma/genetics , Neuroblastoma/pathology , Promoter Regions, Genetic , RNA Caps/genetics , RNA Caps/metabolism , RNA Polymerase II/genetics , Transcription Termination, Genetic
2.
Nature ; 612(7938): 148-155, 2022 12.
Article in English | MEDLINE | ID: mdl-36424410

ABSTRACT

Oncoproteins of the MYC family drive the development of numerous human tumours1. In unperturbed cells, MYC proteins bind to nearly all active promoters and control transcription by RNA polymerase II2,3. MYC proteins can also coordinate transcription with DNA replication4,5 and promote the repair of transcription-associated DNA damage6, but how they exert these mechanistically diverse functions is unknown. Here we show that MYC dissociates from many of its binding sites in active promoters and forms multimeric, often sphere-like structures in response to perturbation of transcription elongation, mRNA splicing or inhibition of the proteasome. Multimerization is accompanied by a global change in the MYC interactome towards proteins involved in transcription termination and RNA processing. MYC multimers accumulate on chromatin immediately adjacent to stalled replication forks and surround FANCD2, ATR and BRCA1 proteins, which are located at stalled forks7,8. MYC multimerization is triggered in a HUWE16 and ubiquitylation-dependent manner. At active promoters, MYC multimers block antisense transcription and stabilize FANCD2 association with chromatin. This limits DNA double strand break formation during S-phase, suggesting that the multimerization of MYC enables tumour cells to proliferate under stressful conditions.


Subject(s)
DNA-Directed RNA Polymerases , Humans , Chromatin/genetics , DNA-Directed RNA Polymerases/metabolism , Promoter Regions, Genetic/genetics , RNA Polymerase II/metabolism , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , DNA Breaks, Double-Stranded , S Phase , Binding Sites , RNA, Messenger/biosynthesis
3.
EMBO J ; 40(19): e107985, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34302370

ABSTRACT

Monoclonal anti-SARS-CoV-2 immunoglobulins represent a treatment option for COVID-19. However, their production in mammalian cells is not scalable to meet the global demand. Single-domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein, we isolated 45 infection-blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS-CoV-2 at 17-50 pM concentration (0.2-0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X-ray and cryo-EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune-escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low-picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such "fold-promoting" nanobodies may allow for simplified production of vaccines and their adaptation to viral escape-mutations.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Mutation/immunology , SARS-CoV-2/immunology , Single-Domain Antibodies/immunology , Animals , COVID-19/virology , Camelids, New World/immunology , Camelids, New World/virology , Cell Line , Escherichia coli/virology , Female , Humans , Spike Glycoprotein, Coronavirus/immunology
4.
Nature ; 567(7749): 545-549, 2019 03.
Article in English | MEDLINE | ID: mdl-30894746

ABSTRACT

MYC is an oncogenic transcription factor that binds globally to active promoters and promotes transcriptional elongation by RNA polymerase II (RNAPII)1,2. Deregulated expression of the paralogous protein MYCN drives the development of neuronal and neuroendocrine tumours and is often associated with a particularly poor prognosis3. Here we show that, similar to MYC, activation of MYCN in human neuroblastoma cells induces escape of RNAPII from promoters. If the release of RNAPII from transcriptional pause sites (pause release) fails, MYCN recruits BRCA1 to promoter-proximal regions. Recruitment of BRCA1 prevents MYCN-dependent accumulation of stalled RNAPII and enhances transcriptional activation by MYCN. Mechanistically, BRCA1 stabilizes mRNA decapping complexes and enables MYCN to suppress R-loop formation in promoter-proximal regions. Recruitment of BRCA1 requires the ubiquitin-specific protease USP11, which binds specifically to MYCN when MYCN is dephosphorylated at Thr58. USP11, BRCA1 and MYCN stabilize each other on chromatin, preventing proteasomal turnover of MYCN. Because BRCA1 is highly expressed in neuronal progenitor cells during early development4 and MYC is less efficient than MYCN in recruiting BRCA1, our findings indicate that a cell-lineage-specific stress response enables MYCN-driven tumours to cope with deregulated RNAPII function.


Subject(s)
BRCA1 Protein/metabolism , N-Myc Proto-Oncogene Protein/metabolism , RNA Polymerase II/metabolism , Transcription Elongation, Genetic , Cell Line, Tumor , Chromatin/genetics , Chromatin/metabolism , Gene Expression Regulation , Humans , Neuroblastoma/genetics , Neuroblastoma/pathology , Protein Stability , Thiolester Hydrolases/metabolism
5.
EMBO Rep ; 23(10): e54136, 2022 10 06.
Article in English | MEDLINE | ID: mdl-35912982

ABSTRACT

N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N-terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor-suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in its active site. Intriguingly, DPP9-depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double-strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N-terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9-deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine-tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.


Subject(s)
DNA Repair , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases , Aminopeptidases , DNA , DNA Damage , Dipeptides , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Proline , Rad51 Recombinase/genetics , Serine
6.
Mol Cell ; 61(1): 68-83, 2016 Jan 07.
Article in English | MEDLINE | ID: mdl-26748827

ABSTRACT

The MDM2 oncoprotein ubiquitinates and antagonizes p53 but may also carry out p53-independent functions. Here we report that MDM2 is required for the efficient generation of induced pluripotent stem cells (iPSCs) from murine embryonic fibroblasts, in the absence of p53. Similarly, MDM2 depletion in the context of p53 deficiency also promoted the differentiation of human mesenchymal stem cells and diminished clonogenic survival of cancer cells. Most of the MDM2-controlled genes also responded to the inactivation of the Polycomb Repressor Complex 2 (PRC2) and its catalytic component EZH2. MDM2 physically associated with EZH2 on chromatin, enhancing the trimethylation of histone 3 at lysine 27 and the ubiquitination of histone 2A at lysine 119 (H2AK119) at its target genes. Removing MDM2 simultaneously with the H2AK119 E3 ligase Ring1B/RNF2 further induced these genes and synthetically arrested cell proliferation. In conclusion, MDM2 supports the Polycomb-mediated repression of lineage-specific genes, independent of p53.


Subject(s)
Chromatin Assembly and Disassembly , Induced Pluripotent Stem Cells/metabolism , Mesenchymal Stem Cells/metabolism , Neoplastic Stem Cells/metabolism , Polycomb Repressive Complex 2/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Cell Differentiation , Cell Lineage , Cell Proliferation , Cell Survival , Gene Expression Regulation, Neoplastic , HCT116 Cells , Histones/metabolism , Humans , MCF-7 Cells , Methylation , Mice , Osteogenesis , Phenotype , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 2/genetics , Proto-Oncogene Proteins c-mdm2/genetics , RNA Interference , Signal Transduction , Time Factors , Transfection , Tumor Suppressor Protein p53/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination
7.
Mol Cell ; 59(2): 243-57, 2015 Jul 16.
Article in English | MEDLINE | ID: mdl-26145175

ABSTRACT

Proteasome inhibition represents a promising strategy of cancer pharmacotherapy, but resistant tumor cells often emerge. Here we show that the microRNA-101 (miR-101) targets the proteasome maturation protein POMP, leading to impaired proteasome assembly and activity, and resulting in accumulation of p53 and cyclin-dependent kinase inhibitors, cell cycle arrest, and apoptosis. miR-101-resistant POMP restores proper turnover of proteasome substrates and re-enables tumor cell growth. In ERα-positive breast cancers, miR-101 and POMP levels are inversely correlated, and high miR-101 expression or low POMP expression associates with prolonged survival. Mechanistically, miR-101 expression or POMP knockdown attenuated estrogen-driven transcription. Finally, suppressing POMP is sufficient to overcome tumor cell resistance to the proteasome inhibitor bortezomib. Taken together, proteasome activity can not only be manipulated through drugs, but is also subject to endogenous regulation through miR-101, which targets proteasome biogenesis to control overall protein turnover and tumor cell proliferation.


Subject(s)
MicroRNAs/genetics , MicroRNAs/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Proteasome Inhibitors/metabolism , 3' Untranslated Regions , Animals , Apoptosis , Boronic Acids/pharmacology , Bortezomib , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor Proteins/metabolism , Drug Resistance, Neoplasm/genetics , Estrogen Receptor alpha/metabolism , Female , Gene Knockdown Techniques , HCT116 Cells , Hep G2 Cells , Humans , MCF-7 Cells , Mice , Molecular Chaperones/antagonists & inhibitors , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Pyrazines/pharmacology , RNA, Small Interfering/genetics , Tumor Suppressor Protein p53/metabolism
8.
Biospektrum (Heidelb) ; 28(1): 39-42, 2022.
Article in German | MEDLINE | ID: mdl-35194331

ABSTRACT

Monoclonal immunoglobulins are widely successful as therapeutics and have also been effective in treating COVID-19. However, their production in mammalian cells is expensive and cannot be scaled to meet the demand in a global pandemic. Camelid VHH antibodies (also called nanobodies), however, can be manufactured cost-efficiently in bacteria or yeast. Here we highlight our progress in developing nanobodies that effectively neutralize SARS-CoV-2 and its variants.

9.
Proc Natl Acad Sci U S A ; 115(48): E11311-E11320, 2018 11 27.
Article in English | MEDLINE | ID: mdl-30413623

ABSTRACT

The p53-Mdm2 system is key to tumor suppression. We have recently reported that p53 as well as Mdm2 are capable of supporting DNA replication fork progression. On the other hand, we found that Mdm2 is a modifier of chromatin, modulating polycomb repressor complex (PRC)-driven histone modifications. Here we show that, similar to Mdm2 knockdown, the depletion of PRC members impairs DNA synthesis, as determined in fiber assays. In particular, the ubiquitin ligase and PRC1 component RNF2/Ring1B is required to support DNA replication, similar to Mdm2. Moreover, the Ring finger domain of Mdm2 is not only essential for its ubiquitin ligase activity, but also for proper DNA replication. Strikingly, Mdm2 overexpression can rescue RNF2 depletion with regard to DNA replication fork progression, and vice versa, strongly suggesting that the two ubiquitin ligases perform overlapping functions in this context. H2A overexpression also rescues fork progression upon depletion of Mdm2 or RNF2, but only when the ubiquitination sites K118/K119 are present. Depleting the H2A deubiquitinating enzyme BAP1 reduces the fork rate, suggesting that both ubiquitination and deubiquitination of H2A are required to support fork progression. The depletion of Mdm2 elicits the accumulation of RNA/DNA hybrids, suggesting R-loop formation as a mechanism of impaired DNA replication. Accordingly, RNase H overexpression or the inhibition of the transcription elongation kinase CDK9 each rescues DNA replication upon depletion of Mdm2 or RNF2. Taken together, our results suggest that chromatin modification by Mdm2 and PRC1 ensures smooth DNA replication through the avoidance of R-loop formation.


Subject(s)
Chromatin/metabolism , DNA Replication , DNA/genetics , Polycomb Repressive Complex 1/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , RNA/genetics , Cell Line, Tumor , Chromatin/genetics , DNA/metabolism , Histones/genetics , Histones/metabolism , Humans , Polycomb Repressive Complex 1/genetics , Protein Domains , Proto-Oncogene Proteins c-mdm2/chemistry , Proto-Oncogene Proteins c-mdm2/genetics , RNA/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin Thiolesterase/metabolism , Ubiquitination
10.
Cancer Sci ; 111(7): 2203-2211, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32335977

ABSTRACT

The Mdm2 oncoprotein and its association with p53 were discovered 30 years ago, and a cornucopia of activities and regulatory pathways have been associated with it. In this review, we will raise questions about Mdm2 and its cousin Mdm4 that we consider worth pursuing in future research, reaching from molecular structures and intracellular activities all the way to development, evolution, and cancer therapy. We anticipate that such research will not only close a few gaps in our knowledge but could add new dimensions to our current view. This compilation of questions contributes to the preparation for the 10th Mdm2 Workshop in Tokyo.


Subject(s)
Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Aging/genetics , Aging/metabolism , Animals , Biological Evolution , Biomarkers, Tumor , Cell Cycle Proteins , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Gene Expression Regulation , Humans , Molecular Targeted Therapy , Multigene Family , Mutation , Protein Binding , Protein Transport , Proto-Oncogene Proteins , Proto-Oncogene Proteins c-mdm2/chemistry , Signal Transduction , Stress, Physiological , Tumor Suppressor Protein p53/metabolism
11.
EMBO Rep ; 17(11): 1609-1623, 2016 11.
Article in English | MEDLINE | ID: mdl-27596623

ABSTRACT

The CHD1 gene, encoding the chromo-domain helicase DNA-binding protein-1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double-strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of CtIP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR-mediated DNA repair but not non-homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects.


Subject(s)
DNA Helicases/metabolism , DNA Repair , DNA-Binding Proteins/metabolism , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Biomarkers , Carrier Proteins/genetics , Cell Line, Tumor , Chromatin , DNA Breaks, Double-Stranded , DNA Helicases/deficiency , DNA Helicases/genetics , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Endodeoxyribonucleases , Humans , Male , Nuclear Proteins/genetics , Poly(ADP-ribose) Polymerase Inhibitors/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Prostatic Neoplasms/genetics , Recombinational DNA Repair
12.
Proc Natl Acad Sci U S A ; 110(42): 16856-61, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-24082115

ABSTRACT

DNA damage can obstruct replication forks, resulting in replicative stress. By siRNA screening, we identified kinases involved in the accumulation of phosphohistone 2AX (γH2AX) upon UV irradiation-induced replication stress. Surprisingly, the strongest reduction of phosphohistone 2AX followed knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation. Moreover, MK2 activity was required for damage response, accumulation of ssDNA, and decreased survival when cells were treated with the nucleoside analogue gemcitabine or when the checkpoint kinase Chk1 was antagonized. By using DNA fiber assays, we found that MK2 inhibition or knockdown rescued DNA replication impaired by gemcitabine or by Chk1 inhibition. This rescue strictly depended on translesion DNA polymerases. In conclusion, instead of being an unavoidable consequence of DNA damage, alterations of replication speed and origin firing depend on MK2-mediated signaling.


Subject(s)
DNA Replication , G2 Phase Cell Cycle Checkpoints , Intracellular Signaling Peptides and Proteins/metabolism , MAP Kinase Signaling System , Protein Serine-Threonine Kinases/metabolism , Animals , Antimetabolites, Antineoplastic/pharmacology , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Damage , DNA, Single-Stranded/genetics , DNA, Single-Stranded/metabolism , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Gene Knockdown Techniques , Histones/genetics , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mice, Knockout , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Ultraviolet Rays , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism , Gemcitabine
14.
Proc Natl Acad Sci U S A ; 108(9): 3624-9, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21300884

ABSTRACT

TAp63, but not its homolog p53, eliminates oocytes that suffered DNA damage. An equivalent gene for guarding the male germ line is currently not known. Here we identify hitherto unknown human p63 transcripts with unique 5'-ends derived from incorporated exons upstream of the currently mapped TP63 gene. These unique p63 transcripts are highly and specifically expressed in testis. Their most upstream region corresponds to a LTR of the human endogenous retrovirus 9 (ERV9). The insertion of this LTR upstream of the TP63 locus occurred only recently in evolution and is unique to humans and great apes (Hominidae). A corresponding p63 protein is the sole p63 species in healthy human testis, and is strongly expressed in spermatogenic precursors but not in mature spermatozoa. In response to DNA damage, this human male germ-cell-encoded TAp63 protein (designated GTAp63) is activated by caspase cleavage near its carboxyterminal domain and induces apoptosis. Human testicular cancer tissues and cell lines largely lost p63 expression. However, pharmacological inhibition of histone deacetylases completely restores p63 expression in testicular cancer cells (>3,000-fold increase). Our data support a model whereby testis-specific GTAp63 protects the genomic integrity of the male germ line and acts as a tumor suppressor. In Hominidae, this guardian function was greatly enhanced by integration of an endogenous retrovirus upstream of the TP63 locus that occurred 15 million years ago. By providing increased germ-line stability, this event may have contributed to the evolution of hominids and enabled their long reproductive periods.


Subject(s)
Apoptosis , Endogenous Retroviruses/metabolism , Hominidae/metabolism , Spermatozoa/metabolism , Trans-Activators/metabolism , Tumor Suppressor Proteins/metabolism , Amino Acid Sequence , Animals , Apoptosis/drug effects , Apoptosis/genetics , DNA Damage , Endogenous Retroviruses/drug effects , Exons/genetics , Gene Expression Regulation/drug effects , Gene Silencing/drug effects , Histone Deacetylase Inhibitors/pharmacology , Humans , Male , Molecular Sequence Data , Mutagenesis, Insertional/genetics , Organ Specificity/drug effects , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Spermatozoa/drug effects , Spermatozoa/pathology , Terminal Repeat Sequences/genetics , Testicular Neoplasms/enzymology , Testicular Neoplasms/pathology , Testis/drug effects , Testis/metabolism , Testis/pathology , Trans-Activators/chemistry , Trans-Activators/genetics , Transcription Factors , Transcription, Genetic , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics
15.
Antiviral Res ; 231: 106006, 2024 Sep 16.
Article in English | MEDLINE | ID: mdl-39293594

ABSTRACT

Monoclonal antibodies targeting the Spike protein of SARS-CoV-2 are effective against COVID-19 and might mitigate future pandemics. However, their efficacy is challenged by the emergence of antibody-resistant virus variants. We developed a method to efficiently identify such resistant mutants based on selection from mutagenized virus pools. By inducing mutations with the active compound of Molnupiravir, N4-hydroxycytidine (NHC), and subsequently passaging the virus in the presence of antibodies, we identified specific Spike mutations linked to resistance. Validation of these mutations was conducted using pseudotypes and immunofluorescence analysis. From a Wuhan-like strain of SARS-CoV-2, we identified the following mutations conferring strong resistance towards the corresponding antibodies: Bamlanivimab - E484K, F490S and S494P; Sotrovimab - E340K; Cilgavimab - K444R/E and N450D. From the Omicron B.1.1.529 variant, the strongly selected mutations were: Bebtelovimab - V445A; Sotrovimab - E340K and K356M; Cilgavimab - K444R, V445A and N450D. We also identified escape mutations in the Wuhan-like Spike for the broadly neutralizing antibodies S2K146 - combined G485S and Q493R - and S2H97 - D428G, K462E and S514F. Structural analysis revealed that the selected mutations occurred at antibody-binding residues within the receptor-binding domains of the Spike protein. Most of the selected mutants largely maintained ACE2 binding and infectivity. Notably, many of the identified resistance-conferring mutations are prevalent in real-world SARS-CoV-2 variants, but some of them (G485S, D428G, and K462E) have not yet been observed in circulating strains. Our approach offers a strategy for predicting the therapeutic efficacy of antibodies against emerging virus variants.

16.
Cell Rep ; 43(8): 114610, 2024 Aug 27.
Article in English | MEDLINE | ID: mdl-39116201

ABSTRACT

The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation.


Subject(s)
Alternative Splicing , Exons , RNA Precursors , Ribosomal Proteins , Tumor Suppressor Protein p53 , Ribosomal Proteins/metabolism , Ribosomal Proteins/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Protein p53/genetics , Humans , Exons/genetics , RNA Precursors/metabolism , RNA Precursors/genetics , Alternative Splicing/genetics , Cell Nucleolus/metabolism , Cell Proliferation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins/genetics , Protein Binding , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Ribosomes/metabolism , Stress, Physiological/genetics , RNA-Binding Proteins
17.
Article in English | MEDLINE | ID: mdl-38741480

ABSTRACT

Molnupiravir, an orally administered prodrug of ß-d-N4-hydroxycytidine (NHC), is incorporated into newly synthesized RNA by viral RNA-dependent RNA polymerase (RdRp). It is used for treatment of SARS-CoV-2 infections. Incorporation of NHC triphosphate into viral RNA inhibits replication of the virus, at least in part by introducing deleterious mutations. However, there is limited information on NHC incorporation into host RNA and reports on the risk of mutagenicity that molnupiravir/NHC pose to the host are conflicting. We used two liquid chromatography-mass spectrometry (LC-MS) methods to evaluate the incorporation of NHC into RNA and DNA of host Vero E6 cells in a SARS-CoV-2 infection model. To test this, host and viral RNA were degraded to their ribonucleosides, while host DNA was degraded to deoxyribonucleosides. Subsequently, nucleic acid constituents were analyzed by LC-MS, which offers specific, direct, and quantitative determination of incorporation. Our findings revealed concentration dependent NHC incorporation into host cell RNA in both infected and uninfected cell cultures, reaching a maximum of 1 in 7,093 bases. Analysis of host DNA revealed no presence of deoxy-N4-hydroxycytidine down to a detection limit of 1 in 133,000 bases. Our findings therefore suggest minimal to no NHC incorporation into host DNA, indicating a low probability of significant host cell mutagenicity associated with its use.

18.
Nat Commun ; 15(1): 411, 2024 Jan 09.
Article in English | MEDLINE | ID: mdl-38195625

ABSTRACT

Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.


Subject(s)
COVID-19 , Cysteine , Humans , SARS-CoV-2 , Drug Design , Oxidation-Reduction
19.
Antiviral Res ; 221: 105778, 2024 01.
Article in English | MEDLINE | ID: mdl-38065245

ABSTRACT

The ongoing threat of COVID-19 has highlighted the need for effective prophylaxis and convenient therapies, especially for outpatient settings. We have previously developed highly potent single-domain (VHH) antibodies, also known as nanobodies, that target the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike protein and neutralize the Wuhan strain of the virus. In this study, we present a new generation of anti-RBD nanobodies with superior properties. The primary representative of this group, Re32D03, neutralizes Alpha to Delta as well as Omicron BA.2.75; other members neutralize, in addition, Omicron BA.1, BA.2, BA.4/5, and XBB.1. Crystal structures of RBD-nanobody complexes reveal how ACE2-binding is blocked and also explain the nanobodies' tolerance to immune escape mutations. Through the cryo-EM structure of the Ma16B06-BA.1 Spike complex, we demonstrated how a single nanobody molecule can neutralize a trimeric spike. We also describe a method for large-scale production of these nanobodies in Pichia pastoris, and for formulating them into aerosols. Exposing hamsters to these aerosols, before or even 24 h after infection with SARS-CoV-2, significantly reduced virus load, weight loss and pathogenicity. These results show the potential of aerosolized nanobodies for prophylaxis and therapy of coronavirus infections.


Subject(s)
COVID-19 , Single-Domain Antibodies , Animals , Cricetinae , Humans , SARS-CoV-2 , Respiratory Aerosols and Droplets , Spike Glycoprotein, Coronavirus , Cell Culture Techniques , Antibodies, Neutralizing , Antibodies, Viral
20.
Circ Res ; 109(7): 758-69, 2011 Sep 16.
Article in English | MEDLINE | ID: mdl-21799151

ABSTRACT

RATIONALE: Telethonin (also known as titin-cap or t-cap) is a 19-kDa Z-disk protein with a unique ß-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardiomechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin's in vivo function. OBJECTIVE: Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation. METHODS AND RESULTS: By using a variety of different genetically altered animal models and biophysical experiments we show that contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin cross-links via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the proapoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis ("mechanoptosis"). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect that may contribute to enhanced rates of apoptosis found in these hearts. CONCLUSIONS: Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect that may also play a role in human heart failure.


Subject(s)
Heart Failure/metabolism , Heart/physiopathology , Mechanotransduction, Cellular , Muscle Proteins/deficiency , Myocardium/metabolism , Adaptation, Physiological , Animals , Animals, Genetically Modified , Apoptosis , Biomechanical Phenomena , Cell Line, Tumor , Connectin , Disease Models, Animal , Echocardiography , Fibrosis , Genotype , Heart Failure/genetics , Heart Failure/pathology , Heart Failure/physiopathology , Humans , Mice , Mice, Knockout , Muscle Proteins/genetics , Myocardium/pathology , Phenotype , RNA Interference , Rats , Sarcomeres/metabolism , Stress, Mechanical , Transfection , Tumor Suppressor Protein p53/metabolism
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