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1.
Nat Biotechnol ; 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38956325

RESUMEN

Antigen discovery technologies have largely focused on major histocompatibility complex (MHC) class I-restricted human T cell receptors (TCRs), leaving methods for MHC class II-restricted and mouse TCR reactivities relatively undeveloped. Here we present TCR mapping of antigenic peptides (TCR-MAP), an antigen discovery method that uses a synthetic TCR-stimulated circuit in immortalized T cells to activate sortase-mediated tagging of engineered antigen-presenting cells (APCs) expressing processed peptides on MHCs. Live, tagged APCs can be directly purified for deconvolution by sequencing, enabling TCRs with unknown specificity to be queried against barcoded peptide libraries in a pooled screening context. TCR-MAP accurately captures self-reactivities or viral reactivities with high throughput and sensitivity for both MHC class I-restricted and class II-restricted TCRs. We elucidate problematic cross-reactivities of clinical TCRs targeting the cancer/testis melanoma-associated antigen A3 and discover targets of myocarditis-inciting autoreactive T cells in mice. TCR-MAP has the potential to accelerate T cell antigen discovery efforts in the context of cancer, infectious disease and autoimmunity.

2.
Cancer Res ; 2024 Jul 18.
Artículo en Inglés | MEDLINE | ID: mdl-39024548

RESUMEN

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer patient deaths due to extensive molecular heterogeneity, high recurrence rates and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. Here, we performed a genome-wide CRISPR/Cas9 screen with PI3Kα and AKT inhibitors to find targetable synthetic lethalities in TNBC. Cholesterol homeostasis was identified as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro, in mouse TNBC xenografts and in patient-derived, estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single agent or combination treatment with AKT inhibitor and pitavastatin, which was rescued by inhibition of the cholesterol trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels, and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. These findings support combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC. .

3.
Neuron ; 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39079530

RESUMEN

The heterogeneity of protein-rich inclusions and its significance in neurodegeneration is poorly understood. Standard patient-derived iPSC models develop inclusions neither reproducibly nor in a reasonable time frame. Here, we developed screenable iPSC "inclusionopathy" models utilizing piggyBac or targeted transgenes to rapidly induce CNS cells that express aggregation-prone proteins at brain-like levels. Inclusions and their effects on cell survival were trackable at single-inclusion resolution. Exemplar cortical neuron α-synuclein inclusionopathy models were engineered through transgenic expression of α-synuclein mutant forms or exogenous seeding with fibrils. We identified multiple inclusion classes, including neuroprotective p62-positive inclusions versus dynamic and neurotoxic lipid-rich inclusions, both identified in patient brains. Fusion events between these inclusion subtypes altered neuronal survival. Proteome-scale α-synuclein genetic- and physical-interaction screens pinpointed candidate RNA-processing and actin-cytoskeleton-modulator proteins like RhoA whose sequestration into inclusions could enhance toxicity. These tractable CNS models should prove useful in functional genomic analysis and drug development for proteinopathies.

4.
J Cell Biol ; 223(8)2024 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-38874443

RESUMEN

N-degrons are short sequences located at protein N-terminus that mediate the interaction of E3 ligases (E3s) with substrates to promote their proteolysis. It is well established that N-degrons can be exposed following protease cleavage to allow recognition by E3s. However, our knowledge regarding how proteases and E3s cooperate in protein quality control mechanisms remains minimal. Using a systematic approach to monitor the protein stability of an N-terminome library, we found that proline residue at the third N-terminal position (hereafter "P+3") promotes instability. Genetic perturbations identified the dipeptidyl peptidases DPP8 and DPP9 and the primary E3s of N-degron pathways, UBR proteins, as regulators of P+3 bearing substrate turnover. Interestingly, P+3 UBR substrates are significantly enriched for secretory proteins. We found that secretory proteins relying on a signal peptide (SP) for their targeting contain a "built-in" N-degron within their SP. This degron becomes exposed by DPP8/9 upon translocation failure to the designated compartments, thus enabling clearance of mislocalized proteins by UBRs to maintain proteostasis.


Asunto(s)
Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Estabilidad Proteica , Ubiquitina-Proteína Ligasas , Humanos , Degrones , Dipeptidasas/metabolismo , Dipeptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Células HEK293 , Señales de Clasificación de Proteína , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética
5.
bioRxiv ; 2024 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-38798327

RESUMEN

Small molecule-mediated proteasomal degradation of proteins is a powerful tool for synthetic regulation of biological activity. To control Cas9 activity in cells, we engineered an anti-CRISPR protein, AcrIIA4, fused to a degradation (dTAG) or small molecule assisted shutoff (SMASh) tag. Co-expression of the tagged AcrIIA4 along with Cas9 and riboswitch-regulated sgRNAs enables precise tunable control of CRISPR activity by small molecule addition.

6.
Nat Commun ; 15(1): 3558, 2024 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-38670995

RESUMEN

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.


Asunto(s)
Microscopía por Crioelectrón , Proteína NEDD8 , Receptores de Interleucina-17 , Ubiquitina-Proteína Ligasas , Ubiquitinación , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/química , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Prolina/metabolismo , Multimerización de Proteína , Células HEK293 , Unión Proteica , Especificidad por Sustrato , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Modelos Moleculares , Proteínas Cullin/metabolismo , Proteínas Cullin/química , Proteínas Cullin/genética , Degrones
7.
JAMA Neurol ; 81(5): 515-524, 2024 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-38497939

RESUMEN

Importance: It remains unclear why only a small proportion of individuals infected with the Epstein-Barr virus (EBV) develop multiple sclerosis (MS) and what the underlying mechanisms are. Objective: To assess the serologic response to all EBV peptides before the first symptoms of MS occur, determine whether the disease is associated with a distinct immune response to EBV, and evaluate whether specific EBV epitopes drive this response. Design, Setting, and Participants: In this prospective, nested case-control study, individuals were selected among US military personnel with serum samples stored in the US Department of Defense Serum Repository. Individuals with MS had serum collected at a median 1 year before onset (reported to the military in 2000-2011) and were matched to controls for age, sex, race and ethnicity, blood collection, and military branch. No individuals were excluded. The data were analyzed between September 1, 2022, and August 31, 2023. Exposure: Antibodies (enrichment z scores) to the human virome measured using VirScan (phage-displayed immunoprecipitation and sequencing). Main Outcome and Measure: Rate ratios (RRs) for MS for antibodies to 2263 EBV peptides (the EBV peptidome) were estimated using conditional logistic regression, adjusting for total anti-EBV nuclear antigen 1 (EBNA-1) antibodies, which have consistently been associated with a higher MS risk. The role of antibodies against other viral peptides was also explored. Results: A total of 30 individuals with MS were matched with 30 controls. Mean (SD) age at sample collection was 27.8 (6.5) years; 46 of 60 participants (76.7%) were male. The antibody response to the EBV peptidome was stronger in individuals with MS, but without a discernible pattern. The antibody responses to 66 EBV peptides, the majority mapping to EBNA antigens, were significantly higher in preonset sera from individuals with MS (RR of highest vs lowest tertile of antibody enrichment, 33.4; 95% CI, 2.5-448.4; P for trend = .008). Higher total anti-EBNA-1 antibodies were also associated with an elevated MS risk (top vs bottom tertile: RR, 27.6; 95% CI, 2.3-327.6; P for trend = .008). After adjusting for total anti-EBNA-1 antibodies, risk estimates from most EBV peptides analyses were attenuated, with 4 remaining significantly associated with MS, the strongest within EBNA-6/EBNA-3C, while the association between total anti-EBNA-1 antibodies and MS persisted. Conclusion and Relevance: These findings suggest that antibody response to EBNA-1 may be the strongest serologic risk factor for MS. No single EBV peptide stood out as being selectively targeted in individuals with MS but not controls. Larger investigations are needed to explore possible heterogeneity of anti-EBV humoral immunity in MS.


Asunto(s)
Infecciones por Virus de Epstein-Barr , Herpesvirus Humano 4 , Esclerosis Múltiple , Humanos , Femenino , Masculino , Herpesvirus Humano 4/inmunología , Esclerosis Múltiple/sangre , Esclerosis Múltiple/inmunología , Estudios de Casos y Controles , Adulto , Infecciones por Virus de Epstein-Barr/inmunología , Infecciones por Virus de Epstein-Barr/sangre , Personal Militar , Anticuerpos Antivirales/sangre , Estudios Prospectivos , Adulto Joven , Antígenos Nucleares del Virus de Epstein-Barr/inmunología , Antígenos Nucleares del Virus de Epstein-Barr/sangre , Péptidos/inmunología , Péptidos/sangre
8.
Nat Genet ; 56(5): 900-912, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38388848

RESUMEN

Whole chromosome and arm-level copy number alterations occur at high frequencies in tumors, but their selective advantages, if any, are poorly understood. Here, utilizing unbiased whole chromosome genetic screens combined with in vitro evolution to generate arm- and subarm-level events, we iteratively selected the fittest karyotypes from aneuploidized human renal and mammary epithelial cells. Proliferation-based karyotype selection in these epithelial lines modeled tissue-specific tumor aneuploidy patterns in patient cohorts in the absence of driver mutations. Hi-C-based translocation mapping revealed that arm-level events usually emerged in multiples of two via centromeric translocations and occurred more frequently in tetraploids than diploids, contributing to the increased diversity in evolving tetraploid populations. Isogenic clonal lineages enabled elucidation of pro-tumorigenic mechanisms associated with common copy number alterations, revealing Notch signaling potentiation as a driver of 1q gain in breast cancer. We propose that intrinsic, tissue-specific proliferative effects underlie tumor copy number patterns in cancer.


Asunto(s)
Aneuploidia , Humanos , Femenino , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Variaciones en el Número de Copia de ADN , Neoplasias/genética , Neoplasias/patología , Translocación Genética , Evolución Molecular , Proliferación Celular/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Especificidad de Órganos/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología
9.
Nat Commun ; 15(1): 1577, 2024 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-38383452

RESUMEN

We investigate a relatively underexplored component of the gut-immune axis by profiling the antibody response to gut phages using Phage Immunoprecipitation Sequencing (PhIP-Seq). To cover large antigenic spaces, we develop Dolphyn, a method that uses machine learning to select peptides from protein sets and compresses the proteome through epitope-stitching. Dolphyn compresses the size of a peptide library by 78% compared to traditional tiling, increasing the antibody-reactive peptides from 10% to 31%. We find that the immune system develops antibodies to human gut bacteria-infecting viruses, particularly E.coli-infecting Myoviridae. Cost-effective PhIP-Seq libraries designed with Dolphyn enable the assessment of a wider range of proteins in a single experiment, thus facilitating the study of the gut-immune axis.


Asunto(s)
Bacteriófagos , Biblioteca de Péptidos , Humanos , Epítopos , Secuencia de Aminoácidos , Péptidos/genética , Anticuerpos , Bacteriófagos/genética , Mapeo Epitopo/métodos
12.
Cell ; 186(25): 5569-5586.e21, 2023 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-38016469

RESUMEN

CD4+ T cells play fundamental roles in orchestrating immune responses and tissue homeostasis. However, our inability to associate peptide human leukocyte antigen class-II (HLA-II) complexes with their cognate T cell receptors (TCRs) in an unbiased manner has hampered our understanding of CD4+ T cell function and role in pathologies. Here, we introduce TScan-II, a highly sensitive genome-scale CD4+ antigen discovery platform. This platform seamlessly integrates the endogenous HLA-II antigen-processing machinery in synthetic antigen-presenting cells and TCR signaling in T cells, enabling the simultaneous screening of multiple HLAs and TCRs. Leveraging genome-scale human, virome, and epitope mutagenesis libraries, TScan-II facilitates de novo antigen discovery and deep exploration of TCR specificity. We demonstrate TScan-II's potential for basic and translational research by identifying a non-canonical antigen for a cancer-reactive CD4+ T cell clone. Additionally, we identified two antigens for clonally expanded CD4+ T cells in Sjögren's disease, which bind distinct HLAs and are expressed in HLA-II-positive ductal cells within affected salivary glands.


Asunto(s)
Linfocitos T CD4-Positivos , Epítopos de Linfocito T , Humanos , Células Presentadoras de Antígenos , Antígenos CD4/metabolismo , Antígenos HLA/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Línea Celular , Genoma Humano
13.
Nat Cell Biol ; 25(10): 1535-1545, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37735597

RESUMEN

Specificity within the ubiquitin-proteasome system is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates-and in particular the molecular features (degrons) that they recognize-remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9/13 and Cul3KLHL15, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the ubiquitin-proteasome system.


Asunto(s)
Complejo de la Endopetidasa Proteasomal , Ubiquitina-Proteína Ligasas , Ubiquitina-Proteína Ligasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Ubiquitina/genética , Ubiquitina/metabolismo
14.
Mol Cell ; 83(18): 3377-3392.e6, 2023 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-37738965

RESUMEN

The ubiquitin-proteasome system plays a critical role in biology by regulating protein degradation. Despite their importance, precise recognition specificity is known for a few of the 600 E3s. Here, we establish a two-pronged strategy for identifying and mapping critical residues of internal degrons on a proteome-scale in HEK-293T cells. We employ global protein stability profiling combined with machine learning to identify 15,800 peptides likely to contain sequence-dependent degrons. We combine this with scanning mutagenesis to define critical residues for over 5,000 predicted degrons. Focusing on Cullin-RING ligase degrons, we generated mutational fingerprints for 219 degrons and developed DegronID, a computational algorithm enabling the clustering of degron peptides with similar motifs. CRISPR analysis enabled the discovery of E3-degron pairs, of which we uncovered 16 pairs that revealed extensive degron variability and structural determinants. We provide the visualization of these data on the public DegronID data browser as a resource for future exploration.


Asunto(s)
Algoritmos , Proteoma , Proteoma/genética , Núcleo Celular , Análisis por Conglomerados , Ubiquitina-Proteína Ligasas/genética
15.
Brain Commun ; 5(5): fcad223, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37731906

RESUMEN

Nodding syndrome is a neglected, disabling and potentially fatal epileptic disorder of unknown aetiology affecting thousands of individuals mostly confined to Eastern sub-Saharan Africa. Previous studies have identified multiple associations-including Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 deficiency and measles virus infection-yet, none is proven causal. We conducted a case-control study of children with early-stage nodding syndrome (symptom onset <1 year). Cases and controls were identified through a household survey in the Greater Mundri area in South Sudan. A wide range of parasitic, bacterial, viral, immune-mediated, metabolic and nutritional risk factors was investigated using conventional and state-of-the-art untargeted assays. Associations were examined by multiple logistic regression analysis, and a hypothetical causal model was constructed using structural equation modelling. Of 607 children with nodding syndrome, 72 with early-stage disease were included as cases and matched to 65 household- and 44 community controls. Mansonella perstans infection (odds ratio 7.04, 95% confidence interval 2.28-21.7), Necator americanus infection (odds ratio 2.33, 95% confidence interval 1.02-5.3), higher antimalarial seroreactivity (odds ratio 1.75, 95% confidence interval 1.20-2.57), higher vitamin E concentration (odds ratio 1.53 per standard deviation increase, 95% confidence interval 1.07-2.19) and lower vitamin B12 concentration (odds ratio 0.56 per standard deviation increase, 95% confidence interval 0.36-0.87) were associated with higher odds of nodding syndrome. In a structural equation model, we hypothesized that Mansonella perstans infection, higher vitamin E concentration and fewer viral exposures increased the risk of nodding syndrome while lower vitamin B12 concentration, Necator americanus and malaria infections resulted from having nodding syndrome. We found no evidence that Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 and other factors were associated with nodding syndrome. Our results argue against several previous causal hypotheses including Onchocerca volvulus. Instead, nodding syndrome may be caused by a complex interplay between multiple pathogens and nutrient levels. Further studies need to confirm these associations and determine the direction of effect.

16.
Science ; 381(6660): eadh5021, 2023 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-37616343

RESUMEN

Cells use ubiquitin to mark proteins for proteasomal degradation. Although the proteasome also eliminates proteins that are not ubiquitinated, how this occurs mechanistically is unclear. Here, we found that midnolin promoted the destruction of many nuclear proteins, including transcription factors encoded by the immediate-early genes. Diverse stimuli induced midnolin, and its overexpression was sufficient to cause the degradation of its targets by a mechanism that did not require ubiquitination. Instead, midnolin associated with the proteasome via an α helix, used its Catch domain to bind a region within substrates that can form a ß strand, and used a ubiquitin-like domain to promote substrate destruction. Thus, midnolin contains three regions that function in concert to target a large set of nuclear proteins to the proteasome for degradation.


Asunto(s)
Genes Inmediatos-Precoces , Proteínas Nucleares , Complejo de la Endopetidasa Proteasomal , Proteolisis , Transcripción Genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ubiquitina , Ubiquitinación , Células HEK293 , Células 3T3 NIH
17.
bioRxiv ; 2023 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-37577562

RESUMEN

We investigated a relatively underexplored component of the gut-immune axis by profiling the antibody response to gut phages using Phage Immunoprecipitation Sequencing (PhIP-Seq). To enhance this approach, we developed Dolphyn, a novel method that uses machine learning to select peptides from protein sets and compresses the proteome through epitope-stitching. Dolphyn improves the fraction of gut phage library peptides bound by antibodies from 10% to 31% in healthy individuals, while also reducing the number of synthesized peptides by 78%. In our study on gut phages, we discovered that the immune system develops antibodies to bacteria-infecting viruses in the human gut, particularly E.coli-infecting Myoviridae. Cost-effective PhIP-Seq libraries designed with Dolphyn enable the assessment of a wider range of proteins in a single experiment, thus facilitating the study of the gut-immune axis.

18.
Sci Transl Med ; 15(706): eadh9917, 2023 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-37494473

RESUMEN

Severe acute hepatitis of unknown etiology in children is under investigation in 35 countries. Although several potential etiologic agents have been investigated, a clear cause for the liver damage observed in these cases remains to be identified. Using VirScan, a high-throughput antibody profiling technology, we probed the antibody repertoires of nine cases of severe acute hepatitis of unknown etiology treated at Children's of Alabama and compared their antibody responses with 38 pediatric and 470 adult controls. We report increased adeno-associated dependoparvovirus A (AAV-A) breadth in cases relative to controls and adeno-associated virus 2 (AAV-2) peptide responses that were conserved in seven of nine cases but rarely observed in pediatric and adult controls. These findings suggest that AAV-2 is a likely etiologic agent of severe acute hepatitis of unknown etiology.


Asunto(s)
Hepatitis , Hepatopatías , Adulto , Humanos , Niño , Dependovirus
19.
Mol Cell ; 83(11): 1921-1935.e7, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-37201526

RESUMEN

Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.


Asunto(s)
Complejo de la Endopetidasa Proteasomal , Ubiquitina , Humanos , Ubiquitina/genética , Ubiquitina/metabolismo , Proteolisis , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas/metabolismo , Ubiquitinación , Proteínas de Ciclo Celular/metabolismo
20.
Nature ; 618(7967): 1024-1032, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37198482

RESUMEN

Focal copy-number amplification is an oncogenic event. Although recent studies have revealed the complex structure1-3 and the evolutionary trajectories4 of oncogene amplicons, their origin remains poorly understood. Here we show that focal amplifications in breast cancer frequently derive from a mechanism-which we term translocation-bridge amplification-involving inter-chromosomal translocations that lead to dicentric chromosome bridge formation and breakage. In 780 breast cancer genomes, we observe that focal amplifications are frequently connected to each other by inter-chromosomal translocations at their boundaries. Subsequent analysis indicates the following model: the oncogene neighbourhood is translocated in G1 creating a dicentric chromosome, the dicentric chromosome is replicated, and as dicentric sister chromosomes segregate during mitosis, a chromosome bridge is formed and then broken, with fragments often being circularized in extrachromosomal DNAs. This model explains the amplifications of key oncogenes, including ERBB2 and CCND1. Recurrent amplification boundaries and rearrangement hotspots correlate with oestrogen receptor binding in breast cancer cells. Experimentally, oestrogen treatment induces DNA double-strand breaks in the oestrogen receptor target regions that are repaired by translocations, suggesting a role of oestrogen in generating the initial translocations. A pan-cancer analysis reveals tissue-specific biases in mechanisms initiating focal amplifications, with the breakage-fusion-bridge cycle prevalent in some and the translocation-bridge amplification in others, probably owing to the different timing of DNA break repair. Our results identify a common mode of oncogene amplification and propose oestrogen as its mechanistic origin in breast cancer.


Asunto(s)
Neoplasias de la Mama , Receptor alfa de Estrógeno , Amplificación de Genes , Oncogenes , Translocación Genética , Femenino , Humanos , Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Oncogenes/genética , Translocación Genética/genética , Genoma Humano/genética , Roturas del ADN de Doble Cadena , Especificidad de Órganos
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