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1.
Mol Omics ; 17(5): 725-739, 2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34636387

RESUMO

Paradoxically, oncogenes that drive cell cycle progression may also trigger pathways leading to senescence, thereby inhibiting the growth of tumorigenic cells. Knowledge of how these pathways operate, and how tumor cells may evade these pathways, is important for understanding tumorigenesis. The Y1 cell line, which harbors an amplification of the proto-oncogene Ras, rapidly senesces in response to the mitogen fibroblast growth factor-2 (FGF-2). To gain a more complete picture of how FGF-2 promotes senescence, we employed a multi-omics approach to analyze histone modifications, mRNA and protein expression, and protein phosphorylation in Y1 cells treated with FGF-2. Compared to control cells treated with serum alone, FGF-2 caused a delayed accumulation of acetylation on histone H4 and higher levels of H3K27me3. Sequencing analysis revealed decreased expression of cell cycle-related genes with concomitant loss of H3K27ac. At the same time, FGF-2 promoted the expression of p21, various cytokines, and MAPK-related genes. Nuclear envelope proteins, particularly lamin B1, displayed increased phosphorylation in response to FGF-2. Proteome analysis suggested alterations in cellular metabolism, as evident by modulated expression of enzymes involved in purine biosynthesis, tRNA aminoacylation, and the TCA cycle. We propose that Y1 cells senesce due to an inability to progress through the cell cycle, which may stem from DNA damage or TGFb signaling. Altogether, the phenotype of Y1 cells is consistent with rapidly established oncogene-induced senescence, demonstrating the synergy between growth factors and oncogenes in driving senescence and bringing additional insight into this tumor suppressor mechanism.

2.
Crit Rev Biochem Mol Biol ; : 1-40, 2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34601997

RESUMO

The etiology of neurodevelopmental disorders (NDDs) remains a challenge for researchers. Human brain development is tightly regulated and sensitive to cellular alterations caused by endogenous or exogenous factors. Intriguingly, the surge of clinical sequencing studies has revealed that many of these disorders are monogenic and monoallelic. Notably, chromatin regulation has emerged as highly dysregulated in NDDs, with many syndromes demonstrating phenotypic overlap, such as intellectual disabilities, with one another. Here we discuss epigenetic writers, erasers, readers, remodelers, and even histones mutated in NDD patients, predicted to affect gene regulation. Moreover, this review focuses on disorders associated with mutations in enzymes involved in histone acetylation and methylation, and it highlights syndromes involving chromatin remodeling complexes. Finally, we explore recently discovered histone germline mutations and their pathogenic outcome on neurological function. Epigenetic regulators are mutated at every level of chromatin organization. Throughout this review, we discuss mechanistic investigations, as well as various animal and iPSC models of these disorders and their usefulness in determining pathomechanism and potential therapeutics. Understanding the mechanism of these mutations will illuminate common pathways between disorders. Ultimately, classifying these disorders based on their effects on the epigenome will not only aid in prognosis in patients but will aid in understanding the role of epigenetic machinery throughout neurodevelopment.

3.
Front Mol Biosci ; 8: 735303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34660696

RESUMO

Methionine is an essential amino acid used, beyond protein synthesis, for polyamine formation and DNA/RNA/protein methylation. Cancer cells require particularly high methionine supply for their homeostasis. A successful approach for decreasing methionine concentration is based on the systemic delivery of methionine γ-lyase (MGL), with in vitro and in vivo studies demonstrating its efficacy in cancer therapy. However, the mechanisms explaining how cancer cells suffer from the absence of methionine more significantly than non-malignant cells are still unclear. We analyzed the outcome of the human colorectal adenocarcinoma cancer cell line HT29 to the exposure of MGL for up to 72 h by monitoring cell viability, proteome expression, histone post-translational modifications, and presence of spurious transcription. The rationale of this study was to verify whether reduced methionine supply would affect chromatin decondensation by changing the levels of histone methylation and therefore increasing genomic instability. MGL treatment showed a time-dependent cytotoxic effect on HT29 cancer cells, with an IC50 of 30 µg/ml, while Hs27 normal cells were less affected, with an IC50 of >460 µg/ml. Although the levels of total histone methylation were not altered, a loss of the silencing histone mark H3K9me2 was observed, as well as a decrease in H4K20me3. Since H3K9me2/3 decorate repetitive DNA elements, we proved by qRT-PCR that MGL treatment leads to an increased expression of major satellite units. Our data indicate that selected histone methylation marks may play major roles in the mechanism of methionine starvation in cancer cells, proving that MGL treatment directly impacts chromatin homeostasis.

4.
ACS Chem Neurosci ; 12(20): 3898-3914, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34607428

RESUMO

Propofol, one of the most commonly used intravenous general anesthetics, modulates neuronal function by interacting with ion channels. The mechanisms that link propofol binding to the modulation of distinct ion channel states, however, are not understood. To tackle this problem, we investigated the prokaryotic ancestors of eukaryotic voltage-gated Na+ channels (Navs) using unbiased photoaffinity labeling (PAL) with a diazirine derivative of propofol (AziPm), electrophysiological methods, and mutagenesis. AziPm inhibits Nav function in a manner that is indistinguishable from that of the parent compound by promoting activation-coupled inactivation. In several replicates (8/9) involving NaChBac and NavMs, we found adducts at residues located at the C-terminal end of the S4 voltage sensor, the S4-S5 linker, and the N-terminal end of the S5 segment. However, the non-inactivating mutant NaChBac-T220A yielded adducts that were different from those found in the wild-type counterpart, which suggested state-dependent changes at the binding site. Then, using molecular dynamics simulations to further elucidate the structural basis of Nav modulation by propofol, we show that the S4 voltage sensors and the S4-S5 linkers shape two distinct propofol binding sites in a conformation-dependent manner. Supporting the PAL and MD simulation results, we also found that Ala mutations of a subset of adducted residues have distinct effects on gating modulation of NaChBac and NavMs by propofol. The results of this study provide direct insights into the structural basis of the mechanism through which propofol binding promotes activation-coupled inactivation to inhibit Nav channel function.

5.
Cureus ; 13(8): e16896, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34513469

RESUMO

Cutaneous Leishmaniasis due to Leishmania mexicana is a common cause of New World protozoal infections endemic to southern Mexico and now the United States (US). We present a case of a 72-year-old male who became infected with cutaneous L. mexicana while participating in numerous diving excursions in the flooded limestone caves, commonly referred to as cenotes, in the Yucatán Peninsula. This unique case of adventure tourism highlights cave diving in endemic regions of leishmaniasis as a possible new risk factor for the acquisition of this disease. We also discuss increasing autochthonous cases of cutaneous leishmaniasis and the different barriers to treatment that occur with this disease.

6.
Neuron ; 109(18): 2943-2966.e8, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34480866

RESUMO

Neuronal alternative splicing is a key gene regulatory mechanism in the brain. However, the spliceosome machinery is insufficient to fully specify splicing complexity. In considering the role of the epigenome in activity-dependent alternative splicing, we and others find the histone modification H3K36me3 to be a putative splicing regulator. In this study, we found that mouse cocaine self-administration caused widespread differential alternative splicing, concomitant with the enrichment of H3K36me3 at differentially spliced junctions. Importantly, only targeted epigenetic editing can distinguish between a direct role of H3K36me3 in splicing and an indirect role via regulation of splice factor expression elsewhere on the genome. We targeted Srsf11, which was both alternatively spliced and H3K36me3 enriched in the brain following cocaine self-administration. Epigenetic editing of H3K36me3 at Srsf11 was sufficient to drive its alternative splicing and enhanced cocaine self-administration, establishing the direct causal relevance of H3K36me3 to alternative splicing of Srsf11 and to reward behavior.


Assuntos
Processamento Alternativo/fisiologia , Comportamento Aditivo/metabolismo , Cromatina/metabolismo , Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Recompensa , Processamento Alternativo/efeitos dos fármacos , Animais , Comportamento Aditivo/genética , Comportamento Aditivo/psicologia , Cromatina/genética , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Autoadministração
7.
EMBO Rep ; 22(9): e52145, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34347354

RESUMO

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.


Assuntos
Chaperonina com TCP-1 , Citidina Desaminase , Chaperonina com TCP-1/genética , Citidina Desaminase/genética , Mutagênese , Proteínas/genética
8.
mSystems ; : e0046821, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34463575

RESUMO

Viral infections are associated with extensive remodeling of the cellular proteome. Viruses encode gene products that manipulate host proteins to redirect cellular processes or subvert antiviral immune responses. Adenovirus (AdV) encodes proteins from the early E4 region which are necessary for productive infection. Some cellular antiviral proteins are known to be targeted by AdV E4 gene products, resulting in their degradation or mislocalization. However, the full repertoire of host proteome changes induced by viral E4 proteins has not been defined. To identify cellular proteins and processes manipulated by viral products, we developed a global, unbiased proteomics approach to analyze changes to the host proteome during infection with adenovirus serotype 5 (Ad5) virus. We used whole-cell proteomics to measure total protein abundances in the proteome during Ad5 infection. Since host antiviral proteins can antagonize viral infection by associating with viral genomes and inhibiting essential viral processes, we used Isolation of Proteins on Nascent DNA (iPOND) proteomics to identify proteins associated with viral genomes during infection with wild-type Ad5 or an E4 mutant virus. By integrating these proteomics data sets, we identified cellular factors that are degraded in an E4-dependent manner or are associated with the viral genome in the absence of E4 proteins. We further show that some identified proteins exert inhibitory effects on Ad5 infection. Our systems-level analysis reveals cellular processes that are manipulated during Ad5 infection and points to host factors counteracted by early viral proteins as they remodel the host proteome to promote efficient infection. IMPORTANCE Viral infections induce myriad changes to the host cell proteome. As viruses harness cellular processes and counteract host defenses, they impact abundance, post-translational modifications, interactions, or localization of cellular proteins. Elucidating the dynamic changes to the cellular proteome during viral replication is integral to understanding how virus-host interactions influence the outcome of infection. Adenovirus encodes early gene products from the E4 genomic region that are known to alter host response pathways and promote replication, but the full extent of proteome modifications they mediate is not known. We used an integrated proteomics approach to quantitate protein abundance and protein associations with viral DNA during virus infection. Systems-level analysis identifies cellular proteins and processes impacted in an E4-dependent manner, suggesting ways that adenovirus counteracts potentially inhibitory host defenses. This study provides a global view of adenovirus-mediated proteome remodeling, which can serve as a model to investigate virus-host interactions of DNA viruses.

9.
Nat Commun ; 12(1): 4365, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272369

RESUMO

Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαß isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinogênese/imunologia , Epitopos de Linfócito T/imunologia , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transferência Adotiva , Alelos , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Peptídeos/genética , Peptídeos/imunologia , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Hepatology ; 2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233020

RESUMO

BACKGROUND AND AIMS: Although germ-free mice are an indispensable tool in studying the gut microbiome and its effects on host physiology, they are phenotypically different than their conventional counterparts. While antibiotic-mediated microbiota depletion in conventional mice leads to physiologic alterations that often mimic the germ-free state, the degree to which the effects of microbial colonization on the host are reversible is unclear. The gut microbiota produce abundant short chain fatty acids (SCFAs), and previous studies have demonstrated a link between microbial-derived SCFAs and global hepatic histone acetylation in germ-free mice. APPROACH AND RESULTS: We demonstrate that global hepatic histone acetylation states measured by mass spectrometry remained largely unchanged despite loss of luminal and portal vein SCFAs after antibiotic-mediated microbiota depletion. In contrast to stable hepatic histone acetylation states, we see robust hepatic transcriptomic alterations after microbiota depletion. Additionally, neither dietary supplementation with supraphysiologic levels of SCFA nor the induction of hepatocyte proliferation in the absence of microbiota-derived SCFAs led to alterations in global hepatic histone acetylation. CONCLUSIONS: These results suggest that microbiota-dependent landscaping of the hepatic epigenome through global histone acetylation is static in nature, while the hepatic transcriptome is responsive to alterations in the gut microbiota.

11.
PLoS Biol ; 19(6): e3001277, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138841

RESUMO

Glycosylation is one of the most complex posttranslational protein modifications. Its importance has been established not only for eukaryotes but also for a variety of prokaryotic cellular processes, such as biofilm formation, motility, and mating. However, comprehensive glycoproteomic analyses are largely missing in prokaryotes. Here, we extend the phenotypic characterization of N-glycosylation pathway mutants in Haloferax volcanii and provide a detailed glycoproteome for this model archaeon through the mass spectrometric analysis of intact glycopeptides. Using in-depth glycoproteomic datasets generated for the wild-type (WT) and mutant strains as well as a reanalysis of datasets within the Archaeal Proteome Project (ArcPP), we identify the largest archaeal glycoproteome described so far. We further show that different N-glycosylation pathways can modify the same glycosites under the same culture conditions. The extent and complexity of the Hfx. volcanii N-glycoproteome revealed here provide new insights into the roles of N-glycosylation in archaeal cell biology.


Assuntos
Proteínas Arqueais/metabolismo , Glicopeptídeos/metabolismo , Glicoproteínas/metabolismo , Haloferax volcanii/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Bioensaio , Forma Celular/efeitos dos fármacos , Bases de Dados de Proteínas , Glicopeptídeos/química , Glicoproteínas/química , Glicosilação/efeitos dos fármacos , Haloferax volcanii/efeitos dos fármacos , Mutação/genética , Fenótipo , Filogenia , Proteômica , Cloreto de Sódio/farmacologia
12.
Nat Commun ; 12(1): 3338, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099686

RESUMO

The versatile nucleotide excision repair (NER) pathway initiates as the XPC-RAD23B-CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4-Rad23-Rad33 (yeast homologue of XPC-RAD23B-CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9-9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.


Assuntos
Microscopia Crioeletrônica , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/química , Adutos de DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/genética
13.
Nat Commun ; 12(1): 3487, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108468

RESUMO

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.


Assuntos
Alquil e Aril Transferases/química , Diterpenos/metabolismo , Proteínas Fúngicas/química , Alquil e Aril Transferases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Catálise , Domínio Catalítico , Microscopia Crioeletrônica , Ciclização , Dimetilaliltranstransferase/química , Dimetilaliltranstransferase/metabolismo , Proteínas Fúngicas/metabolismo , Glicosídeos/biossíntese , Liases/química , Liases/metabolismo , Enzimas Multifuncionais , Fosfatos de Poli-Isoprenil/metabolismo , Conformação Proteica
14.
Mol Cell ; 81(11): 2332-2348.e9, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-33974912

RESUMO

Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.


Assuntos
Carcinogênese/genética , DNA Helicases/genética , Proteínas Intrinsicamente Desordenadas/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Sequência de Bases , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Análise de Sobrevida , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo
15.
Front Cell Dev Biol ; 9: 630067, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33816475

RESUMO

Cell fate decisions during development are governed by multi-factorial regulatory mechanisms including chromatin remodeling, DNA methylation, binding of transcription factors to specific loci, RNA transcription and protein synthesis. However, the mechanisms by which such regulatory "dimensions" coordinate cell fate decisions are currently poorly understood. Here we quantified the multi-dimensional molecular changes that occur in mouse embryonic stem cells (mESCs) upon depletion of Estrogen related receptor beta (Esrrb), a key pluripotency regulator. Comparative analyses of expression changes subsequent to depletion of Esrrb or Nanog, indicated that a system of interlocked feed-forward loops involving both factors, plays a central part in regulating the timing of mESC fate decisions. Taken together, our meta-analyses support a hierarchical model in which pluripotency is maintained by an Oct4-Sox2 regulatory module, while the timing of differentiation is regulated by a Nanog-Esrrb module.

16.
J Am Soc Mass Spectrom ; 32(6): 1300-1311, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-33818074

RESUMO

The cell cycle is a highly regulated and evolutionary conserved process that results in the duplication of cell content and the equal distribution of the duplicated chromosomes into a pair of daughter cells. Histones are fundamental structural components of chromatin in eukaryotic cells, and their post-translational modifications (PTMs) benchmark DNA readout and chromosome condensation. Aberrant regulation of the cell cycle associated with dysregulation of histone PTMs is the cause of critical diseases such as cancer. Monitoring changes of histone PTMs could pave the way to understanding the molecular mechanisms associated with epigenetic regulation of cell proliferation. Previously, our lab established a novel middle-down workflow using porous graphitic carbon (PGC) as a stationary phase to analyze histone PTMs, which utilizes the same reversed-phase chromatography for gradient separation as canonical proteomics coupled with online mass spectrometry (MS). Here, we applied this novel workflow for high-throughput analysis of histone modifications of H3.1 and H3.2 during the cell cycle. Collectively, we identified 1133 uniquely modified canonical histone H3 N-terminal tails. Consistent with previous findings, histone H3 phosphorylation increased significantly during the mitosis (M) phase. Histone H3 variant-specific and cell-cycle-dependent expressions of PTMs were observed, underlining the need to not combine H3.1 and H3.2 together as H3. We confirmed previously known H3 PTM crosstalk (e.g., K9me-S10ph) and revealed new information in this area as well. These findings imply that the combinatorial PTMs play a role in cell cycle control, and they may serve as markers for proliferation.

17.
Sci Adv ; 7(15)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33827808

RESUMO

During transcription initiation, the general transcription factor TFIIH marks RNA polymerase II by phosphorylating Ser5 of the carboxyl-terminal domain (CTD) of Rpb1, which is followed by extensive modifications coupled to transcription elongation, mRNA processing, and histone dynamics. We have determined a 3.5-Å resolution cryo-electron microscopy (cryo-EM) structure of the TFIIH kinase module (TFIIK in yeast), which is composed of Kin28, Ccl1, and Tfb3, yeast homologs of CDK7, cyclin H, and MAT1, respectively. The carboxyl-terminal region of Tfb3 was lying at the edge of catalytic cleft of Kin28, where a conserved Tfb3 helix served to stabilize the activation loop in its active conformation. By combining the structure of TFIIK with the previous cryo-EM structure of the preinitiation complex, we extend the previously proposed model of the CTD path to the active site of TFIIK.

19.
J Proteome Res ; 20(4): 1918-1927, 2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33764077

RESUMO

Stable isotope labeling by amino acids in cell culture (SILAC) coupled to data-dependent acquisition (DDA) is a common approach to quantitative proteomics with the desirable benefit of reducing batch effects during sample processing and data acquisition. More recently, using data-independent acquisition (DIA/SWATH) to systematically measure peptides has gained popularity for its comprehensiveness, reproducibility, and accuracy of quantification. The complementary advantages of these two techniques logically suggests combining them. Here we develop a SILAC-DIA-MS workflow using free, open-source software. We empirically determine that using DIA achieves similar peptide detection numbers as DDA and that DIA improves the quantitative accuracy and precision of SILAC by an order of magnitude. Finally, we apply SILAC-DIA-MS to determine protein turnover rates of cells treated with bortezomib, an FDA-approved 26S proteasome inhibitor for multiple myeloma and mantle cell lymphoma. We observe that SILAC-DIA produces more sensitive protein turnover models. Of the proteins determined to be differentially degraded by both acquisition methods, we find known proteins that are degraded by the ubiquitin-proteasome pathway, such as HNRNPK, EIF3A, and IF4A1/EIF4A-1, and a slower turnover for CATD, a protein implicated in invasive breast cancer. With improved quantification from DIA, we anticipate that this workflow will make SILAC-based experiments like protein turnover more sensitive.


Assuntos
Proteoma , Espectrometria de Massas em Tandem , Bortezomib/farmacologia , Proteólise , Reprodutibilidade dos Testes
20.
FASEB J ; 35(4): e21523, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33734487

RESUMO

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, there is increasing concern about the long-term and sex-specific health implications. Augmenting our IVF mouse model to longitudinally investigate metabolic outcomes in offspring from optimal neonatal litter sizes, we found sex-specific metabolic outcomes in IVF offspring. IVF-conceived females had higher body weight and cholesterol levels compared to naturally conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through adult liver transcriptomics and proteomics, we identified sexually dimorphic dysregulation of the sterol regulatory element-binding protein (SREBP) pathways that are associated with the sex-specific phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.


Assuntos
Fertilização/fisiologia , Proteoma/metabolismo , Fatores Sexuais , Transcriptoma/fisiologia , Animais , Composição Corporal/fisiologia , Metilação de DNA/fisiologia , Feminino , Fígado/metabolismo , Camundongos , Placenta/metabolismo , Gravidez
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