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1.
Neuromolecular Med ; 26(1): 23, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38861223

RESUMO

Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disease affecting motor neurons. Pathological forms of Tar-DNA binding protein-43 (TDP-43), involving its mislocalisation to the cytoplasm and the formation of misfolded inclusions, are present in almost all ALS cases (97%), and ~ 50% cases of the related condition, frontotemporal dementia (FTD), highlighting its importance in neurodegeneration. Previous studies have shown that endoplasmic reticulum protein 57 (ERp57), a member of the protein disulphide isomerase (PDI) family of redox chaperones, is protective against ALS-linked mutant superoxide dismutase (SOD1) in neuronal cells and transgenic SOD1G93A mouse models. However, it remains unclear whether ERp57 is protective against pathological TDP-43 in ALS. Here, we demonstrate that ERp57 is protective against key features of TDP-43 pathology in neuronal cells. ERp57 inhibited the mislocalisation of TDP-43M337V from the nucleus to the cytoplasm. In addition, ERp57 inhibited the number of inclusions formed by ALS-associated variant TDP-43M337V and reduced the size of these inclusions. ERp57 was also protective against ER stress and induction of apoptosis. Furthermore, ERp57 modulated the steady-state expression levels of TDP-43. This study therefore demonstrates a novel mechanism of action of ERp57 in ALS. It also implies that ERp57 may have potential as a novel therapeutic target to prevent the TDP-43 pathology associated with neurodegeneration.


Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Ligação a DNA , Corpos de Inclusão , Isomerases de Dissulfetos de Proteínas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/genética , Animais , Camundongos , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Superóxido Dismutase-1/genética , Mutação
2.
Cell Mol Life Sci ; 81(1): 253, 2024 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-38852108

RESUMO

Post-transcriptional regulation of cytokine/chemokine mRNA turnover is critical for immune processes and contributes to the mammalian cellular response to diverse inflammatory stimuli. The ubiquitous RNA-binding protein human antigen R (HuR) is an integral regulator of inflammation-associated mRNA fate. HuR function is regulated by various post-translational modifications that alter its subcellular localization and ability to stabilize target mRNAs. Both poly (ADP-ribose) polymerase 1 (PARP1) and p38 mitogen-activated protein kinases (MAPKs) have been reported to regulate the biological function of HuR, but their specific regulatory and crosstalk mechanisms remain unclear. In this study, we show that PARP1 acts via p38 to synergistically promote cytoplasmic accumulation of HuR and stabilization of inflammation-associated mRNAs in cells under inflammatory conditions. Specifically, p38 binds to auto-poly ADP-ribosylated (PARylated) PARP1 resulting in the covalent PARylation of p38 by PARP1, thereby promoting the retention and activity of p38 in the nucleus. In addition, PARylation of HuR facilitates the phosphorylation of HuR at the serine 197 site mediated by p38, which then increases the translocation of HuR to the cytoplasm, ultimately stabilizing the inflammation-associated mRNA expression at the post-transcriptional level.


Assuntos
Citoplasma , Proteína Semelhante a ELAV 1 , Inflamação , Poli(ADP-Ribose) Polimerase-1 , RNA Mensageiro , Proteínas Quinases p38 Ativadas por Mitógeno , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Citoplasma/metabolismo , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fosforilação , Regulação da Expressão Gênica , Animais , Poli ADP Ribosilação/genética , Células HEK293 , Núcleo Celular/metabolismo , Camundongos
3.
J Cell Sci ; 137(11)2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38841902

RESUMO

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.


Assuntos
Citoplasma , Homeostase , RNA Mensageiro , Grânulos de Estresse , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Grânulos de Estresse/metabolismo , Citoplasma/metabolismo , Capuzes de RNA/metabolismo , Arsenitos/farmacologia , Estresse Oxidativo , Transporte Ativo do Núcleo Celular , RNA Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/genética , Compostos de Sódio/farmacologia , Proteína Exportina 1 , Carioferinas/metabolismo , Carioferinas/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Grânulos Citoplasmáticos/metabolismo , Estabilidade de RNA , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , Nucleotidiltransferases
4.
Nat Commun ; 15(1): 4986, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862544

RESUMO

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P2-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.


Assuntos
Membrana Celular , Adesões Focais , Talina , Vinculina , Talina/metabolismo , Talina/química , Adesões Focais/metabolismo , Membrana Celular/metabolismo , Vinculina/metabolismo , Vinculina/química , Humanos , Animais , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Integrinas/metabolismo , Integrinas/química , Citoplasma/metabolismo , Ligação Proteica , Separação de Fases
5.
Biochem Biophys Res Commun ; 718: 149981, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-38735134

RESUMO

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.


Assuntos
Endossomos , PTEN Fosfo-Hidrolase , Fosfatidilinositóis , Vacúolos , Vacúolos/metabolismo , Vacúolos/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/efeitos dos fármacos , Humanos , Fosfatidilinositóis/metabolismo , Animais , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Camundongos , Morfolinas/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/genética , Citoplasma/metabolismo , Células HeLa , Aminopiridinas , Compostos Heterocíclicos com 3 Anéis
6.
Curr Protoc ; 4(5): e1042, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38767195

RESUMO

Biochemical fractionation is a technique used to isolate and separate distinct cellular compartments, critical for dissecting cellular mechanisms and molecular pathways. Herein we outline a biochemical fraction methodology for isolation of ultra-pure nuclei and cytoplasm. This protocol utilizes hypotonic lysis buffer to suspend cells, coupled with a calibrated centrifugation strategy, for enhanced separation of cytoplasm from the nuclear fraction. Subsequent purification steps ensure the integrity of the isolated nuclear fraction. Overall, this method facilitates accurate protein localization, essential for functional studies, demonstrating its efficacy in separating cellular compartments. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Biochemical fractionation Support Protocol 1: Protein quantification using Bradford assay Support Protocol 2: SDS/PAGE and Western blotting.


Assuntos
Fracionamento Celular , Núcleo Celular , Citoplasma , Citoplasma/metabolismo , Citoplasma/química , Núcleo Celular/metabolismo , Núcleo Celular/química , Fracionamento Celular/métodos , Humanos , Eletroforese em Gel de Poliacrilamida , Western Blotting
7.
Reprod Biol Endocrinol ; 22(1): 55, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38745305

RESUMO

The role of cytoplasmic fragmentation in human embryo development and reproductive potential is widely recognized, albeit without standard definition nor agreed upon implication. While fragmentation is best understood to be a natural process across species, the origin of fragmentation remains incompletely understood and likely multifactorial. Several factors including embryo culture condition, gamete quality, aneuploidy, and abnormal cytokinesis seem to have important role in the etiology of cytoplasmic fragmentation. Fragmentation reduces the volume of cytoplasm and depletes embryo of essential organelles and regulatory proteins, compromising the developmental potential of the embryo. While it has been shown that degree of fragmentation and embryo implantation potential are inversely proportional, the degree, pattern, and distribution of fragmentation as it relates to pregnancy outcome is debated in the literature. This review highlights some of the challenges in analysis of fragmentation, while revealing trends in our evolving knowledge of how fragmentation may relate to functional development of the human embryos, implantation, and pregnancy outcome.


Assuntos
Citoplasma , Desenvolvimento Embrionário , Resultado da Gravidez , Humanos , Feminino , Gravidez , Desenvolvimento Embrionário/fisiologia , Citoplasma/metabolismo , Citoplasma/fisiologia , Implantação do Embrião/fisiologia
8.
Mol Brain ; 17(1): 28, 2024 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-38790036

RESUMO

The aggregated alpha-synuclein (αsyn) in oligodendrocytes (OLGs) is one of the pathological hallmarks in multiple system atrophy (MSA). We have previously reported that αsyn accumulates not only in neurons but also in OLGs long after the administration of αsyn preformed fibrils (PFFs) in mice. However, detailed spatial and temporal analysis of oligodendroglial αsyn aggregates was technically difficult due to the background neuronal αsyn aggregates. The aim of this study is to create a novel mouse that easily enables sensitive and specific detection of αsyn aggregates in OLGs and the comparable analysis of the cellular tropism of αsyn aggregates in MSA brains. To this end, we generated transgenic (Tg) mice expressing human αsyn-green fluorescent protein (GFP) fusion proteins in OLGs under the control of the 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) promoter (CNP-SNCAGFP Tg mice). Injection of αsyn PFFs in these mice induced distinct GFP-positive aggregates in the processes of OLGs as early as one month post-inoculation (mpi), and their number and size increased in a centripetal manner. Moreover, MSA-brain homogenates (BH) induced significantly more oligodendroglial αsyn aggregates than neuronal αsyn aggregates compared to DLB-BH in CNP-SNCAGFP Tg mice, suggestive of their potential tropism of αsyn seeds for OLGs. In conclusion, CNP-SNCAGFP Tg mice are useful for studying the development and tropism of αsyn aggregates in OLGs and could contribute to the development of therapeutics targeting αsyn aggregates in OLGs.


Assuntos
Modelos Animais de Doenças , Corpos de Inclusão , Camundongos Transgênicos , Atrofia de Múltiplos Sistemas , Oligodendroglia , Agregados Proteicos , alfa-Sinucleína , Animais , alfa-Sinucleína/metabolismo , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Atrofia de Múltiplos Sistemas/patologia , Atrofia de Múltiplos Sistemas/metabolismo , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Humanos , Proteínas de Fluorescência Verde/metabolismo , Citoplasma/metabolismo , Camundongos , Encéfalo/patologia , Encéfalo/metabolismo , Agregação Patológica de Proteínas/metabolismo
9.
Planta ; 260(1): 6, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38780795

RESUMO

MAIN CONCLUSION: TaAGL66, a MADS-box transcription factor highly expressed in fertile anthers of KTM3315A, regulates anther and/or pollen development, as well as male fertility in wheat with Aegilops kotschyi cytoplasm. Male sterility, as a string of sophisticated biological processes in higher plants, is commonly regulated by transcription factors (TFs). Among them, MADS-box TFs are mainly participated in the processes of floral organ formation and pollen development, which are tightly related to male sterility, but they have been little studied in the reproductive development in wheat. In our study, TaAGL66, a gene that was specifically expressed in spikes and highly expressed in fertile anthers, was identified by RNA sequencing and the expression profiles data of these genes, and qRT-PCR analyses, which was localized to the nucleus. Silencing of TaAGL66 under fertility condition in KTM3315A, a thermo-sensitive male sterile line with Ae. kotschyi cytoplasm, displayed severe fertility reduction, abnormal anther dehiscence, defective pollen development, decreased viability, and low seed-setting. It can be concluded that TaAGL66 plays an important role in wheat pollen development in the presence of Ae. kotschyi cytoplasm, providing new insights into the utilization of male sterility.


Assuntos
Aegilops , Citoplasma , Fertilidade , Regulação da Expressão Gênica de Plantas , Infertilidade das Plantas , Proteínas de Plantas , Pólen , Triticum , Triticum/genética , Triticum/crescimento & desenvolvimento , Triticum/fisiologia , Citoplasma/metabolismo , Citoplasma/genética , Pólen/genética , Pólen/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Aegilops/genética , Infertilidade das Plantas/genética , Fertilidade/genética , Flores/genética , Flores/crescimento & desenvolvimento , Proteínas de Domínio MADS/genética , Proteínas de Domínio MADS/metabolismo , Genes de Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Comput Methods Programs Biomed ; 252: 108215, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38781811

RESUMO

BACKGROUND AND OBJECTIVE: Cell segmentation in bright-field histological slides is a crucial topic in medical image analysis. Having access to accurate segmentation allows researchers to examine the relationship between cellular morphology and clinical observations. Unfortunately, most segmentation methods known today are limited to nuclei and cannot segment the cytoplasm. METHODS: We present a new network architecture Cyto R-CNN that is able to accurately segment whole cells (with both the nucleus and the cytoplasm) in bright-field images. We also present a new dataset CytoNuke, consisting of multiple thousand manual annotations of head and neck squamous cell carcinoma cells. Utilizing this dataset, we compared the performance of Cyto R-CNN to other popular cell segmentation algorithms, including QuPath's built-in algorithm, StarDist, Cellpose and a multi-scale Attention Deeplabv3+. To evaluate segmentation performance, we calculated AP50, AP75 and measured 17 morphological and staining-related features for all detected cells. We compared these measurements to the gold standard of manual segmentation using the Kolmogorov-Smirnov test. RESULTS: Cyto R-CNN achieved an AP50 of 58.65% and an AP75 of 11.56% in whole-cell segmentation, outperforming all other methods (QuPath 19.46/0.91%; StarDist 45.33/2.32%; Cellpose 31.85/5.61%, Deeplabv3+ 3.97/1.01%). Cell features derived from Cyto R-CNN showed the best agreement to the gold standard (D¯=0.15) outperforming QuPath (D¯=0.22), StarDist (D¯=0.25), Cellpose (D¯=0.23) and Deeplabv3+ (D¯=0.33). CONCLUSION: Our newly proposed Cyto R-CNN architecture outperforms current algorithms in whole-cell segmentation while providing more reliable cell measurements than any other model. This could improve digital pathology workflows, potentially leading to improved diagnosis. Moreover, our published dataset can be used to develop further models in the future.


Assuntos
Algoritmos , Processamento de Imagem Assistida por Computador , Redes Neurais de Computação , Humanos , Processamento de Imagem Assistida por Computador/métodos , Núcleo Celular , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico por imagem , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Citoplasma , Reprodutibilidade dos Testes , Carcinoma de Células Escamosas/diagnóstico por imagem , Carcinoma de Células Escamosas/patologia
11.
Nat Commun ; 15(1): 3901, 2024 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-38724505

RESUMO

Activation of the NF-κB pathway is strictly regulated to prevent excessive inflammatory and immune responses. In a well-known negative feedback model, IκBα-dependent NF-κB termination is a delayed response pattern in the later stage of activation, and the mechanisms mediating the rapid termination of active NF-κB remain unclear. Here, we showed IκBα-independent rapid termination of nuclear NF-κB mediated by CLK2, which negatively regulated active NF-κB by phosphorylating the RelA/p65 subunit of NF-κB at Ser180 in the nucleus to limit its transcriptional activation through degradation and nuclear export. Depletion of CLK2 increased the production of inflammatory cytokines, reduced viral replication and increased the survival of the mice. Mechanistically, CLK2 phosphorylated RelA/p65 at Ser180 in the nucleus, leading to ubiquitin‒proteasome-mediated degradation and cytoplasmic redistribution. Importantly, a CLK2 inhibitor promoted cytokine production, reduced viral replication, and accelerated murine psoriasis. This study revealed an IκBα-independent mechanism of early-stage termination of NF-κB in which phosphorylated Ser180 RelA/p65 turned off posttranslational modifications associated with transcriptional activation, ultimately resulting in the degradation and nuclear export of RelA/p65 to inhibit excessive inflammatory activation. Our findings showed that the phosphorylation of RelA/p65 at Ser180 in the nucleus inhibits early-stage NF-κB activation, thereby mediating the negative regulation of NF-κB.


Assuntos
Citoplasma , Inibidor de NF-kappaB alfa , NF-kappa B , Proteínas Tirosina Quinases , Fator de Transcrição RelA , Animais , Fosforilação , Inibidor de NF-kappaB alfa/metabolismo , Inibidor de NF-kappaB alfa/genética , Camundongos , Fator de Transcrição RelA/metabolismo , Humanos , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , NF-kappa B/metabolismo , Citoplasma/metabolismo , Proteólise , Núcleo Celular/metabolismo , Replicação Viral , Células HEK293 , Transdução de Sinais , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Serina-Treonina Quinases
12.
Cryobiology ; 115: 104901, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38754687

RESUMO

While cryopreservation of cauda epididymal sperm (SpCau) allows the preservation of post-mortem bulls' gametes, the process triggers sperm damage. Although improving post-thaw sperm quality, using egg yolk extenders (EY) raises biosafety concerns which forces the use of EY-free extenders (EYFE). Since EYFE are less efficient in preserving post-thaw sperm quality, a strategy for ejaculated sperm (SpEj) frozen with EYFE is to add an Equilibrium Time (ET) step period to the cryopreservation process. However, the ET effect on the quality of SpCau cryopreserved in EYFE remains unknown. Distinct from SpEJ, SpCau physiologically displays cytoplasmic droplets (CDs) in the flagellum that may benefit cell exchange during ET. We hypothesized that using ET in SpCau cryopreserved with EYFE impacts sperm morphofunctional features, CD area, and in vitro fertility ability. Extender nanoparticles were also assessed. Following collection from the cauda epididymis of six Nellore bulls by retrograde flow, SpCau were cryopreserved in EYFE BoviFree® (Minitube, Germany) using three ET protocols: ET0 (no-ET); ET2.5 (2.5 h-ET); and ET5 (5 h-ET). SpCau from ET2.5 and ET5 showed a higher (P ≤ 0.05) percentage of motility and integrity of plasma and acrosome membranes and a smaller (P ≤ 0.05) distal CD area. There are no differences in sperm abnormalities, oxidative stress, capacitation-like events, and in vitro fertility ability. However, a better sperm recovery was found after Percoll® selection for ET2.5 and ET5. Interestingly, the number of nanoparticles in the extender decreased in post-thawed samples. In conclusion, an ET of 2.5 or 5 h is required for an efficient SpCau cryopreservation using an EYFE.


Assuntos
Criopreservação , Crioprotetores , Epididimo , Nanopartículas , Preservação do Sêmen , Motilidade dos Espermatozoides , Espermatozoides , Masculino , Animais , Criopreservação/métodos , Criopreservação/veterinária , Preservação do Sêmen/métodos , Preservação do Sêmen/veterinária , Crioprotetores/farmacologia , Espermatozoides/citologia , Epididimo/citologia , Bovinos , Nanopartículas/química , Gema de Ovo/química , Análise do Sêmen , Citoplasma
13.
J Biotechnol ; 390: 62-70, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38761885

RESUMO

Human serum albumin (HSA), a polypeptide featuring 17 disulfide bonds, acts as a crucial transport protein in human blood plasma. Its extended circulation half-life, mediated by FcRn (neonatal Fc receptor)-facilitated recycling, positions HSA as an excellent carrier for long-acting drug delivery. However, the conventional method of obtaining HSA from human blood faces limitations due to availability and potential contamination risks, such as blood-borne diseases. This study introduced SHuffle, an oxidative Escherichia coli (E. coli) expression system, for the production of recombinant HSA (rHSA) that spontaneously self-folds into its native conformation. This system ensures precise disulfide bond formation and correct folding of cysteine-rich rHSA, eliminating the need for chaperone co-expression or domain fusion of a folding enhancer. The purified rHSA underwent thorough physicochemical characterization, including mass spectrometry, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, esterase-like activity assay, and size exclusion chromatography, to assess critical quality attributes. Importantly, rHSA maintained native binding affinity to FcRn and the albumin-binding domain. Collectively, our analyses demonstrated a high comparability between rHSA and plasma-derived HSA. The expression of rHSA in E. coli with an oxidizing cytosol provides a secure and cost-effective approach, enhancing the potential of rHSA for diverse medical applications.


Assuntos
Escherichia coli , Oxirredução , Dobramento de Proteína , Proteínas Recombinantes , Albumina Sérica Humana , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Albumina Sérica Humana/metabolismo , Albumina Sérica Humana/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Citoplasma/metabolismo , Receptores Fc/metabolismo , Receptores Fc/química , Antígenos de Histocompatibilidade Classe I/metabolismo
14.
Biochemistry ; 63(8): 1000-1015, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38577872

RESUMO

PI31 (Proteasome Inhibitor of 31,000 Da) is a 20S proteasome binding protein originally identified as an in vitro inhibitor of 20S proteasome proteolytic activity. Recently reported cryo-electron microscopy structures of 20S-PI31 complexes have revealed that the natively disordered proline-rich C-terminus of PI31 enters the central chamber in the interior of the 20S proteasome and interacts directly with the proteasome's multiple catalytic threonine residues in a manner predicted to inhibit their enzymatic function while evading its own proteolysis. Higher eukaryotes express an alternative form of the 20S proteasome (termed "immuno-proteasome") that features genetically and functionally distinct catalytic subunits. The effect of PI31 on immuno-proteasome function is unknown. We examine the relative inhibitory effects of PI31 on purified constitutive (20Sc) and immuno-(20Si) 20S proteasomes in vitro and show that PI31 inhibits 20Si hydrolytic activity to a significantly lesser degree than that of 20Sc. Unlike 20Sc, 20Si hydrolyzes the carboxyl-terminus of PI31 and this effect contributes to the reduced inhibitory activity of PI31 toward 20Si. Conversely, loss of 20Sc inhibition by PI31 point mutants leads to PI31 degradation by 20Sc. These results demonstrate unexpected differential interactions of PI31 with 20Sc and 20Si and document their functional consequences.


Assuntos
Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Microscopia Crioeletrônica , Proteínas/química , Citoplasma/metabolismo , Antivirais
15.
Biochemistry (Mosc) ; 89(Suppl 1): S205-S223, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38621751

RESUMO

The term "biomolecular condensates" is used to describe membraneless compartments in eukaryotic cells, accumulating proteins and nucleic acids. Biomolecular condensates are formed as a result of liquid-liquid phase separation (LLPS). Often, they demonstrate properties of liquid-like droplets or gel-like aggregates; however, some of them may appear to have a more complex structure and high-order organization. Membraneless microcompartments are involved in diverse processes both in cytoplasm and in nucleus, among them ribosome biogenesis, regulation of gene expression, cell signaling, and stress response. Condensates properties and structure could be highly dynamic and are affected by various internal and external factors, e.g., concentration and interactions of components, solution temperature, pH, osmolarity, etc. In this review, we discuss variety of biomolecular condensates and their functions in live cells, describe their structure variants, highlight domain and primary sequence organization of the constituent proteins and nucleic acids. Finally, we describe current advances in methods that characterize structure, properties, morphology, and dynamics of biomolecular condensates in vitro and in vivo.


Assuntos
Fenômenos Bioquímicos , Ácidos Nucleicos , Condensados Biomoleculares , Proteínas , Citoplasma
16.
World J Microbiol Biotechnol ; 40(5): 153, 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38564115

RESUMO

Ralstonia solanacearum, the bacterium that causes bacterial wilt, is a destructive phytopathogen that can infect over 450 different plant species. Several agriculturally significant crop plants, including eggplant, tomato, pepper, potato, and ginger, are highly susceptible to this plant disease, which has a global impact on crop quality and yield. There is currently no known preventive method that works well for bacterial wilt. Bacteria use two-component systems (TCSs) to sense their environment constantly and react appropriately. This is achieved by an extracellular sensor kinase (SK) capable of sensing a suitable signal and a cytoplasmic response regulator (RR) which gives a downstream response. Moreover, our investigation revealed that R. solanacearum GMI1000 possesses a substantial count of TCSs, specifically comprising 36 RRs and 27 SKs. While TCSs are known targets for various human pathogenic bacteria, such as Salmonella, the role of TCSs in R. solanacearum remains largely unexplored in this context. Notably, numerous inhibitors targeting TCSs have been identified, including GHL (Gyrase, Hsp, and MutL) compounds, Walk inhibitors, and anti-TCS medications like Radicicol. Consequently, the investigation into the involvement of TCSs in virulence and pathogenesis has gained traction; however, further research is imperative to ascertain whether TCSs could potentially supplant conventional anti-wilt therapies. This review delves into the prospective utilization of TCSs as an alternative anti-wilt therapy, focusing on the lethal phytopathogen R. solanacearum.


Assuntos
Ralstonia solanacearum , Humanos , Estudos Prospectivos , Bactérias , Citoplasma , Citosol
17.
Proc Natl Acad Sci U S A ; 121(15): e2313004121, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38564631

RESUMO

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.


Assuntos
Polifosfatos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Polifosfatos/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo
18.
In Vivo ; 38(3): 1316-1324, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38688649

RESUMO

BACKGROUND/AIM: Our objectives in this study were to (i) evaluate the clinical significance of X-box-binding protein 1 (XBP1) expression in cases of hepatocellular carcinoma (HCC) and (ii) assess the potential of XBP1 to be used as a prognostic biomarker. PATIENTS AND METHODS: The expression of XBP1 protein in 267 HCC tissue specimens was measured using immunohistochemistry in order to characterize the associations among XBP1 expression, clinicopathological factors and survival outcomes. Survival analysis using follow-up data was used to assess the prognostic value of XBP1 in cases of HCC. Immunohistochemistry revealed a significant decrease in cytoplasmic XBP1 protein expression in HCC tumor tissue. RESULTS: Immunoreactivity results showed that low cytoplasmic XBP1 expression was significantly associated with vascular invasion, as well as poor 5-year overall survival and long-term disease-specific (DSS) and disease-free (DFS) survival rates. Kaplan-Meier survival curves further confirmed a significant association between low cytoplasmic XBP1 protein expression and poor DSS and DFS. Univariate and multivariate analyses revealed that XBP1 expression, tumor differentiation, vascular invasion, tumor stage, and the rate of recurrence were linked to DSS, while low cytoplasmic XBP1 expression remained an independent predictor of poor DSS. Our analysis also revealed that XBP1 expression, tumor differentiation, vascular invasion, and T classification were linked to DFS, while low cytoplasmic XBP1 expression remained an independent predictor of poor DFS. CONCLUSION: Low cytoplasmic XBP1 protein expression may play an important role in the pathogenesis of HCC, which suggests that XBP1 could potentially be targeted to benefit therapeutic strategies for HCC.


Assuntos
Biomarcadores Tumorais , Carcinoma Hepatocelular , Citoplasma , Neoplasias Hepáticas , Proteína 1 de Ligação a X-Box , Humanos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/genética , Proteína 1 de Ligação a X-Box/metabolismo , Proteína 1 de Ligação a X-Box/genética , Masculino , Feminino , Pessoa de Meia-Idade , Citoplasma/metabolismo , Prognóstico , Biomarcadores Tumorais/metabolismo , Idoso , Adulto , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Estadiamento de Neoplasias
19.
J Math Biol ; 88(6): 72, 2024 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-38678110

RESUMO

In this work, we formulate a random Wolbachia invasion model incorporating the effects of imperfect maternal transmission and incomplete cytoplasmic incompatibility (CI). Under constant environments, we obtain the following results: Firstly, the complete invasion equilibrium of Wolbachia does not exist, and thus the population replacement is not achievable in the case of imperfect maternal transmission; Secondly, imperfect maternal transmission or incomplete CI may obliterate bistability and backward bifurcation, which leads to the failure of Wolbachia invasion, no matter how many infected mosquitoes would be released; Thirdly, the threshold number of the infected mosquitoes to be released would increase with the decrease of the maternal transmission rate or the intensity of CI effect. In random environments, we investigate in detail the Wolbachia invasion dynamics of the random mosquito population model and establish the initial release threshold of infected mosquitoes for successful invasion of Wolbachia into the wild mosquito population. In particular, the existence and stability of invariant probability measures for the establishment and extinction of Wolbachia are determined.


Assuntos
Conceitos Matemáticos , Modelos Biológicos , Mosquitos Vetores , Wolbachia , Wolbachia/fisiologia , Wolbachia/patogenicidade , Animais , Feminino , Mosquitos Vetores/microbiologia , Dinâmica Populacional/estatística & dados numéricos , Citoplasma/microbiologia , Culicidae/microbiologia , Masculino , Simulação por Computador , Herança Materna
20.
Int Immunopharmacol ; 133: 112065, 2024 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-38608448

RESUMO

Signal transducer and activator of transcription 3 (STAT3) functions to regulate inflammation and immune response, but its mechanism is not fully understood. We report here that STAT3 inhibitors Stattic and Niclosamide up-regulated IL-1ß-induced IL-8 production in C33A, CaSki, and Siha cervical cancer cells. As expected, IL-1ß-induced IL-8 production was also up-regulated through the molecular inhibition of STAT3 by use of CRISPR/Cas9 technology. Unexpectedly, IL-1ß induced IL-8 production via activating ERK and P38 signal pathways, but neither STAT3 inhibitors nor STAT3 knockout affected IL-1ß-induced signal transduction, suggesting that STAT3 decreases IL-8 production not via inhibition of signal transduction. To our surprise, STAT3 inhibition increased the stabilization, and decreased the degradation of IL-8 mRNA, suggesting a post-transcriptional regulation of IL-1ß-induced IL-8. Moreover, Dihydrotanshinone I, an inhibitor of RNA-binding protein HuR, down-regulated IL-1ß-induced IL-8 dose-dependently. HuR inhibition by CRISPR/Cas9 also decreased IL-8 production induced by IL-1ß. Mechanistically, co-immunoprecipitation results showed that STAT3 did not react with HuR directly, but STAT3 inhibition increased the protein levels of HuR in cytoplasm. And IL-6 activation of STAT3 induced HuR cytoplasmic-nuclear transport. Taken together, these results suggest that STAT3 contributes to HuR nuclear localization and inhibits Il-1ß-induced IL-8 production through this non-transcriptional mechanism.


Assuntos
Núcleo Celular , Citoplasma , Proteína Semelhante a ELAV 1 , Interleucina-1beta , Interleucina-8 , Fator de Transcrição STAT3 , Humanos , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT3/genética , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Interleucina-8/genética , Proteína Semelhante a ELAV 1/metabolismo , Proteína Semelhante a ELAV 1/genética , Citoplasma/metabolismo , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , Óxidos S-Cíclicos/farmacologia , Transporte Proteico , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Sistemas CRISPR-Cas
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