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1.
Adv Exp Med Biol ; 1148: 131-150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482498

RESUMO

Enzymes are biocatalysts that have found profound applications in the current biotherapeutic industry and play a crucial role in diagnosis, prevention, and biochemical analysis of major diseases. However, stability, protein degradation and immunogenicity in the body present unique challenges that are faced upon sustained use of such enzymes. The present chapter is an attempt to dissect the state-of-the-art in relation to the challenges of development of therapeutic enzymes and the recent advances to address them. At the very outset, diseases where enzymes have found effective applications and the various causes of enzyme instability have been discussed. In recent times, polymer or nano- conjugated resistant delivery methods, as well as mutagenesis have led to manifold increase in enzyme stability against thermal denaturation, acidic gut environment, proteolysis and immunogenicity. Further, methods of analytical characterization of proteins have been highlighted and explored to shape future research directions.


Assuntos
Estabilidade Enzimática , Enzimas/química , Enzimas/farmacologia , Proteólise
2.
Nihon Yakurigaku Zasshi ; 154(3): 108-113, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31527359

RESUMO

Similar to calcium (Ca2+) and chloride (Cl-) ion channels/transporters, potassium (K+) channels have been recognized as a crucial cancer treatment target. Recent studies have provided convincing evidences of positive correlation between elevated expression levels of Ca2+-activated K+ (KCa) channels and cancer proliferation, metastasis, and poor patient prognosis. In cancer cells, KCa1.1 and KCa3.1 KCa channels are co-localized with Ca2+-permeable Orai/TRP channels to provide a positive-feedback loop for Ca2+ entry. They are responsible for the promotion of cell growth and metastasis in the different types of cancer, and are therefore potential therapeutic targets and biomarkers for cancer. We determined the epigenetic and post-transcriptional dysregulation of KCa3.1 by class I histone deacetylase inhibitors in breast and prostate cancer cells. We further determined the transcriptional repression and protein degradation of KCa1.1 by vitamin D receptor agonists and androgen receptor antagonists, which are expected as potential therapeutic drugs for triple-negative breast cancer. The anti-inflammatory cytokine, interleukin-10 (IL-10) is an immunosuppressive factor involved in tumorigenesis, and plays a crucial role in escape from tumor immune surveillance. We determined KCa3.1 activators are a possible therapeutic option to suppress the tumor-promoting activities of IL-10. These results may provide new insights into cancer treatment focused on Ca2+-activated K+ channels.


Assuntos
Neoplasias da Mama/patologia , Inibidores de Histona Desacetilases/farmacologia , Canais de Potássio Cálcio-Ativados/metabolismo , Neoplasias da Próstata/patologia , Antagonistas de Receptores de Andrógenos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Epigênese Genética , Feminino , Humanos , Vigilância Imunológica , Interleucina-10/metabolismo , Masculino , Proteólise , Processamento Pós-Transcricional do RNA , Receptores de Calcitriol/agonistas
3.
Adv Exp Med Biol ; 1152: 401-411, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31456196

RESUMO

Understanding breast cancer cell proteolysis and migration is crucial for developing novel therapies to prevent local and distant metastases. Human cancer cells utilize many biological functions comparable to those observed during embryogenesis conferring the cancer cells with survival advantages. One such advantage is the ability to secrete proteases into the tumor microenvironment in order to remodel the extracellular matrix to facilitate migration. These proteases degrade the extracellular matrix, which initially functions as a barrier to cancer cell escape from their site of origin. The extracellular matrix also functions as a reservoir for growth factors that can be released by the secreted proteases and thereby further aid tumor growth and progression. Other survival advantages of tumor cells include: the ability to utilize multiple modes of motility, thrive in acidic microenvironments, and the tumor cell's ability to hijack stromal and immune cells to foster their own migration and survival. In order to reduce metastasis, we must focus our efforts on addressing the survival advantages that tumor cells have acquired.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Proteólise , Matriz Extracelular , Feminino , Humanos , Peptídeo Hidrolases/metabolismo , Microambiente Tumoral
4.
Life Sci ; 234: 116788, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31445935

RESUMO

Livin is an important member of the human inhibitor of apoptosis proteins (IAPs) family. IAPs are proteins with antiapoptotic abilities, and their functions are different from the Bcl-2 (B-cell lymphoma-2) family proteins. However, the precise role of Livin in colon cancer progression remains unclear. The purpose of this study is to assess the effect of overexpression Livin in colon cancer cells and to examine its molecular mechanism. We demonstrated that Livin induced a colon cancer phenotype, including proliferation and migration, by regulating H2A.XY39ph (histone family 2A variant (H2AX) phosphorylated on the 39th serine site). We elucidated that Livin degraded Jumonji-C domain-containing 6 protein (JMJD6), which was mediated by the proteasome murine double minute 2 (MDM2), thereby regulating H2A.XY39ph. Above all, the overexpression of JMJD6 recovered H2A.XY39ph in colon cancer cells with a high level of Livin, thus inhibiting colon cancer malignancy progression. These results reveal a previously unrecognized role for Livin in regulating the tumor-initiating capacity in colon cancer and provide a novel treatment strategy in cancer via the interruption of H2A.XY39ph function and the interaction between H2A.XY39ph and JMJD6.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias do Colo/patologia , Histonas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interação de Proteínas , Proteínas Adaptadoras de Transdução de Sinal/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Humanos , Proteínas Inibidoras de Apoptose/genética , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas de Neoplasias/genética , Proteólise
5.
Mol Biol (Mosk) ; 53(4): 638-647, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397437

RESUMO

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.


Assuntos
Resposta ao Choque Térmico , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Proteólise , Células U937 , Ubiquitina/metabolismo
6.
J Agric Food Chem ; 67(28): 7898-7907, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31282664

RESUMO

This study aimed to explore the effects of silencing HB12 and TT8 genes on protein utilization characteristics of alfalfa. Ground samples of 11 HB12-silenced (HB12i), 5 TT8-silenced (TT8i) and 4 wild type (WT) were incubated in a Daisy II incubator with N15 labeled ammonium sulfate for 0, 4, 8, 12, and 24 h. CP degradation and degradational kinetics, microbial nitrogen fractions, and protein metabolic profiles were determined. Moreover, relationships between protein profiles and FTIR spectral parameters were estimated. Results showed that transgenic alfalfa had lower CP degradation, microbial protein, and total available protein compared with WT, especially for HB12i. In addition, CP degradation and protein metabolic profiles were closely correlated with FTIR spectral parameters and thereby could be predicted from spectral parameters. In conclusion, silencing of HB12 and TT8 genes in alfalfa decreased protein degradational and metabolic profiles, which were predictable with FTIR spectral parameters.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Inativação Gênica , Proteínas de Homeodomínio/genética , Medicago sativa/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rúmen/metabolismo , Ração Animal/análise , Animais , Bactérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bovinos , Digestão , Proteínas de Homeodomínio/metabolismo , Cinética , Medicago sativa/química , Medicago sativa/metabolismo , Proteínas de Plantas/química , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteólise , Rúmen/química , Rúmen/microbiologia
7.
Genes Dev ; 33(15-16): 1083-1094, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31296559

RESUMO

The orphan nuclear receptor SHP (small heterodimer partner) is a well-known transcriptional corepressor of bile acid and lipid metabolism in the liver; however, its function in other tissues is poorly understood. Here, we report an unexpected role for SHP in the exocrine pancreas as a modulator of the endoplasmic reticulum (ER) stress response. SHP expression is induced in acinar cells in response to ER stress and regulates the protein stability of the spliced form of X-box-binding protein 1 (XBP1s), a key mediator of ER stress response. Loss of SHP reduces XBP1s protein level and transcriptional activity, which in turn attenuates the ER stress response during the fasting-feeding cycle. Consequently, SHP-deficient mice also are more susceptible to cerulein-induced pancreatitis. Mechanistically, we show that SHP physically interacts with the transactivation domain of XBP1s, thereby inhibiting the polyubiquitination and degradation of XBP1s by the Cullin3-SPOP (speckle-type POZ protein) E3 ligase complex. Together, our data implicate SHP in governing ER homeostasis and identify a novel posttranslational regulatory mechanism for the key ER stress response effector XBP1.


Assuntos
Estresse do Retículo Endoplasmático/genética , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Células Acinares/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas Exócrino/metabolismo , Pancreatite/genética , Processamento de Proteína , Estabilidade Proteica , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Ubiquitinação/genética
8.
J Chromatogr A ; 1602: 284-299, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31230875

RESUMO

A great number of protein-binding peptides are known and utilized as drugs, diagnostic reagents, and affinity ligands. Recently, however, peptide mimetics have been proposed as valuable alternative to peptides by virtue of their excellent biorecognition activity and higher biochemical stability. This poses the need to develop a strategy for translating known protein-binding peptides into peptoid analogues with comparable or better affinity. This work proposes a route for translation utilizing the IgG-binding peptide HWRGWV as reference sequence. An ensemble of peptoid analogues of HWRGWV were produced by adjusting the number and sequence arrangement of residues containing functional groups that resemble both natural and non-natural amino acids. The variants were initially screened via IgG binding tests in non-competitive mode to select candidate ligands. A set of selected peptoids were studied in silico by docking onto putative binding sites identified on the crystal structures of human IgG1, IgG2, IgG3, and IgG4 subclasses, returning values of predicted binding energy that aligned well with the binding data. Selected peptoids PL-16 and PL-22 were further characterized by binding isotherm analysis to determine maximum capacity (Qmax ˜ 48-57 mg of IgG per mL of adsorbent) and binding strength on solid phase (KD ˜ 5.4-7.8 10-7 M). Adsorbents PL-16-Workbeads and PL-22-Workbeads were used for purifying human IgG from a cell culture supernatant added with bovine serum, affording high values of IgG recovery (up to 85%) and purity (up to 98%) under optimized binding and elution conditions. Both peptoid ligands also proved to be stable against proteolytic enzymes and strong alkaline agents. Collectively, these studies form a method guiding the design of peptoid variants of cognate peptide ligands, and help addressing the challenges that, despite the structural similarity, the peptide-to-peptoid translation presents.


Assuntos
Anticorpos/metabolismo , Afinidade de Anticorpos , Peptídeos/química , Peptoides/química , Adsorção , Álcalis/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Bovinos , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Proteólise , Temperatura Ambiente
9.
Food Chem ; 297: 125012, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253295

RESUMO

To obtain better understanding of the formation mechanisms of bitterness and adhesiveness, protease activities, proteolysis index and protein degradation were investigated among raw, normal and defective hams. Normal and defective hams both showed a decrease in cathepsin B and B + L activities compared with raw ham, while higher residual activities were observed in defective ham. Approximate 1.2-fold values of proteolysis index were observed in defective ham than in normal ham, indicating that cathepsin B and B + L activities were key contributors in degrading muscle proteins of dry-cured ham. 322 proteins were identified by label-free proteomics, and 49 down-regulated proteins were found in the comparison between normal and defective hams. Creatine kinase, myosin, α-actinin and troponin-T showed the most intense response to bitterness and adhesiveness of dry-cured ham, confirmed by partial least squares regression analysis. Myosin could be a suitable biomarker to monitor bitterness and adhesiveness of dry-cured ham.


Assuntos
Produtos da Carne/análise , Proteômica/métodos , Paladar/fisiologia , Adesividade , Animais , Catepsina B/metabolismo , Catepsina L/metabolismo , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Regulação para Baixo , Análise dos Mínimos Quadrados , Proteínas/análise , Proteólise , Suínos , Espectrometria de Massas em Tandem , Troponina T/análise
10.
J Mass Spectrom ; 54(8): 716-727, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31254303

RESUMO

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) is a molecular imaging technology uniquely capable of untargeted measurement of proteins, lipids, and metabolites while retaining spatial information about their location in situ. This powerful combination of capabilities has the potential to bring a wealth of knowledge to the field of molecular histology. Translation of this innovative research tool into clinical laboratories requires the development of reliable sample preparation protocols for the analysis of proteins from formalin-fixed paraffin-embedded (FFPE) tissues, the standard preservation process in clinical pathology. Although ideal for stained tissue analysis by microscopy, the FFPE process cross-links, disrupts, or can remove proteins from the tissue, making analysis of the protein content challenging. To date, reported approaches differ widely in process and efficacy. This tutorial presents a strategy derived from systematic testing and optimization of key parameters, for reproducible in situ tryptic digestion of proteins in FFPE tissue and subsequent MALDI IMS analysis. The approach describes a generalized method for FFPE tissues originating from virtually any source.


Assuntos
Proteínas/análise , Manejo de Espécimes/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise Serial de Tecidos/métodos , Formaldeído/química , Humanos , Inclusão em Parafina , Proteólise , Fixação de Tecidos , Tripsina/química
11.
Cell Mol Life Sci ; 76(16): 3229-3248, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31197404

RESUMO

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.


Assuntos
Encéfalo/metabolismo , Matriz Extracelular/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Matriz Extracelular/química , Humanos , Ácido Hialurônico/metabolismo , Proteólise , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Receptores de Superfície Celular/metabolismo , Sinapses/metabolismo , Tenascina/metabolismo
12.
Cell Mol Life Sci ; 76(16): 3193-3206, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31201463

RESUMO

Alzheimer's Disease (AD) is the sixth-leading cause of death in industrialized countries. Neurotoxic amyloid-ß (Aß) plaques are one of the pathological hallmarks in AD patient brains. Aß accumulates in the brain upon sequential, proteolytic processing of the amyloid precursor protein (APP) by ß- and γ-secretases. However, so far disease-modifying drugs targeting ß- and γ-secretase pathways seeking a decrease in the production of toxic Aß peptides have failed in clinics. It has been demonstrated that the metalloproteinase meprin ß acts as an alternative ß-secretase, capable of generating truncated Aß2-x peptides that have been described to be increased in AD patients. This indicates an important ß-site cleaving enzyme 1 (BACE-1)-independent contribution of the metalloprotease meprin ß within the amyloidogenic pathway and may lead to novel drug targeting avenues. However, meprin ß itself is embedded in a complex regulatory network. Remarkably, the anti-amyloidogenic α-secretase a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is a direct competitor for APP at the cell surface, but also a sheddase of inactive pro-meprin ß. Overall, we highlight the current cellular, molecular and structural understanding of meprin ß as alternative ß-secretase within the complex protease web, regulating APP processing in health and disease.


Assuntos
Proteína ADAM10/metabolismo , Metaloendopeptidases/metabolismo , Proteína ADAM10/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Metaloendopeptidases/química , Presenilina-1/metabolismo , Proteólise , Serina Endopeptidases/metabolismo
13.
Cell Mol Life Sci ; 76(16): 3055-3081, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31236626

RESUMO

'A disintegrin and metalloproteases' (ADAMs) are a family of transmembrane proteins with diverse functions in multicellular organisms. About half of the ADAMs are active metalloproteases and cleave numerous cell surface proteins, including growth factors, receptors, cytokines and cell adhesion proteins. The other ADAMs have no catalytic activity and function as adhesion proteins or receptors. Some ADAMs are ubiquitously expressed, others are expressed tissue specifically. This review highlights functions of ADAMs in the mammalian nervous system, including their links to diseases. The non-proteolytic ADAM11, ADAM22 and ADAM23 have key functions in neural development, myelination and synaptic transmission and are linked to epilepsy. Among the proteolytic ADAMs, ADAM10 is the best characterized one due to its substrates Notch and amyloid precursor protein, where cleavage is required for nervous system development or linked to Alzheimer's disease (AD), respectively. Recent work demonstrates that ADAM10 has additional substrates and functions in the nervous system and its substrate selectivity may be regulated by tetraspanins. New roles for other proteolytic ADAMs in the nervous system are also emerging. For example, ADAM8 and ADAM17 are involved in neuroinflammation. ADAM17 additionally regulates neurite outgrowth and myelination and its activity is controlled by iRhoms. ADAM19 and ADAM21 function in regenerative processes upon neuronal injury. Several ADAMs, including ADAM9, ADAM10, ADAM15 and ADAM30, are potential drug targets for AD. Taken together, this review summarizes recent progress concerning substrates and functions of ADAMs in the nervous system and their use as drug targets for neurological and psychiatric diseases.


Assuntos
Proteínas ADAM/metabolismo , Sistema Nervoso/metabolismo , Proteínas ADAM/química , Animais , Transporte Biológico , Epilepsia/metabolismo , Epilepsia/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Bainha de Mielina/fisiologia , Sistema Nervoso/crescimento & desenvolvimento , Canais de Potássio/metabolismo , Proteólise
14.
Nat Commun ; 10(1): 2524, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175306

RESUMO

Deterioration of biomolecules in clinical tissues is an inevitable pre-analytical process, which affects molecular measurements and thus potentially confounds conclusions from cohort analyses. Here, we investigate the degradation of mRNA and protein in 68 pairs of adjacent prostate tissue samples using RNA-Seq and SWATH mass spectrometry, respectively. To objectively quantify the extent of protein degradation, we develop a numerical score, the Proteome Integrity Number (PIN), that faithfully measures the degree of protein degradation. Our results indicate that protein degradation only affects 5.9% of the samples tested and shows negligible correlation with mRNA degradation in the adjacent samples. These findings are confirmed by independent analyses on additional clinical sample cohorts and across different mass spectrometric methods. Overall, the data show that the majority of samples tested are not compromised by protein degradation, and establish the PIN score as a generic and accurate indicator of sample quality for proteomic analyses.


Assuntos
Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas/metabolismo , Proteólise , Estabilidade de RNA , RNA Mensageiro/metabolismo , Idoso , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Análise de Sequência de RNA
15.
Nat Commun ; 10(1): 2629, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201355

RESUMO

Light initiates chloroplast biogenesis by activating photosynthesis-associated genes encoded by not only the nuclear but also the plastidial genome, but how photoreceptors control plastidial gene expression remains enigmatic. Here we show that the photoactivation of phytochromes triggers the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) by stimulating the assembly of the bacterial-type plastidial RNA polymerase (PEP) into a 1000-kDa complex. Using forward genetic approaches, we identified REGULATOR OF CHLOROPLAST BIOGENESIS (RCB) as a dual-targeted nuclear/plastidial phytochrome signaling component required for PEP assembly. Surprisingly, RCB controls PhAPG expression primarily from the nucleus by interacting with phytochromes and promoting their localization to photobodies for the degradation of the transcriptional regulators PIF1 and PIF3. RCB-dependent PIF degradation in the nucleus signals the plastids for PEP assembly and PhAPG expression. Thus, our findings reveal the framework of a nucleus-to-plastid anterograde signaling pathway by which phytochrome signaling in the nucleus controls plastidial transcription.


Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fitocromo/metabolismo , Tiorredoxinas/metabolismo , Transcrição Genética/fisiologia , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Plastídeos/genética , Plastídeos/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Transcrição Genética/efeitos da radiação
16.
Nat Commun ; 10(1): 2666, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209254

RESUMO

Ran is a nucleocytoplasmic shuttle protein that is involved in cell cycle regulation, nuclear-cytoplasmic transport, and cell transformation. Ran plays an important role in cancer cell survival and cancer progression. Here, we show that, in addition to the nucleocytoplasmic localization of Ran, this GTPase is specifically associated with the plasma membrane/ruffles of ovarian cancer cells. Ran depletion has a drastic effect on RhoA stability and inhibits RhoA localization to the plasma membrane/ruffles and RhoA activity. We further demonstrate that the DEDDDL domain of Ran is required for the interaction with serine 188 of RhoA, which prevents RhoA degradation by the proteasome pathway. Moreover, the knockdown of Ran leads to a reduction of ovarian cancer cell invasion by impairing RhoA signalling. Our findings provide advanced insights into the mode of action of the Ran-RhoA signalling axis and may represent a potential therapeutic avenue for drug development to prevent ovarian tumour metastasis.


Assuntos
Carcinoma Epitelial do Ovário/patologia , Membrana Celular/metabolismo , Neoplasias Ovarianas/patologia , Proteína ran de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Feminino , Técnicas de Silenciamento de Genes , Humanos , Invasividade Neoplásica/patologia , Domínios Proteicos , Estabilidade Proteica , Proteólise , Serina/metabolismo , Transdução de Sinais , Proteína ran de Ligação ao GTP/genética
17.
Nat Commun ; 10(1): 2576, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189900

RESUMO

Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca2+ handling.


Assuntos
Dinaminas/metabolismo , Mitocôndrias Musculares/patologia , Dinâmica Mitocondrial/fisiologia , Miopatias Mitocondriais/patologia , Músculo Esquelético/patologia , Animais , Cálcio/metabolismo , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Dinaminas/genética , Homeostase/fisiologia , Humanos , Camundongos , Camundongos Knockout , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/mortalidade , Músculo Esquelético/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitinas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
18.
Nat Commun ; 10(1): 2572, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189917

RESUMO

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities.


Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Retroalimentação Fisiológica/fisiologia , Chaperonas Moleculares/metabolismo , Ataxias Espinocerebelares/patologia , Animais , Retroalimentação Fisiológica/efeitos dos fármacos , Fibroblastos , Células HEK293 , Proteínas de Choque Térmico HSP70/antagonistas & inibidores , Hipocampo/patologia , Holoenzimas/metabolismo , Humanos , Leupeptinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Cultura Primária de Células , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Nucleosídeos de Purina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ataxias Espinocerebelares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia
19.
Nat Commun ; 10(1): 2738, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227696

RESUMO

The breeding of cereals with altered gibberellin (GA) signaling propelled the 'Green Revolution' by generating semidwarf plants with increased tiller number. The mechanism by which GAs promote shoot height has been studied extensively, but it is not known what causes the inverse relationship between plant height and tiller number. Here we show that rice tiller number regulator MONOCULM 1 (MOC1) is protected from degradation by binding to the DELLA protein SLENDER RICE 1 (SLR1). GAs trigger the degradation of SLR1, leading to stem elongation and also to the degradation of MOC1, and hence a decrease in tiller number. This discovery provides a molecular explanation for the coordinated control of plant height and tiller number in rice by GAs, SLR1 and MOC1.


Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Giberelinas/metabolismo , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Brotos de Planta/fisiologia , Grão Comestível , Giberelinas/genética , Mutação , Melhoramento Vegetal/métodos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteólise , Transdução de Sinais/fisiologia
20.
Food Chem ; 295: 456-465, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174782

RESUMO

Kefir is a fermented dairy product, associated to health benefits because of being a probiotic and due to the presence of molecules with biological activity. In this work, we have profiled the peptide composition of goat milk kefir at three different fermentation times using a peptidomics approach, in order to study changes in peptide concentrations and patterns of protein digestion throughout the fermentation time. We identified 2328 unique peptides corresponding to 22 protein annotations, with a maximum of peptides found after 24 h fermentation. We established different digestion patterns according to the nature of the proteins, and quantified the changes in the peptides appearing in all the fermentation times. We also identified 11 peptides that matched exactly to sequences with biological activity in databases, almost all of them belonging to caseins. This is the most comprehensive proteomic analysis of goat milk kefir to date.


Assuntos
Kefir/análise , Proteínas do Leite/análise , Peptídeos/análise , Peptídeos/farmacologia , Animais , Caseínas/análise , Caseínas/metabolismo , Fermentação , Cabras , Proteínas do Leite/metabolismo , Proteínas do Leite/farmacologia , Mapeamento de Peptídeos/métodos , Peptídeos/metabolismo , Probióticos , Proteólise , Proteômica/métodos , Fatores de Tempo
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