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1.
Int J Mol Sci ; 24(2)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36674445

RESUMO

Abnormal functions of the cell adhesion molecule L1 are linked to several neural diseases. Proteolytic L1 fragments were reported to interact with nuclear and mitochondrial proteins to regulate events in the developing and the adult nervous system. Recently, we identified a 55 kDa L1 fragment (L1-55) that interacts with methyl CpG binding protein 2 (MeCP2) and heterochromatin protein 1 (HP1) via the KDET motif. We now show that L1-55 also interacts with histone H1.4 (HistH1e) via this motif. Moreover, we show that this motif binds to NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), splicing factor proline/glutamine-rich (SFPQ), the non-POU domain containing octamer-binding protein (NonO), paraspeckle component 1 (PSPC1), WD-repeat protein 5 (WDR5), heat shock cognate protein 71 kDa (Hsc70), and synaptotagmin 1 (SYT1). Furthermore, applications of HistH1e, NDUFV2, SFPQ, NonO, PSPC1, WDR5, Hsc70, or SYT1 siRNAs or a cell-penetrating KDET-carrying peptide decrease L1-dependent neurite outgrowth and the survival of cultured neurons. These findings indicate that L1's KDET motif binds to an unexpectedly large number of molecules that are essential for nervous system-related functions, such as neurite outgrowth and neuronal survival. In summary, L1 interacts with cytoplasmic, nuclear and mitochondrial proteins to regulate development and, in adults, the formation, maintenance, and flexibility of neural functions.


Assuntos
Proteínas Mitocondriais , Molécula L1 de Adesão de Célula Nervosa , Citoplasma/metabolismo , Citosol/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Molécula L1 de Adesão de Célula Nervosa/química , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Humanos , Camundongos
2.
Viruses ; 15(1)2023 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-36680194

RESUMO

Herpes simplex virus (HSV) and varicella zoster virus (VZV) rely on transport of virus particles in neuronal axons to spread from sites of viral latency in sensory ganglia to peripheral tissues then on to other hosts. This process of anterograde axonal transport involves kinesin motors that move virus particles rapidly along microtubules. α-herpesvirus anterograde transport has been extensively studied by characterizing the porcine pseudorabies virus (PRV) and HSV, with most studies focused on two membrane proteins: gE/gI and US9. It was reported that PRV and HSV US9 proteins bind to kinesin motors, promoting tethering of virus particles on the motors, and furthering anterograde transport within axons. Alternatively, other models have argued that HSV and PRV US9 and gE/gI function in the cytoplasm and not in neuronal axons. Specifically, HSV gE/gI and US9 mutants are defective in the assembly of virus particles in the cytoplasm of neurons and the subsequent sorting of virus particles to cell surfaces and into axons. However, PRV US9 and gE/gI mutants have not been characterized for these cytoplasmic defects. We examined neurons infected with PRV mutants, one lacking both gE/gI and US9 and the other lacking just US9, by electron microscopy. Both PRV mutants exhibited similar defects in virus assembly and cytoplasmic sorting of virus particles to cell surfaces. As well, the mutants exhibited reduced quantities of infectious virus in neurons and in cell culture supernatants. We concluded that PRV US9 primarily functions in neurons to promote cytoplasmic steps in anterograde transport.


Assuntos
Herpesvirus Suídeo 1 , Animais , Suínos , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/metabolismo , Transporte Axonal/fisiologia , Proteínas do Envelope Viral/metabolismo , Cinesinas/metabolismo , Linhagem Celular , Axônios , Simplexvirus/fisiologia , Citoplasma/metabolismo , Vírion/metabolismo
3.
Int J Mol Sci ; 24(2)2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36675134

RESUMO

Acute myeloid leukemia (AML) with a nucleophosmin 1 (NPM1) mutation is a unique subtype of adult leukemia. Recent studies show that NPM1-mutated AML has high autophagy activity. However, the mechanism for upholding the high autophagic level is still not fully elucidated. In this study, we first identified that tumor protein p53 inducible nuclear protein 2 (TP53INP2) was highly expressed and cytoplasmically localized in NPM1-mutated AML cells. Subsequent data showed that the expression of TP53INP2 was upregulated by fat mass and obesity-associated protein (FTO)-mediated m6A modification. Meanwhile, TP53INP2 was delocalized to the cytoplasm by interacting with NPM1 mutants. Functionally, cytoplasmic TP53INP2 enhanced autophagy activity by promoting the interaction of microtubule-associated protein 1 light chain 3 (LC3) - autophagy-related 7 (ATG7) and further facilitated the survival of leukemia cells. Taken together, our study indicates that TP53INP2 plays an oncogenic role in maintaining the high autophagy activity of NPM1-mutated AML and provides further insight into autophagy-targeted therapy of this leukemia subtype.


Assuntos
Leucemia Mieloide Aguda , Proteínas Nucleares , Adulto , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Citoplasma/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Autofagia/genética , Mutação , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética
4.
Methods Mol Biol ; 2623: 73-85, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36602680

RESUMO

Several light-inducible hetero-dimerization tools have been developed to spatiotemporally control subcellular localization and activity of target proteins or their downstream signaling. In contrast to other genetic technologies, such as CRISPR-mediated genome editing, these optogenetic tools can locally control protein localization on the second timescale. In addition, these tools can be used to understand the sufficiency of target proteins' function and manipulate downstream events. In this chapter, I will present methods for locally activating cytoplasmic dynein at the mitotic cell cortex in human cells, with a focus on how to generate knock-in cell lines and set up a microscope system.


Assuntos
Dineínas , Optogenética , Humanos , Dineínas/genética , Dineínas/metabolismo , Optogenética/métodos , Luz , Edição de Genes , Citoplasma/metabolismo
5.
Cell Death Dis ; 14(1): 20, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36635270

RESUMO

The carcinogenic role of FASN by regulating lipid metabolism reprogramming has been well-established in multiple tumors. However, whether mechanisms during intrahepatic cholangiocarcinoma (ICC) progression, such as circRNAs, regulate FASN expression remains unknown. Here we demonstrate a lipid metabolism-related circRNA, circMBOAT2 (hsa_circ_0007334 in circBase), frequently upregulated in ICC tissues, and positively correlated with ICC malignant features. CircMBOAT2 knockdown inhibits the growth and metastasis of ICC cells. Mechanistically, circMBOAT2 combines with PTBP1 and protects PTBP1 from ubiquitin/proteasome-dependent degradation, impairing the function of PTBP1 to transfer FASN mRNA from the nucleus to the cytoplasm. Moreover, circMBOAT2 and FASN have the same effect on fatty acid profile, unsaturated fatty acids instead of saturated fatty acids are primarily regulated and associated with malignant behaviors of ICC cells. The levels of lipid peroxidation and ROS were significantly higher when FASN was knocked down and recovered when circMBOAT2 was overexpressed. Our results identified that circMBOAT2 was upregulated in ICC and promoted progression by stabilizing PTBP1 to facilitate FASN mRNA cytoplasmic export, which altered lipid metabolic profile and regulated redox homeostasis in ICC, suggesting that circMBOAT2 may serve as an available therapeutic target for ICC with active lipid metabolism.


Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Metabolismo dos Lipídeos/genética , RNA Circular/genética , RNA Circular/metabolismo , Colangiocarcinoma/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias dos Ductos Biliares/patologia , Citoplasma/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo
6.
J Phys Chem B ; 127(1): 6-17, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36594654

RESUMO

Purple phototrophic bacteria are ancient anoxygenic phototrophs and attractive research tools because they capture light energy in the near-infrared (NIR) region of the spectrum and transform it into chemical energy by way of uphill energy transfers. The heart of this reaction occurs in light-harvesting 1-reaction center (LH1-RC) complexes, which are the simplest model systems for understanding basic photosynthetic reactions within type-II (quinone-utilizing) reaction centers. In this Perspective, we highlight structure-function relationships concerning unresolved fundamental processes in purple bacterial photosynthesis, including the diversified light-harvesting capacity of LH1-associated BChl molecules, energies necessary for photoelectric conversion in the RC special pairs, and quinone transport mechanisms. Based on recent progress in the spectroscopic and structural analysis of LH1-RC complexes from a variety of purple phototrophs, we discuss several key factors for understanding how purple bacteria resource light energy in the inherently energy-poor NIR region of the electromagnetic spectrum.


Assuntos
Complexos de Proteínas Captadores de Luz , Proteobactérias , Proteobactérias/metabolismo , Complexos de Proteínas Captadores de Luz/química , Fotossíntese , Análise Espectral , Citoplasma/metabolismo , Proteínas de Bactérias/química
7.
J Med Chem ; 66(1): 875-889, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36594812

RESUMO

Platinum drugs as primary chemotherapy drugs have been applied to various cancer patients. However, their therapeutic applicability is limited due to the adverse effects and immunosuppression. To minimize the side effects and boost the immune response, we designed and synthesized platinum(IV) prodrugs that introduced BRD4 inhibitor JQ-1. Among them, CJ2 had the most potent therapeutic activity and less toxicity. With the introduction of ligand JQ-1, CJ2-reduced PD-L1 protein was found in the cytoplasm and cytomembrane for the first time. By interfering with the PD-L1 synthesis, CJ2 could arouse the immune system and promote CD8+ T cell infiltration. Meanwhile, CJ2 could accelerate PD-L1 degradation in the cytoplasm to block DNA damage repair. In vivo, CJ2 markedly suppressed tumor growth by reversing the immunosuppression microenvironment and enhancing DNA damage. These findings provide an effective approach to improve the selectivity and activity of the platinum drugs with elevated immune response.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neoplasias , Pró-Fármacos , Humanos , Antígeno B7-H1 , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Platina/farmacologia , Platina/uso terapêutico , Proteínas Nucleares , Fatores de Transcrição , Imunoterapia , Citoplasma/metabolismo , Microambiente Tumoral , Linhagem Celular Tumoral , Proteínas de Ciclo Celular
8.
Cell Death Dis ; 14(1): 26, 2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36639679

RESUMO

Splicing factor 3B subunit 4 (SF3B4) plays important functional roles not only in pre-mRNA splicing, but also in the regulation of transcription, translation, and cell signaling, and its dysregulation contributes to various diseases including Nager syndrome and tumorigenesis. However, the role of SF3B4 and underlying mechanisms in clear cell renal cell carcinoma (ccRCC) remain obscure. In the present study, we found that the expression of SF3B4 was significantly elevated in ccRCC tissues and negatively correlated with the overall survival of ccRCC patients. Upregulation of SF3B4 promotes migration and invasion of ccRCC cells in vitro and in vivo. The promoting effect of SF3B4 on cell migration and invasion is mediated by Twist1, a key transcription factor to mediate EMT. Interestingly, SF3B4, a component of the pre-mRNA spliceosome, is able to promote KLF16 expression by facilitating the transport of KLF16 mRNA into the cytoplasm. Mechanistically, SF3B4 promotes the export of KLF16 mRNA from the nucleus to the cytoplasm and thus enhances KLF16 expression, and in turn elevated KLF16 directly binds to the Twist1 promoter to activate its transcription, leading to EMT and ccRCC progression. Our findings provide evidence that the SF3B4-KLF16-Twist1 axis plays important functional roles in the development and progression of ccRCC, and manipulating this pathway may be a novel therapeutic target for the treatment of ccRCC.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/genética , Citoplasma/metabolismo , Linhagem Celular Tumoral , Neoplasias Renais/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo
9.
Synapse ; 77(1): e22253, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36121749

RESUMO

Anorexia nervosa (AN) is a mental illness with the highest rates of mortality and relapse, and no approved pharmacological treatment. Using an animal model of AN, called activity-based anorexia (ABA), we showed earlier that a single intraperitoneal injection of ketamine at a dose of 30 mg/kg (30mgKET), but not 3 mg/kg (3mgKET), has a long-lasting effect upon adolescent females of ameliorating anorexia-like symptoms through the following changes: enhanced food consumption and body weight; reduced running and anxiety-like behavior. However, there were also individual differences in the drug's efficacy. We hypothesized that individual differences in ketamine's ameliorative effects involve drebrin A, an F-actin-binding protein known to be required for the activity-dependent trafficking of NMDA receptors (NMDARs). We tested this hypothesis by electron microscopic quantifications of drebrin A immunoreactivity at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (GABA-IN) in deep layer 1 of prefrontal cortex (PFC) of these mice. Results reveal that (1) the areal density of excitatory synapses on GABA-IN is greater for the 30mgKET group than the 3mgKET group; (2) the proportion of drebrin A+ excitatory synapses is greater for both PN and GABA-IN of 30mgKET than 3mgKET group. Correlation analyses with behavioral measurements revealed that (3) 30mgKET's protection is associated with reduced levels of drebrin A in the cytoplasm of GABA-IN and higher levels at extrasynaptic membranous sites of PN and GABA-IN; (5) altogether pointing to 30mgKET-induced homeostatic plasticity that engages drebrin A at excitatory synapses of both PN and GABA-IN.


Assuntos
Anorexia Nervosa , Ketamina , Camundongos , Feminino , Animais , Ketamina/farmacologia , Anorexia Nervosa/tratamento farmacológico , Anorexia Nervosa/metabolismo , Anorexia/tratamento farmacológico , Anorexia/metabolismo , Individualidade , Sinapses/metabolismo , Modelos Animais de Doenças , Córtex Pré-Frontal/metabolismo , Citoplasma/metabolismo , Ácido gama-Aminobutírico/metabolismo
10.
Nature ; 613(7942): 187-194, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36544021

RESUMO

R-loops are RNA-DNA-hybrid-containing nucleic acids with important cellular roles. Deregulation of R-loop dynamics can lead to DNA damage and genome instability1, which has been linked to the action of endonucleases such as XPG2-4. However, the mechanisms and cellular consequences of such processing have remained unclear. Here we identify a new population of RNA-DNA hybrids in the cytoplasm that are R-loop-processing products. When nuclear R-loops were perturbed by depleting the RNA-DNA helicase senataxin (SETX) or the breast cancer gene BRCA1 (refs. 5-7), we observed XPG- and XPF-dependent cytoplasmic hybrid formation. We identify their source as a subset of stable, overlapping nuclear hybrids with a specific nucleotide signature. Cytoplasmic hybrids bind to the pattern recognition receptors cGAS and TLR3 (ref. 8), activating IRF3 and inducing apoptosis. Excised hybrids and an R-loop-induced innate immune response were also observed in SETX-mutated cells from patients with ataxia oculomotor apraxia type 2 (ref. 9) and in BRCA1-mutated cancer cells10. These findings establish RNA-DNA hybrids as immunogenic species that aberrantly accumulate in the cytoplasm after R-loop processing, linking R-loop accumulation to cell death through the innate immune response. Aberrant R-loop processing and subsequent innate immune activation may contribute to many diseases, such as neurodegeneration and cancer.


Assuntos
Citoplasma , DNA , Ácidos Nucleicos Heteroduplexes , Estruturas R-Loop , RNA , Humanos , Apoptose , Citoplasma/imunologia , Citoplasma/metabolismo , DNA/química , DNA/imunologia , DNA Helicases/genética , DNA Helicases/metabolismo , Genes BRCA1 , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Mutação , Neoplasias , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/imunologia , Estruturas R-Loop/imunologia , RNA/química , RNA/imunologia , RNA Helicases/genética , RNA Helicases/metabolismo , Ataxias Espinocerebelares/genética
11.
Curr Biol ; 33(1): 122-133.e4, 2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36565699

RESUMO

Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events occurring in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along the pre-existing green microtubule within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. Lateral distance from the microtubule center was similar to that in incorporation sites and in growing ends. The saturation of the size and spatial frequency of incorporations as a function of injected tubulin concentration and post-injection delay suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 µm of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations.


Assuntos
Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Microtúbulos/metabolismo , Citoplasma/metabolismo , Polímeros/metabolismo , Citoesqueleto de Actina/metabolismo , Guanosina Trifosfato/metabolismo
12.
Artigo em Inglês | MEDLINE | ID: mdl-36586340

RESUMO

The exact levels of some DNA adducts, like N7-deoxyguanosine (N7-dG), can be under-calculated since these adducts may depurinate due to their chemical instability, leading to corresponding nucleobase adducts being released into the cytoplasm. To accurately quantify the levels of DNA adducts, it is necessary to consider those modified nucleobases. However, high levels and diversity of cytoplasmic small molecule metabolites (SMMs) can strongly interfere with the detection of adducts, and it is almost impossible to remove them with nucleobase adducts being well retained. Therefore, we aimed to establish an optimized enrichment method based on solid-phase extraction (SPE) to reduce the co-elution of SMMs with target analytes. In this vein, we employed three bisphenols (BPA, BPF, and BPAF) as examples, prepared corresponding N7-guanine (N7-Gua) adducts, loaded on an Oasis hydrophilic-lipophilic balance ® (HLB) cartridge, used a series of mobile phases containing different percentage of methanol for elution, and evaluated the levels of these adducts in each eluent. First, we found that neutral samples led to the best retention for all three adducts compared with acidified or basified ones. We next employed normal distribution fitting model to characterize the elution of analytes from H2O/methanol with different pHs and observed that neutral mobile phases resulted in more hydrophobic elution for all three adducts. Besides, N7-BPA-Gua and N7-BPF-Gua obtained narrow fitted peaks at neutral pH, while N7-BPAF-Gua had minimized elution windows at low pH. After optimization, we exposed 293T cells to the aforementioned bisphenols and quantified the N7-Gua adducts in the cytoplasm and the corresponding N7-dG adducts in genomic DNA. The results revealed that with the same levels of BPs exposure, BPAF led to the highest levels of adducts in both cytoplasm and genomic DNA samples, followed by BPA and BPF in order. In summary, our research established an appropriate model to describe the elution of DNA adducts in the SPE, applied it to optimize the loading and elution conditions, and discussed the genotoxicity of bisphenols by accurate quantification of both cleaved and uncleaved N7-dG adducts.


Assuntos
Adutos de DNA , Metanol , DNA/química , Guanina/química , Indicadores e Reagentes , Citoplasma/metabolismo , Extração em Fase Sólida , Genômica
13.
Elife ; 112022 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-36503602

RESUMO

Microtubules are tubes of about 25 nm in diameter that are critically involved in a variety of cellular functions, including motility, compartmentalization, and division. They are considered as pseudo-helical polymers whose constituent αß-tubulin heterodimers share lateral homotypic interactions, except at one unique region called the seam. Here, we used a segmented sub-tomogram averaging strategy to reassess this paradigm and analyze the organization of the αß-tubulin heterodimers in microtubules assembled from purified porcine brain tubulin in the presence of GTP and GMPCPP, and in Xenopus egg cytoplasmic extracts. We find that in almost all conditions, microtubules incorporate variable protofilament and/or tubulin subunit helical-start numbers, as well as variable numbers of seams. Strikingly, the seam number and location vary along individual microtubules, generating holes of one to a few subunits in size within their lattices. Together, our results reveal that the formation of mixed and discontinuous microtubule lattices is an intrinsic property of tubulin that requires the formation of unique lateral interactions without longitudinal ones. They further suggest that microtubule assembly is tightly regulated in a cytoplasmic environment.


Assuntos
Microtúbulos , Tubulina (Proteína) , Animais , Suínos , Tubulina (Proteína)/metabolismo , Xenopus laevis/metabolismo , Microtúbulos/metabolismo , Citoplasma/metabolismo , Encéfalo/metabolismo
14.
Int J Mol Sci ; 23(24)2022 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-36555789

RESUMO

Proteasome is a large proteolytic complex that consists of a 20S core particle (20SP) and 19S regulatory particle (19SP) in eukaryotes. The proteasome degrades most cellular proteins, thereby controlling many key processes, including gene expression and protein quality control. Proteasome dysfunction in plants leads to abnormal development and reduced adaptability to environmental stresses. Previous studies have shown that proteasome dysfunction upregulates the gene expression of proteasome subunits, which is known as the proteasome bounce-back response. However, the proteasome bounce-back response cannot explain the damaging effect of proteasome dysfunction on plant growth and stress adaptation. To address this question, we focused on downregulated genes caused by proteasome dysfunction. We first confirmed that the 20SP subunit PBE is an essential proteasome subunit in Arabidopsis and that PBE1 mutation impaired the function of the proteasome. Transcriptome analyses showed that hypoxia-responsive genes were greatly enriched in the downregulated genes in pbe1 mutants. Furthermore, we found that the pbe1 mutant is hypersensitive to waterlogging stress, a typical hypoxic condition, and hypoxia-related developments are impaired in the pbe1 mutant. Meanwhile, the 19SP subunit rpn1a mutant seedlings are also hypersensitive to waterlogging stress. In summary, our results suggested that proteasome dysfunction downregulated the hypoxia-responsive pathway and impaired plant growth and adaptability to hypoxia stress.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica de Plantas , Hipóxia , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo
15.
Biochem J ; 479(24): 2477-2495, 2022 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-36534469

RESUMO

Reproductive success of metazoans relies on germ cells. These cells develop early during embryogenesis, divide and undergo meiosis in the adult to make sperm and oocytes. Unlike somatic cells, germ cells are immortal and transfer their genetic material to new generations. They are also totipotent, as they differentiate into different somatic cell types. The maintenance of immortality and totipotency of germ cells depends on extensive post-transcriptional and post-translational regulation coupled with epigenetic remodeling, processes that begin with the onset of embryogenesis [1, 2]. At the heart of this regulation lie germ granules, membraneless ribonucleoprotein condensates that are specific to the germline cytoplasm called the germ plasm. They are a hallmark of all germ cells and contain several proteins and RNAs that are conserved across species. Interestingly, germ granules are often structured and tend to change through development. In this review, we describe how the structure of germ granules becomes established and discuss possible functional outcomes these structures have during development.


Assuntos
Oócitos , Sêmen , Masculino , Animais , Sêmen/metabolismo , Oócitos/metabolismo , Células Germinativas/metabolismo , Citoplasma/metabolismo , Ribonucleoproteínas/metabolismo
16.
Int J Mol Sci ; 23(23)2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36499069

RESUMO

Cost-effective production of therapeutic proteins in microbial hosts is an indispensable tool towards accessible healthcare. Many of these heterologously expressed proteins, including all antibody formats, require disulfide bond formation to attain their native and functional state. A system for catalyzed disulfide bond formation (CyDisCo) has been developed allowing efficient production of recombinant proteins in the cytoplasm of one of the most used microbial expression systems, Escherichia coli. Here, we report high-yield production (up to 230 mg/L from 3 mL cultures) of in-demand therapeutics such as IgG1-based Fc fusion proteins in the E. coli cytoplasm. However, the production of this drug class using the CyDisCo system faces bottlenecks related to redox heterogeneity during oxidative folding. Our investigations identified and addressed one of the major causes of redox heterogeneity during CyDisCo-based production of Fc fusion proteins, i.e., disulfide bond formation in the IgG1 CH3 domain. Here, we communicate that mutating the cysteines in the CH3 domain of target Fc fusion proteins allows their production in a homogeneous redox state in the cytoplasm of E. coli without compromising on yields and thermal stability.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Dissulfetos/química , Proteínas Recombinantes/metabolismo , Citoplasma/metabolismo , Imunoglobulina G/metabolismo , Proteínas Recombinantes de Fusão/química
17.
ASN Neuro ; 14: 17590914221136662, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36383501

RESUMO

Depression is a common psychiatric comorbidity in patients with epilepsy, especially those with temporal lobe epilepsy (TLE). The aim of this study was to assess changes in high mobility group box protein 1 (HMGB1) expression in epileptic patients with and without comorbid depression. Sixty patients with drug-resistant TLE who underwent anterior temporal lobectomy were enrolled. Anterior hippocampal samples were collected after surgery and analyzed by immunofluorescence (n = 7/group). We also evaluated the expression of HMGB1 in TLE patients with hippocampal sclerosis and measured the level of plasma HMGB1 by enzyme-linked immunosorbent assay. The results showed that 28.3% of the patients (17/60) had comorbid depression. HMGB1 was ubiquitously expressed in all subregions of the anterior hippocampus. The ratio of HMGB1-immunoreactive neurons and astrocytes was significantly increased in both TLE patients with hippocampal sclerosis and TLE patients with comorbid depression compared to patients with TLE only. The ratio of cytoplasmic to nuclear HMGB1-positive neurons in the hippocampus was higher in depressed patients with TLE than in nondepressed patients, which suggested that more HMGB1 translocated from the nucleus to the cytoplasm in the depressed group. There was no significant difference in the plasma level of HMGB1 among patients with TLE alone, TLE with hippocampal sclerosis, and TLE with comorbid depression. The results of the study revealed that the translocation of HMGB1 from the nucleus to the cytoplasm in hippocampal neurons may play a previously unrecognized role in the initiation and amplification of epilepsy and comorbid depression. The direct targeting of neural HMGB1 is a promising approach for anti-inflammatory therapy.


Assuntos
Epilepsia do Lobo Temporal , Epilepsia , Proteína HMGB1 , Humanos , Esclerose/metabolismo , Esclerose/patologia , Proteína HMGB1/metabolismo , Epilepsia do Lobo Temporal/cirurgia , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/patologia , Hipocampo/patologia , Epilepsia/cirurgia , Epilepsia/metabolismo , Gliose/patologia , Citoplasma/metabolismo
18.
Results Probl Cell Differ ; 70: 443-467, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36348118

RESUMO

Irregularities in nuclear shape and/or alterations to nuclear size are a hallmark of malignancy in a broad range of cancer types. Though these abnormalities are commonly used for diagnostic purposes and are often used to assess cancer progression in the clinic, the mechanisms through which they occur are not well understood. Nuclear size alterations in cancer could potentially arise from aneuploidy, changes in osmotic coupling with the cytoplasm, and perturbations to nucleocytoplasmic transport. Nuclear shape changes may occur due to alterations to cell-generated mechanical stresses and/or alterations to nuclear structural components, which balance those stresses, such as the nuclear lamina and chromatin. A better understanding of the mechanisms underlying abnormal nuclear morphology and size may allow the development of new therapeutics to target nuclear aberrations in cancer.


Assuntos
Núcleo Celular , Neoplasias , Humanos , Citoplasma/metabolismo , Transporte Ativo do Núcleo Celular , Cromatina/metabolismo , Neoplasias/metabolismo
19.
Proc Natl Acad Sci U S A ; 119(47): e2211637119, 2022 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-36343219

RESUMO

Cytoplasmic incompatibility (CI) is the most common reproductive manipulation produced by Wolbachia, obligately intracellular alphaproteobacteria that infect approximately half of all insect species. Once infection frequencies within host populations approach 10%, intense CI can drive Wolbachia to near fixation within 10 generations. However, natural selection among Wolbachia variants within individual host populations does not favor enhanced CI. Indeed, variants that do not cause CI but increase host fitness or are more reliably maternally transmitted are expected to spread if infected females remain protected from CI. Nevertheless, approximately half of analyzed Wolbachia infections cause detectable CI. Why? The frequency and persistence of CI are more plausibly explained by preferential spread to new host species (clade selection) rather than by natural selection among variants within host populations. CI-causing Wolbachia lineages preferentially spread into new host species because 1) CI increases equilibrium Wolbachia frequencies within host populations, and 2) CI-causing variants can remain at high frequencies within populations even when conditions change so that initially beneficial Wolbachia infections become harmful. An epidemiological model describing Wolbachia acquisition and loss by host species and the loss of CI-induction within Wolbachia lineages yields simple expressions for the incidence of Wolbachia infections and the fraction of those infections causing CI. Supporting a determinative role for differential interspecific spread in maintaining CI, many Wolbachia infections were recently acquired by their host species, many show evidence for contemporary spatial spread or retreat, and rapid evolution of CI-inducing loci, especially degradation, is common.


Assuntos
Wolbachia , Feminino , Humanos , Wolbachia/genética , Fertilidade , Citoplasma/metabolismo , Reprodução , Seleção Genética , Simbiose
20.
Mol Biol Cell ; 33(14): ae4, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36399622

RESUMO

In 1956, referring to the emerging application of electron microscopy to the study of eukaryotic cells, Keith R. Porter wrote, "For those of us who are fortunate to be part of this new development, these are days of great interest and opportunity." Those early days left us a rich legacy of knowledge on the internal organization of eukaryotic cells that provides a framework for current research on cell structure and function. In this vein, my long-time quest has been to understand how proteins and organelles travel through the cytoplasm to reach their respective destinations within the cell. This research has led us to elucidate various mechanisms of protein sorting and organelle transport and how defects in these mechanisms cause human disease.


Assuntos
Organelas , Humanos , Organelas/metabolismo , Microscopia Eletrônica , Transporte Biológico/fisiologia , Citoplasma/metabolismo , Transporte Proteico
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