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2.
AoB Plants ; 15(4): plad053, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37608926

RESUMO

Data on protein post-translational modifications (PTMs) increased exponentially in the last years due to the refinement of mass spectrometry techniques and the development of databases to store and share datasets. Nevertheless, these data per se do not create comprehensive biochemical knowledge. Complementary studies on protein biochemistry are necessary to fully understand the function of these PTMs at the molecular level and beyond, for example, designing rational metabolic engineering strategies to improve crops. Phosphoenolpyruvate carboxykinases (PEPCKs) are critical enzymes for plant metabolism with diverse roles in plant development and growth. Multiple lines of evidence showed the complex regulation of PEPCKs, including PTMs. Herein, we present PEPCKs as an example of the integration of combined mechanisms modulating enzyme activity and metabolic pathways. PEPCK studies strongly advanced after the production of the recombinant enzyme and the establishment of standardized biochemical assays. Finally, we discuss emerging open questions for future research and the challenges in integrating all available data into functional biochemical models.

3.
Plant J ; 114(5): 1037-1058, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37092344

RESUMO

Plant metabolism is finely orchestrated to allow the occurrence of complementary and sometimes opposite metabolic pathways. In part this is achieved by the allosteric regulation of enzymes, which has been a cornerstone of plant research for many decades. The completion of the Arabidopsis genome and the development of the associated toolkits for Arabidopsis research moved the focus of many researchers to other fields. This is reflected by the increasing number of high-throughput proteomic studies, mainly focused on post-translational modifications. However, follow-up 'classical' biochemical studies to assess the functions and upstream signaling pathways responsible for such modifications have been scarce. In this work, we review the basic concepts of allosteric regulation of enzymes involved in plant carbon metabolism, comprising photosynthesis and photorespiration, starch and sucrose synthesis, glycolysis and gluconeogenesis, the oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Additionally, we revisit the latest results on the allosteric control of the enzymes involved in these pathways. To conclude, we elaborate on the current methods for studying protein-metabolite interactions, which we consider will become crucial for discoveries in the future.


Assuntos
Arabidopsis , Carbono , Carbono/metabolismo , Arabidopsis/metabolismo , Proteômica , Fotossíntese , Via de Pentose Fosfato , Processamento de Proteína Pós-Traducional
4.
Curr Opin Plant Biol ; 72: 102347, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36806837

RESUMO

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. Our current understanding of Tre6P metabolism and signaling pathways in plants is based almost entirely on studies performed with Arabidopsis thaliana, a model plant that performs C3 photosynthesis. Conversely, our knowledge on the molecular mechanisms involved in Tre6P regulation of carbon partitioning and metabolism in C4 plants is scarce. This topic is especially relevant due to the agronomic importance of crops performing C4 photosynthesis, such as maize, sorghum and sugarcane. In this review, we focused our attention on recent developments related to Tre6P metabolism in C4 species and raised some open questions that should be addressed in the near future to improve the yield of economically important crops.


Assuntos
Arabidopsis , Trealose , Trealose/metabolismo , Plantas/metabolismo , Arabidopsis/metabolismo , Fotossíntese , Carbono/metabolismo , Fosfatos/metabolismo
5.
Plant Physiol Biochem ; 194: 461-469, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36508780

RESUMO

Sugar-alcohols are major photosynthates in plants from the Rosaceae family. Expression of the gene encoding aldose-6-phosphate reductase (Ald6PRase), the critical enzyme for glucitol synthesis in rosaceous species, is regulated by physiological and environmental cues. Additionally, Ald6PRase is inhibited by small molecules (hexose-phosphates and inorganic orthophosphate) and oxidizing compounds. This work demonstrates that Ald6PRase from peach leaves is phosphorylated in planta at the N-terminus. We also show in vitro phosphorylation of recombinant Ald6PRase by a partially purified kinase extract from peach leaves containing Ca2+-dependent protein kinases (CDPKs). Moreover, phosphorylation of recombinant Ald6PRase was inhibited by hexose-phosphates, phosphoenolpyruvate and pyrophosphate. We further show that phosphorylation of recombinant Ald6PRase was maximal using recombinant CDPKs. Overall, our results suggest that phosphorylation could fine-tune the activity of Ald6PRase.


Assuntos
Prunus persica , Fosforilação , Prunus persica/metabolismo , Fosfatos/metabolismo , Folhas de Planta/metabolismo , Hexoses/metabolismo
6.
New Phytol ; 235(5): 1780-1795, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35637555

RESUMO

During germination, seed reserves are mobilised to sustain the metabolic and energetic demands of plant growth. Mitochondrial respiration is presumably required to drive germination in several species, but only recently its role in this process has begun to be elucidated. Using Arabidopsis thaliana lines with changes in the levels of the respiratory chain component cytochrome c (CYTc), we investigated the role of this protein in germination and its relationship with hormonal pathways. Cytochrome c deficiency causes delayed seed germination, which correlates with decreased cyanide-sensitive respiration and ATP production at the onset of germination. In addition, CYTc affects the sensitivity of germination to abscisic acid (ABA), which negatively regulates the expression of CYTC-2, one of two CYTc-encoding genes in Arabidopsis. CYTC-2 acts downstream of the transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4), which binds to a region of the CYTC-2 promoter required for repression by ABA and regulates its expression. The results show that CYTc is a main player during seed germination through its role in respiratory metabolism and energy production. In addition, the direct regulation of CYTC-2 by ABI4 and its effect on ABA-responsive germination establishes a link between mitochondrial and hormonal functions during this process.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação/genética , Mitocôndrias/metabolismo , Sementes/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Phys Med ; 89: 1-10, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34339928

RESUMO

PURPOSE: The aims of this study were to develop and apply a method to correct for the differences in partial volume effects of pre-therapy Technetium-99 m (99mTc)-MAA SPECT and post-therapy Yttrium-90 (90Y) bremsstrahlung SPECT imaging in selective internal radiation therapy, and to use this method to improve quantitative comparison of predicted and delivered 90Y absorbed doses. METHODS: The spatial resolution of 99mTc SPECT data was converted to that of 90Y SPECT data using a function calculated from 99mTc and 90Y point spread functions. This resolution conversion method (RCM) was first applied to 99mTc and 90Y SPECT phantom data to validate the method, and then to clinical data to assess the power of 99mTc SPECT imaging to predict the therapeutic absorbed dose. RESULTS: The maximum difference between absorbed doses to phantom spheres was 178%. This was reduced to 27% after the RCM was applied. The clinical data demonstrated differences within 38% for mean absorbed doses delivered to the normal liver, which were reduced to 20% after application of the RCM. Analysis of clinical data showed that therapeutic absorbed doses delivered to tumours greater than 100 cm3 were predicted to within 52%, although there were differences of up to 210% for smaller tumours, even after the RCM was applied. CONCLUSIONS: The RCM was successfully verified using phantom data. Analysis of the clinical data established that the 99mTc pre-therapy imaging was predictive of the 90Y absorbed dose to the normal liver to within 20%, but had poor predictability for tumours smaller than 100 cm3.


Assuntos
Embolização Terapêutica , Neoplasias Hepáticas , Humanos , Microesferas , Agregado de Albumina Marcado com Tecnécio Tc 99m , Tomografia Computadorizada de Emissão de Fóton Único , Radioisótopos de Ítrio/uso terapêutico
8.
Br J Radiol ; 94(1126): 20210547, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34433005

RESUMO

The Internal Dosimetry User Group (IDUG) is an independent, non-profit group of medical professionals dedicated to the promotion of dosimetry in molecular radiotherapy (www.IDUG.org.uk). The Ionising Radiation (Medical Exposure) Regulations 2017, IR(ME)R, stipulate a requirement for optimisation and verification of molecular radiotherapy treatments, ensuring doses to non-target organs are as low as reasonably practicable. For many molecular radiotherapy treatments currently undertaken within the UK, this requirement is not being fully met. The growth of this field is such that we risk digressing further from IR(ME)R compliance potentially delivering suboptimal therapies that are not in the best interest of our patients. For this purpose, IDUG proposes ten points of action to aid in the successful implementation of this legislation. We urge stakeholders to support these proposals and ensure national provision is sufficient to meet the criteria necessary for compliance, and for the future advancement of molecular radiotherapy within the UK.


Assuntos
Monitoramento de Radiação/legislação & jurisprudência , Monitoramento de Radiação/normas , Radioterapia (Especialidade)/normas , Proteção Radiológica/legislação & jurisprudência , Proteção Radiológica/normas , Humanos , Objetivos Organizacionais , Órgãos em Risco , Radiação Ionizante , Dosagem Radioterapêutica , Sociedades Médicas , Reino Unido
9.
EMBO J ; 40(15): e106800, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34156108

RESUMO

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.


Assuntos
Arabidopsis/efeitos dos fármacos , Dipeptídeos/farmacologia , Gliceraldeído-3-Fosfato Desidrogenases/antagonistas & inibidores , Nicotiana/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Simulação por Computador , Dipeptídeos/química , Dipeptídeos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/química , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NADP/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Plântula/efeitos dos fármacos , Plântula/metabolismo , Nicotiana/metabolismo
10.
J Exp Bot ; 72(7): 2514-2524, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33315117

RESUMO

Phosphoenolpyruvate carboxykinase (PEPCK) plays a crucial role in gluconeogenesis. In this work, we analyze the proteolysis of Arabidopsis thaliana PEPCK1 (AthPEPCK1) in germinating seedlings. We found that the amount of AthPEPCK1 protein peaks at 24-48 h post-imbibition. Concomitantly, we observed shorter versions of AthPEPCK1, putatively generated by metacaspase-9 (AthMC9). To study the impact of AthMC9 cleavage on the kinetic and regulatory properties of AthPEPCK1, we produced truncated mutants based on the reported AthMC9 cleavage sites. The Δ19 and Δ101 truncated mutants of AthPEPCK1 showed similar kinetic parameters and the same quaternary structure as the wild type. However, activation by malate and inhibition by glucose 6-phosphate were abolished in the Δ101 mutant. We propose that proteolysis of AthPEPCK1 in germinating seedlings operates as a mechanism to adapt the sensitivity to allosteric regulation during the sink-to-source transition.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fosfoenolpiruvato Carboxiquinase (ATP) , Regulação Alostérica , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Gluconeogênese , Fosfoenolpiruvato , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Proteólise
11.
Phys Med Biol ; 65(24): 245038, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33142274

RESUMO

Despite a growth in molecular radiotherapy treatment (MRT) and an increase in interest, centres still rarely perform MRT dosimetry. The aims of this report were to assess the main reasons why centres are not performing MRT dosimetry and provide advice on the resources required to set-up such a service. A survey based in the United Kingdom was developed to establish how many centres provide an MRT dosimetry service and the main reasons why it is not commonly performed. Twenty-eight per cent of the centres who responded to the survey performed some form of dosimetry, with 88% of those centres performing internal dosimetry. The survey showed that a 'lack of clinical evidence', a 'lack of guidelines' and 'not current UK practice' were the largest obstacles to setting up an MRT dosimetry service. More practical considerations, such as 'lack of software' and 'lack of staff training/expertise', were considered to be of lower significance by the respondents. Following on from the survey, this report gives an overview of the current guidelines, and the evidence available demonstrating the benefits of performing MRT dosimetry. The resources required to perform such techniques are detailed with reference to guidelines, training resources and currently available software. It is hoped that the information presented in this report will allow MRT dosimetry to be performed more frequently and in more centres, both in routine clinical practice and in multicentre trials. Such trials are required to harmonise dosimetry techniques between centres, build on the current evidence base, and provide the data necessary to establish the dose-response relationship for MRT.


Assuntos
Guias de Prática Clínica como Assunto , Radiometria/métodos , Relatório de Pesquisa , Humanos , Planejamento da Radioterapia Assistida por Computador , Inquéritos e Questionários
12.
Front Plant Sci ; 11: 1058, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32754189

RESUMO

Starch is the dominant reserve polysaccharide accumulated in the seed of grasses (like wheat). It is the most common carbohydrate in the human diet and a material applied to the bioplastics and biofuels industry. Hence, the complete understanding of starch metabolism is critical to design rational strategies to improve its allocation in plant reserve tissues. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the key (regulated) step in the synthetic starch pathway. The enzyme comprises a small (S) and a large (L) subunit forming an S2L2 heterotetramer, which is allosterically regulated by orthophosphate, fructose-6P, and 3P-glycerate. ADP-Glc PPase was found in a phosphorylated state in extracts from wheat seeds. The amount of the phosphorylated protein increased along with the development of the seed and correlated with relative increases of the enzyme activity and starch content. Conversely, this post-translational modification was absent in seeds from Ricinus communis. In vitro, the recombinant ADP-Glc PPase from wheat endosperm was phosphorylated by wheat seed extracts as well as by recombinant Ca2+-dependent plant protein kinases. Further analysis showed that the preferential phosphorylation takes place on the L subunit. Results suggest that the ADP-Glc PPase is a phosphorylation target in seeds from grasses but not from oleaginous plants. Accompanying seed maturation and starch accumulation, a combined regulation of ADP-Glc PPase by metabolites and phosphorylation may provide an enzyme with stable levels of activity. Such concerted modulation would drive carbon skeletons to the synthesis of starch for its long-term storage, which later support seed germination.

13.
Cancer Biother Radiopharm ; 35(7): 530-539, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32429699

RESUMO

Introduction: Thorium-227 is an alpha-emitting radioisotope with potential therapeutic applications in targeted alpha therapy. Thorium-227 decays to Radium-223, which may have an independent biodistribution to that of the parent Thorium-227 radiopharmaceutical. Quantitative in vivo imaging with sodium iodide (NaI) detectors is challenging due to cross-talk between neighboring γ-photopeaks as well as scattered γ-photons. The aim of this work was to validate the use of a spectral analysis technique to estimate the activity of each isotope within a region of interest applied to a pair of conjugate view planar acquisitions, acquired at multiple energy windows. Methods: Energy spectra per unit activity arising from unscattered Thorium-227 photons and Radium-223 photons as well as from scattered photons were modeled. These spectra were scaled until the combination of these component spectra resulted in the closest match to the measured data in four energy windows. Results: Measured estimates of activity followed the known decay curves in phantoms representative of a human torso. The mean errors in estimating Thorium-227 and Radium-223 were 5.1% (range -8.0% to 40.0%) and 3.4% (range -50.0% to 48.7%), respectively. The differences between the integrals of the theoretical and estimated time activity curve were <10% for both Thorium-227 and Radium-223. Conclusion: γ-camera quantification of Thorium-227 and Radium-223 can be achieved by using multiple energy window acquisitions.


Assuntos
Modelos Teóricos , Compostos Radiofarmacêuticos/farmacocinética , Rádio (Elemento)/farmacocinética , Tório/farmacocinética , Câmaras gama , Humanos , Modelos Anatômicos , Imagens de Fantasmas , Radiometria/métodos , Espectrometria gama/instrumentação , Espectrometria gama/métodos , Distribuição Tecidual , Tronco/diagnóstico por imagem
14.
Biochem J ; 476(20): 2939-2952, 2019 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-31548269

RESUMO

ATP-dependent phosphoenolpyruvate carboxykinases (PEPCKs, EC 4.1.1.49) from C4 and CAM plants have been widely studied due to their crucial role in photosynthetic CO2 fixation. However, our knowledge on the structural, kinetic and regulatory properties of the enzymes from C3 species is still limited. In this work, we report the recombinant production and biochemical characterization of two PEPCKs identified in Arabidopsis thaliana: AthPEPCK1 and AthPEPCK2. We found that both enzymes exhibited high affinity for oxaloacetate and ATP, reinforcing their role as decarboxylases. We employed a high-throughput screening for putative allosteric regulators using differential scanning fluorometry and confirmed their effect on enzyme activity by performing enzyme kinetics. AthPEPCK1 and AthPEPCK2 are allosterically modulated by key intermediates of plant metabolism, namely succinate, fumarate, citrate and α-ketoglutarate. Interestingly, malate activated and glucose 6-phosphate inhibited AthPEPCK1 but had no effect on AthPEPCK2. Overall, our results demonstrate that the enzymes involved in the critical metabolic node constituted by phosphoenolpyruvate are targets of fine allosteric regulation.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Fosfoenolpiruvato Carboxiquinase (ATP)/química , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Ácido Cítrico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fluorometria/métodos , Fumaratos/metabolismo , Cinética , Malatos/metabolismo , Manganês/metabolismo , Ácido Oxaloacético/metabolismo , Fotossíntese , Ligação Proteica , Proteínas Recombinantes/metabolismo , Ácido Succínico/metabolismo , Temperatura de Transição
16.
Anal Biochem ; 557: 120-122, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30036499

RESUMO

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD+, which is quantified by its fluorescent properties after alkaline treatment (linear range 0-10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases.


Assuntos
Fluorometria/métodos , Proteínas Quinases/análise , Proteínas Quinases/metabolismo , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Solanum tuberosum/enzimologia
19.
Virol J ; 9: 222, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-23021128

RESUMO

BACKGROUND: DNA viruses, such as herpes simplex virus type 1 (HSV-1), Simian virus 40 (SV40), and Cytomegaloviruses (CMV), start their replicative processes and transcription at specific nuclear domains known as ND10 (nuclear domain 10, also called PML bodies). It has been previously determined that for HSV-1 and SV40, a short DNA sequence and its binding protein are required and sufficient for cell localization of viral DNA replication and gene transcription. RESULTS: Our recent observations provide evidence that a foreign (not endogenous) DNA/protein complex in the nucleus recruits ND10 proteins. First, the complexes formed from the bacterial lac operator DNA and its binding protein (lac repressor), or from HPV11 (human papillomavirus 11) origin DNA and its binding protein (E2), co-localized with different ND10 proteins. Second, the HSV-1 amplicon without inserted lac operator DNA repeats distributed in the nucleus randomly, whereas the amplicon with lac operator DNA repeats associated with ND10, suggesting that DNA-binding proteins are required to localize at ND10. The cellular intrinsic DNA/protein complex (as detected for U2 DNA) showed no association with ND10. Furthermore, our examination of PML-/-, Daxx-/-, and Sp100-negative cells led to our discovering that DNA/protein complexes recruit ND10 protein independently. Using the GFP-LacI/Operator system, we were able to direct the transfected DNA to ND10 and found that gene expression was significantly repressed when the transfected DNA was directed to ND10. CONCLUSION: Taken together, the results suggest that cells recognize DNA/protein complexes through a mechanism that involves interaction with the ND10-associated proteins.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Linhagem Celular , Herpesvirus Humano 1/genética , Papillomavirus Humano 11/genética , Humanos , Óperon Lac , Regiões Operadoras Genéticas , Ligação Proteica , Vírus 40 dos Símios/genética
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