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1.
Heredity (Edinb) ; 129(4): 225-232, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35764697

RESUMO

Mitochondria are organelles that produce cellular energy in the form of ATP through oxidative phosphorylation, and this primary function is conserved among many taxa. Locomotion is a trait that is highly reliant on metabolic function and expected to be greatly affected by disruptions to mitochondrial performance. To this end, we aimed to examine how activity and sleep vary between Drosophila melanogaster strains with different geographic origins, how these patterns are affected by mitochondrial DNA (mtDNA) variation, and how breaking up co-evolved mito-nuclear gene combinations affect the studied activity traits. Our results demonstrate that Drosophila strains from different locations differ in sleep and activity, and that females are generally more active than males. By comparing activity and sleep of mtDNA variants introgressed onto a common nuclear background in cytoplasmic hybrid (cybrid) strains, we were able to quantify the among-line variance attributable to mitochondrial DNA, and we establish that mtDNA variation affects both activity and sleep, in a sex-specific manner. Altogether our study highlights the important role that mitochondrial genome variation plays on organismal physiology and behaviour.


Assuntos
DNA Mitocondrial , Drosophila melanogaster , Trifosfato de Adenosina/metabolismo , Animais , DNA Mitocondrial/genética , Drosophila/genética , Drosophila melanogaster/genética , Feminino , Locomoção/genética , Masculino , Mitocôndrias/genética , Sono/genética
2.
Elife ; 102021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34477104

RESUMO

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations. Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined. Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents. Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues. Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).


Assuntos
Amina Oxidase (contendo Cobre) , Motivos de Aminoácidos/genética , Produtos Biológicos , Heparina/metabolismo , Antagonistas dos Receptores Histamínicos , Amina Oxidase (contendo Cobre)/química , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Antagonistas dos Receptores Histamínicos/química , Antagonistas dos Receptores Histamínicos/metabolismo , Humanos , Camundongos , Mutação/genética , Ligação Proteica/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Biomolecules ; 11(6)2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-34199986

RESUMO

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein-protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1α, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC50 for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Chaperoninas/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Macrolídeos/farmacologia , Nanoconjugados , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/metabolismo , Galinhas , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Macrolídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
4.
J Biol Chem ; 296: 100593, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33775697

RESUMO

Dysregulation of the developmentally important Notch signaling pathway is implicated in several types of cancer, including breast cancer. However, the specific roles and regulation of the four different Notch receptors have remained elusive. We have previously reported that the oncogenic PIM kinases phosphorylate Notch1 and Notch3. Phosphorylation of Notch1 within the second nuclear localization sequence of its intracellular domain (ICD) enhances its transcriptional activity and tumorigenicity. In this study, we analyzed Notch3 phosphorylation and its functional impact. Unexpectedly, we observed that the PIM target sites are not conserved between Notch1 and Notch3. Notch3 ICD (N3ICD) is phosphorylated within a domain, which is essential for formation of a transcriptionally active complex with the DNA-binding protein CSL. Through molecular modeling, X-ray crystallography, and isothermal titration calorimetry, we demonstrate that phosphorylation of N3ICD sterically hinders its interaction with CSL and thereby inhibits its CSL-dependent transcriptional activity. Surprisingly however, phosphorylated N3ICD still maintains tumorigenic potential in breast cancer cells under estrogenic conditions, which support PIM expression. Taken together, our data indicate that PIM kinases modulate the signaling output of different Notch paralogs by targeting distinct protein domains and thereby promote breast cancer tumorigenesis via both CSL-dependent and CSL-independent mechanisms.


Assuntos
Neoplasias da Mama/patologia , Carcinogênese , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Receptor Notch3/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Camundongos , Modelos Moleculares , Proteínas Musculares/metabolismo , Fosforilação , Domínios Proteicos , Receptor Notch3/química
5.
Cancers (Basel) ; 13(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672199

RESUMO

The ATP-competitive inhibitors of Hsp90 have been tested predominantly in kinase addicted cancers; however, they have had limited success. A mechanistic connection between Hsp90 and oncogenic K-Ras is not known. Here, we show that K-Ras selectivity is enabled by the loss of the K-Ras membrane nanocluster modulator galectin-3 downstream of the Hsp90 client HIF-1α. This mechanism suggests a higher drug sensitivity in the context of KRAS mutant, HIF-1α-high and/or Gal3-high cancer cells, such as those found, in particular, in pancreatic adenocarcinoma. The low toxicity of conglobatin further indicates a beneficial on-target toxicity profile for Hsp90/Cdc37 interface inhibitors. We therefore computationally screened >7 M compounds, and identified four novel small molecules with activities of 4 µM-44 µM in vitro. All of the compounds were K-Ras selective, and potently decreased the Hsp90 client protein levels without inducing the heat shock response. Moreover, they all inhibited the 2D proliferation of breast, pancreatic, and lung cancer cell lines. The most active compounds from each scaffold, furthermore, significantly blocked 3D spheroids and the growth of K-Ras-dependent microtumors. We foresee new opportunities for improved Hsp90/Cdc37 interface inhibitors in cancer and other aging-associated diseases.

6.
Glycobiology ; 31(4): 444-458, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-32985651

RESUMO

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.


Assuntos
Amina Oxidase (contendo Cobre) , Proteoglicanas de Heparan Sulfato , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Células CHO , Cricetinae , Glicosaminoglicanos , Heparitina Sulfato/metabolismo , Humanos , Camundongos , Ratos
7.
Molecules ; 25(6)2020 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-32178384

RESUMO

Two members of the copper-containing amine oxidase family are physiologically important proteins: (1) Diamine oxidase (hDAO; AOC1) with a preference for diamines is involved in degradation of histamine and (2) Vascular adhesion protein-1 (hVAP-1; AOC3) with a preference for monoamines is a multifunctional cell-surface receptor and an enzyme. hVAP-1-targeted inhibitors are designed to treat inflammatory diseases and cancer, whereas the off-target binding of the designed inhibitors to hDAO might result in adverse drug reactions. The X-ray structures for both human enzymes are solved and provide the basis for computer-aided inhibitor design, which has been reported by several research groups. Although the putative off-target effect of hDAO is less studied, computational methods could be easily utilized to avoid the binding of VAP-1-targeted inhibitors to hDAO. The choice of the model organism for preclinical testing of hVAP-1 inhibitors is not either trivial due to species-specific binding properties of designed inhibitors and different repertoire of copper-containing amine oxidase family members in mammalian species. Thus, the facts that should be considered in hVAP-1-targeted inhibitor design are discussed in light of the applied structural bioinformatics and structural biology approaches.


Assuntos
Amina Oxidase (contendo Cobre)/química , Moléculas de Adesão Celular/genética , Desenho de Fármacos , Desenvolvimento de Medicamentos/tendências , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/uso terapêutico , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/uso terapêutico , Histamina/química , Humanos
8.
FEBS J ; 287(14): 2998-3011, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31876382

RESUMO

Microbes are competent chemists that are able to generate thousands of chemically complex natural products with potent biological activities. The key to the formation of this chemical diversity has been the rapid evolution of secondary metabolism. Many enzymes residing on these metabolic pathways have acquired atypical catalytic properties in comparison with their counterparts found in primary metabolism. The biosynthetic pathway of the anthracycline nogalamycin contains two such proteins, SnoK and SnoN, belonging to nonheme iron and 2-oxoglutarate-dependent mono-oxygenases. In spite of structural similarity, the two proteins catalyze distinct chemical reactions; SnoK is a C2-C5″ carbocyclase, whereas SnoN catalyzes stereoinversion at the adjacent C4″ position. Here, we have identified four structural regions involved in the functional differentiation and generated 30 chimeric enzymes to probe catalysis. Our analyses indicate that the carbocyclase SnoK is the ancestral form of the enzyme from which SnoN has evolved to catalyze stereoinversion at the neighboring carbon. The critical step in the appearance of epimerization activity has likely been the insertion of three residues near the C-terminus, which allow repositioning of the substrate in front of the iron center. The loss of the original carbocyclization activity has then occurred with changes in four amino acids near the iron center that prohibit alignment of the substrate for the formation of the C2-C5″ bond. Our study provides detailed insights into the evolutionary processes that have enabled Streptomyces soil bacteria to become the major source of antibiotics and antiproliferative agents. ENZYMES: EC number 1.14.11.


Assuntos
Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Evolução Molecular , Engenharia Genética/métodos , Nogalamicina/biossíntese , Ferroproteínas não Heme/metabolismo , Streptomyces/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ferroproteínas não Heme/química , Ferroproteínas não Heme/genética , Conformação Proteica
9.
SLAS Discov ; 24(10): 953-968, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31503511

RESUMO

Physiological nucleosides are used for the synthesis of DNA, RNA, and ATP in the cell and serve as universal mammalian signaling molecules that regulate physiological processes such as vasodilation and platelet aggregation by engaging with cell surface receptors. The same pathways that allow uptake of physiological nucleosides mediate the cellular import of synthetic nucleoside analogs used against cancer, HIV, and other viral diseases. Physiological nucleosides and nucleoside drugs are imported by two families of nucleoside transporters: the SLC28 concentrative nucleoside transporters (CNTs) and SLC29 equilibrative nucleoside transporters (ENTs). The four human ENT paralogs are expressed in distinct tissues, localize to different subcellular sites, and transport a variety of different molecules. Here we provide an overview of the known structure-function relationships of the ENT family with a focus on ligand binding and transport in the context of a new hENT1 homology model. We provide a generic residue numbering system for the different ENTs to facilitate the interpretation of mutational data produced using different ENT homologs. The discovery of paralog-selective small-molecule modulators is highly relevant for the design of new therapies and for uncovering the functions of poorly characterized ENT family members. Here, we discuss recent developments in the discovery of new paralog-selective small-molecule ENT inhibitors, including new natural product-inspired compounds. Recent progress in the ability to heterologously produce functional ENTs will allow us to gain insight into the structure and functions of different ENT family members as well as the rational discovery of highly selective inhibitors.


Assuntos
Desenho de Fármacos , Descoberta de Drogas , Proteínas de Transporte de Nucleosídeo Equilibrativas/química , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Descoberta de Drogas/métodos , Proteínas de Transporte de Nucleosídeo Equilibrativas/antagonistas & inibidores , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Humanos , Ligantes , Estrutura Molecular , Mutação , Ligação Proteica , Relação Estrutura-Atividade
10.
Eur J Pharm Sci ; 137: 104963, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31226387

RESUMO

Transport proteins of the ATP-binding cassette (ABC) family are found in all kingdoms of life. In humans, several ABC efflux transporters play a role in drug disposition and excretion. Therefore, in vitro methods have been developed to characterize the substrate and inhibitor properties of drugs with respect to these transporters. In the vesicular transport assay, transport is studied using inverted membrane vesicles produced from transporter overexpressing cell lines of both mammalian and insect origin. Insect cell expression systems benefit from a higher expression compared to background, but are not as well characterized as their mammalian counterparts regarding endogenous transport. Therefore, the contribution of this transport in the assay might be underappreciated. In this study, endogenous transport in membrane vesicles from Spodoptera frugiperda -derived Sf9 cells was characterized using four typical substrates of human ABC transporters: 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), estradiol-17ß-glucuronide, estrone sulfate and N-methyl-quinidine. Significant ATP-dependent transport was observed for three of the substrates with cholesterol-loading of the vesicles, which is sometimes used to improve the activity of human transporters expressed in Sf9 cells. The highest effect of cholesterol was on CDCF transport, and this transport in the cholesterol-loaded Sf9 vesicles was time and concentration dependent with a Km of 8.06 ±â€¯1.11 µM. The observed CDCF transport was inhibited by known inhibitors of human ABCC transporters, but not by ABCB1 and ABCG2 inhibitors verapamil and Ko143, respectively. Two candidate genes for ABCC-type transporters in the S. frugiperda genome (SfABCC2 and SfABCC3) were identified based on sequence analysis as a hypothesis to explain the observed endogenous ABCC-type transport in Sf9 vesicles. Although further studies are needed to verify the role of SfABCC2 and SfABCC3 in Sf9 vesicles, the findings of this study highlight the need to carefully characterize background transport in Sf9 derived membrane vesicles to avoid false positive substrate findings for human ABC transporters studied with this overexpression system.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/farmacologia , Estradiol/análogos & derivados , Estrona/análogos & derivados , Fluoresceínas/farmacologia , Quinidina/análogos & derivados , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Estradiol/farmacologia , Estrona/farmacologia , Filogenia , Quinidina/farmacologia , Alinhamento de Sequência , Células Sf9 , Spodoptera
11.
Biochem J ; 476(6): 1009-1020, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30877192

RESUMO

Spermidine is a ubiquitous polyamine synthesized by spermidine synthase (SPDS) from the substrates, putrescine and decarboxylated S-adenosylmethionine (dcAdoMet). SPDS is generally active as homodimer, but higher oligomerization states have been reported in SPDS from thermophiles, which are less specific to putrescine as the aminoacceptor substrate. Several crystal structures of SPDS have been solved with and without bound substrates and/or products as well as inhibitors. Here, we determined the crystal structure of SPDS from the cyanobacterium Synechococcus (SySPDS) that is a homodimer, which we also observed in solution. Unlike crystal structures reported for bacterial and eukaryotic SPDS with bound ligands, SySPDS structure has not only bound putrescine substrate taken from the expression host, but also spermidine product most probably as a result of an enzymatic reaction. Hence, to the best of our knowledge, this is the first structure reported with both amino ligands in the same structure. Interestingly, the gate-keeping loop is disordered in the putrescine-bound monomer while it is stabilized in the spermidine-bound monomer of the SySPDS dimer. This confirms the gate-keeping loop as the key structural element that prepares the active site upon binding of dcAdoMet for the catalytic reaction of the amine donor and putrescine.


Assuntos
Proteínas de Bactérias/química , Putrescina/química , Espermidina Sintase/química , Synechococcus/enzimologia , Cristalografia por Raios X , Domínios Proteicos , Estrutura Secundária de Proteína
12.
Scand J Work Environ Health ; 45(2): 183-193, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30614502

RESUMO

Objective Studies of loud noise exposure and vestibular schwannomas (VS) have shown conflicting results. The population-based INTERPHONE case‒control study was conducted in 13 countries during 2000-2004. In this paper, we report the results of analyses on the association between VS and self-reported loud noise exposure. Methods Self-reported noise exposure was analyzed in 1024 VS cases and 1984 matched controls. Life-long noise exposure was estimated through detailed questions. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using adjusted conditional logistic regression for matched sets. Results The OR for total work and leisure noise exposure was 1.6 (95% CI 1.4-1.9). OR were 1.5 (95% CI 1.3-1.9) for only occupational noise, 1.9 (95% CI 1.4-2.6) for only leisure noise and 1.7 (95% CI 1.2-2.2) for exposure in both contexts. OR increased slightly with increasing lag-time. For occupational exposures, duration, time since exposure start and a metric combining lifetime duration and weekly exposure showed significant trends of increasing risk with increasing exposure. OR did not differ markedly by source or other characteristics of noise. Conclusion The consistent associations seen are likely to reflect either recall bias or a causal association, or potentially indicate a mixture of both.


Assuntos
Neuroma Acústico/epidemiologia , Ruído Ocupacional/estatística & dados numéricos , Exposição Ocupacional/estatística & dados numéricos , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
13.
Cell Death Differ ; 25(3): 600-615, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29305585

RESUMO

The Notch signaling pathway is a key regulator of stem cells during development, and its deregulated activity is linked to developmental defects and cancer. Transcriptional activation of Notch target genes requires cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD1), NICD1 migration into the nucleus, and assembly of a transcriptional complex. Post-translational modifications of Notch regulate its trafficking, turnover, and transcriptional activity. Here, we show that NICD1 is modified by small ubiquitin-like modifier (SUMO) in a stress-inducible manner. Sumoylation occurs in the nucleus where NICD1 is sumoylated in the RBPJ-associated molecule (RAM) domain. Although stress and sumoylation enhance nuclear localization of NICD1, its transcriptional activity is attenuated. Molecular modeling indicates that sumoylation can occur within the DNA-bound ternary transcriptional complex, consisting of NICD1, the transcription factor Suppressor of Hairless (CSL), and the co-activator Mastermind-like (MAML) without its disruption. Mechanistically, sumoylation of NICD1 facilitates the recruitment of histone deacetylase 4 (HDAC4) to the Notch transcriptional complex to suppress Notch target gene expression. Stress-induced sumoylation decreases the NICD1-mediated induction of Notch target genes, which was abrogated by expressing a sumoylation-defected mutant in cells and in the developing central nervous system of the chick in vivo. Our findings of the stress-inducible sumoylation of NICD1 reveal a novel context-dependent regulatory mechanism of Notch target gene expression.


Assuntos
Regulação da Expressão Gênica , Receptor Notch1/química , Receptor Notch1/metabolismo , Estresse Fisiológico , Sumoilação , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Estresse Oxidativo , Transdução de Sinais
14.
Adv Healthc Mater ; 6(21)2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28892296

RESUMO

Approaches to increase the efficiency in developing drugs and diagnostics tools, including new drug delivery and diagnostic technologies, are needed for improved diagnosis and treatment of major diseases and health problems such as cancer, inflammatory diseases, chronic wounds, and antibiotic resistance. Development within several areas of research ranging from computational sciences, material sciences, bioengineering to biomedical sciences and bioimaging is needed to realize innovative drug development and diagnostic (DDD) approaches. Here, an overview of recent progresses within key areas that can provide customizable solutions to improve processes and the approaches taken within DDD is provided. Due to the broadness of the area, unfortunately all relevant aspects such as pharmacokinetics of bioactive molecules and delivery systems cannot be covered. Tailored approaches within (i) bioinformatics and computer-aided drug design, (ii) nanotechnology, (iii) novel materials and technologies for drug delivery and diagnostic systems, and (iv) disease models to predict safety and efficacy of medicines under development are focused on. Current developments and challenges ahead are discussed. The broad scope reflects the multidisciplinary nature of the field of DDD and aims to highlight the convergence of biological, pharmaceutical, and medical disciplines needed to meet the societal challenges of the 21st century.


Assuntos
Modelos Biológicos , Biologia Computacional , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Nanomedicina , Nanotecnologia , Neoplasias/diagnóstico , Proteínas/química , Proteínas/metabolismo , Relação Quantitativa Estrutura-Atividade
15.
World J Microbiol Biotechnol ; 33(4): 72, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28299555

RESUMO

Spermidine synthase (Spds) catalyzes the formation of spermidine by transferring the aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. The Synechococcus spds gene encoding Spds was expressed in Escherichia coli. The purified recombinant enzyme had a molecular mass of 33 kDa and showed optimal activity at pH 7.5, 37 °C. The enzyme had higher affinity for dcSAM (K m, 20 µM) than for putrescine (K m, 111 µM) and was highly specific towards the diamine putrescine with no activity observed towards longer chain diamines. The three-dimensional structural model for Synechococcus Spds revealed that most of the ligand binding residues in Spds from Synechococcus sp. PCC 7942 are identical to those of human and parasite Spds. Based on the model, the highly conserved acidic residues, Asp89, Asp159 and Asp162, are involved in the binding of substrates putrescine and dcSAM and Pro166 seems to confer substrate specificity towards putrescine.


Assuntos
Putrescina/metabolismo , S-Adenosilmetionina/metabolismo , Espermidina Sintase/química , Espermidina Sintase/metabolismo , Synechococcus/enzimologia , Asparagina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Peso Molecular , Prolina/metabolismo , Ligação Proteica , Homologia de Sequência do Ácido Nucleico , Espermidina Sintase/genética , Homologia Estrutural de Proteína , Especificidade por Substrato , Synechococcus/química , Synechococcus/genética
16.
PLoS One ; 11(11): e0166935, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27893774

RESUMO

Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C22) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C21 and C22 domains, modeled its 3D structure and characterized the hAOC3-Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C22 domains. The V domain is responsible for the unusually tight Siglec-9-hAOC3 interactions whereas the intact C22 domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C22 are involved in the interactions with the active site channel of hAOC3. Furthermore, the C22 domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C22 domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9-hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools.


Assuntos
Amina Oxidase (contendo Cobre)/metabolismo , Antígenos CD/química , Antígenos CD/metabolismo , Moléculas de Adesão Celular/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/química , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Amina Oxidase (contendo Cobre)/química , Animais , Antígenos CD/genética , Arginina , Moléculas de Adesão Celular/química , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Semicarbazidas/farmacocinética , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Spodoptera/genética , Ressonância de Plasmônio de Superfície
17.
PLoS One ; 11(8): e0161852, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27571415

RESUMO

BACKGROUND: A wide range of insects living at higher latitudes enter diapause at the end of the warm season, which increases their chances of survival through harsh winter conditions. In this study we used RNA sequencing to identify genes involved in adult reproductive diapause in a northern fly species, Drosophila montana. Both diapausing and non-diapausing flies were reared under a critical day length and temperature, where about half of the emerging females enter diapause enabling us to eliminate the effects of varying environmental conditions on gene expression patterns of the two types of female flies. RESULTS: RNA sequencing revealed large differences between gene expression patterns of diapausing and non-diapausing females, especially in genes involved with metabolism, fatty acid biosynthesis, and metal and nucleotide binding. Differently expressed genes included several gene groups, including myosin, actin and cytochromeP450 genes, which have been previously associated with diapause. This study also identified new candidate genes, including some involved in cuticular hydrocarbon synthesis or regulation (desat1 and desat2), and acyl-CoA Δ11-desaturase activity (CG9747), and few odorant-binding protein genes (e.g. Obp44A). Also, several transposable elements (TEs) showed differential expression between the two female groups motivating future research on their roles in diapause. CONCLUSIONS: Our results demonstrate that the adult reproductive diapause in D. montana involves changes in the expression level of a variety of genes involved in key processes (e.g. metabolism and fatty acid biosynthesis) which help diapausing females to cope with overwintering. This is consistent with the view that diapause is a complex adaptive phenotype where not only sexual maturation is arrested, but also changes in adult physiology are required in order to survive over the winter.


Assuntos
Diapausa de Inseto/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/genética , Actinas/genética , Actinas/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Drosophila/genética , Feminino , Metamorfose Biológica , Miosinas/genética , Miosinas/metabolismo , Fotoperíodo , Reprodução/genética , Reprodução/fisiologia , Temperatura
18.
Methods Mol Biol ; 1351: 199-210, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26530684

RESUMO

Mitochondrial gene expression is essential in all organisms. Our understanding of mitochondrial transcription on a biochemical level has been limited by the inability to purify the individual protein components involved in mitochondrial gene expression. Recently, new systems have been identified that permit purification of these proteins from bacteria. However, the generalizability of these systems is not clear. Here, we have applied the technology from the Cameron lab to express and purify mitochondrial RNA polymerase and transcription factor A from Drosophila melanogaster. We show that the use of SUMO system to produce SUMO fusion proteins in bacteria is effective not only for the human and mouse proteins, but also for the fly proteins. The application of this system to produce the mitochondrial proteins from other organisms should permit detailed understanding of mitochondrial transcription from any organism.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Proteínas de Drosophila/genética , RNA/biossíntese , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/genética , Animais , Bactérias/genética , Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , RNA Polimerases Dirigidas por DNA/biossíntese , Proteínas de Drosophila/biossíntese , Drosophila melanogaster , Mitocôndrias/genética , RNA/genética , RNA Mitocondrial , Fatores de Transcrição/biossíntese , Transcrição Gênica/genética
19.
J Theor Biol ; 386: 78-88, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26393783

RESUMO

Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A.


Assuntos
Autoantígenos/química , Proteínas de Membrana/química , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Sequência de Aminoácidos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Relação Quantitativa Estrutura-Atividade , Alinhamento de Sequência , Eletricidade Estática
20.
Acta Oncol ; 54(8): 1159-65, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25982941

RESUMO

BACKGROUND: The number of mobile phone users has grown rapidly, which has generated mounting public concern regarding possible health hazards. This study aims to assess pituitary tumor risk, as it has rarely been investigated. MATERIAL AND METHODS: A case-control study was conducted with 80 eligible cases identified from all five university hospitals in Finland and frequency-matched 240 controls from the national population register. Controls were matched to cases by age, sex, region of residence and date of interview. A detailed history of mobile phone use was obtained using a structured interview. Several indicators of mobile phone use were assessed using conditional logistic regression. RESULTS: A reduced odds ratio was seen among regular mobile phone users [OR 0.39, 95% confidence interval (CI) 0.21, 0.72] relative to never/non-regular users, possibly reflecting methodological limitations. Pituitary tumor risk was not increased after 10 or more years since first use (OR 0.69, 95% CI 0.25, 1.89). The risk was not increased in relation to duration, cumulative hours of use, or cumulative number of calls. The results were similar for analog and digital phones. CONCLUSIONS: We found no excess risk associated with self-reported short- or medium-term use of mobile phones. This is consistent with most of the published studies. However, uncertainties remained for longer duration of use, as a very small proportion of study participants reported use beyond 10 years.


Assuntos
Telefone Celular , Neoplasias Hipofisárias/epidemiologia , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Finlândia/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Adulto Jovem
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