RESUMO
A case of accidental carbon monoxide poisoning in several occupants of two neighboring residential buildings in Hamburg-Harburg (Germany) caused by a defective gas central heating system is described. Because of leaks in one of the residential buildings and the directly adjacent wall of the neighboring house, the gas could spread and accumulated in both residential buildings, which resulted in a highly dangerous situation. Exposure to the toxic gas caused mild to severe intoxication in 15 persons. Three victims died still at the site of the accident. Measures to protect the occupants were taken only with a great delay. As symptoms were unspecific, it was not realized that the various alarms given by persons involved in the accident were related to the same cause. In order to take appropriate measures in time it is indispensible to recognize, assess and check potential risks, which can be done by using carbon monoxide warning devices and performing immediate COHb measurements with special pulse oximeters on site. Moreover, the COHb content in the blood should be routinely determined in all patients admitted to an emergency department with unspecific symptoms.
Assuntos
Gasometria/métodos , Intoxicação por Monóxido de Carbono/etiologia , Intoxicação por Monóxido de Carbono/prevenção & controle , Monóxido de Carbono/sangue , Patologia Legal/métodos , Calefação/instrumentação , Monóxido de Carbono/toxicidade , Falha de Equipamento , Alemanha , Calefação/efeitos adversos , HumanosRESUMO
Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)-benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three Hâ isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing (2) H and (3) H at their benzylic Câ atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA-thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.
Assuntos
Carbono-Carbono Liases/metabolismo , Succinatos/metabolismo , Tolueno/química , Fumaratos/química , Oxirredução , Estereoisomerismo , Succinatos/química , Tolueno/metabolismo , Trítio/químicaRESUMO
4-Hydroxybutyryl-coenzyme A (CoA) dehydratase (4HBD) from Clostridium aminobutyricum catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA and the irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. 4HBD is an oxygen-sensitive homotetrameric enzyme with one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) in each subunit. Upon the addition of crotonyl-CoA or the analogues butyryl-CoA, acetyl-CoA, and CoA, UV-visible light and electron paramagnetic resonance (EPR) spectroscopy revealed an internal one-electron transfer to FAD and the [4Fe-4S](2+) cluster prior to hydration. We describe an active recombinant 4HBD and variants produced in Escherichia coli. The variants of the cluster ligands (H292C [histidine at position 292 is replaced by cysteine], H292E, C99A, C103A, and C299A) had no measurable dehydratase activity and were composed of monomers, dimers, and tetramers. Variants of other potential catalytic residues were composed only of tetramers and exhibited either no measurable (E257Q, E455Q, and Y296W) hydratase activity or <1% (Y296F and T190V) dehydratase activity. The E455Q variant but not the Y296F or E257Q variant displayed the same spectral changes as the wild-type enzyme after the addition of crotonyl-CoA but at a much lower rate. The results suggest that upon the addition of a substrate, Y296 is deprotonated by E455 and reduces FAD to FADH·, aided by protonation from E257 via T190. In contrast to FADH·, the tyrosyl radical could not be detected by EPR spectroscopy. FADH· appears to initiate the radical dehydration via an allylic ketyl radical that was proposed 19 years ago. The mode of radical generation in 4HBD is without precedent in anaerobic radical chemistry. It differs largely from that in enzymes, which use coenzyme B12, S-adenosylmethionine, ATP-driven electron transfer, or flavin-based electron bifurcation for this purpose.
Assuntos
Clostridium/enzimologia , Radicais Livres/metabolismo , Hidroliases/metabolismo , Acetilcoenzima A/metabolismo , Acil Coenzima A/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise EspectralRESUMO
According to the literature, physicians have an increased risk of committing suicide, which can partly be explained by occupational stress. A retrospective analysis of the deaths investigated by the Hamburg office of Criminal Investigation and documents of the Hamburg Institute of Forensic Medicine for the years 2001 to 2013 showed that the average suicide rate among physicians is not significantly higher than that of the total population, it is 0.021% for physicians, 0.023% for dentists and 0.013% for the general public; thus the difference is not statistically significant (p: 0.57 for physicians and 0.74 for dentists). Drug intoxication has been found to be the most common method of suicide committed by physicians. However, these results must be interpreted with caution in view of the fact that a detailed evaluation of data was often not possible, especially because information as to the profession of the deceased and the motive for the suicide were missing or insufficiently documented.
Assuntos
Odontólogos/estatística & dados numéricos , Médicos/estatística & dados numéricos , Suicídio/estatística & dados numéricos , População Urbana/estatística & dados numéricos , Adulto , Idoso , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Fatores de RiscoRESUMO
BACKGROUND: The steadily increasing field of radical biochemistry is dominated by the large family of S-adenosylmethionine dependent enzymes, the so-called radical SAM enzymes, of which several new members are discovered every year. Here we report on 2- and 4-hydroxyacyl-CoA dehydratases which apply a very different method of radical generation. In these enzymes ketyl radicals are formed by one-electron reduction or oxidation and are recycled after each turnover without further energy input. Earlier reviews on 2-hydroxyacyl-CoA dehydratases were published in 2004 [J. Kim, M. Hetzel, C.D. Boiangiu, W. Buckel, FEMS Microbiol. Rev. 28 (2004) 455-468. W. Buckel, M. Hetzel, J. Kim, Curr. Opin. Chem. Biol. 8 (2004) 462-467.] SCOPE OF REVIEW: The review focuses on four types of 2-hydroxyacyl-CoA dehydratases that are involved in the fermentation of amino acids by anaerobic bacteria, especially clostridia. These enzymes require activation by one-electron transfer from an iron-sulfur protein driven by hydrolysis of ATP. The review further describes the proposed mechanism that is highlighted by the identification of the allylic ketyl radical intermediate and the elucidation of the crystal structure of 2-hydroxyisocapryloyl-CoA dehydratase. With 4-hydroxybutyryl-CoA dehydratase the crystal structure, the complete stereochemistry and the function of several conserved residues around the active site could be identified. Finally potential biotechnological applications of the radical dehydratases are presented. GENERAL SIGNIFICANCE: The action of the activator as an 'Archerase' shooting electrons into difficultly reducible acceptors becomes an emerging principle in anaerobic metabolism. The dehydratases may provide useful tools in biotechnology. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
Assuntos
Proteínas de Bactérias/metabolismo , Hidroliases/metabolismo , Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Anaerobiose , Bactérias Anaeróbias/metabolismo , Proteínas de Bactérias/química , Biocatálise , Domínio Catalítico , Radicais Livres/química , Radicais Livres/metabolismo , Hidroliases/química , Proteínas Ferro-Enxofre/química , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Oxirredução , S-Adenosilmetionina/química , EstereoisomerismoRESUMO
BACKGROUND: Calpains are calcium regulated intracellular cysteine proteases implicated in a variety of physiological functions and pathological conditions. The Drosophila melanogaster genome contains only two genes, CalpA and CalpB coding for canonical, active calpain enzymes. The movement of the border cells in Drosophila egg chambers is a well characterized model of the eukaryotic cell migration. Using this genetically pliable model we can investigate the physiological role of calpains in cell motility. RESULTS: We demonstrate at the whole organism level that CalpB is implicated in cell migration, while the structurally related CalpA paralog can not fulfill the same function. The downregulation of the CalpB gene by mutations or RNA interference results in a delayed migration of the border cells in Drosophila egg chambers. This phenotype is significantly enhanced when the focal adhesion complex genes encoding for α-PS2 integrin ( if), ß-PS integrin (mys) and talin (rhea) are silenced. The reduction of CalpB activity diminishes the release of integrins from the rear end of the border cells. The delayed migration and the reduced integrin release phenotypes can be suppressed by expressing wild-type talin-head in the border cells but not talin-head(R367A), a mutant form which is not able to bind ß-PS integrin. CalpB can cleave talin in vitro, and the two proteins coimmunoprecipitate from Drosophila extracts. CONCLUSIONS: The physiological function of CalpB in border cell motility has been demonstrated in vivo. The genetic interaction between the CalpB and the if, mys, as well as rhea genes, the involvement of active talin head-domains in the process, and the fact that CalpB and talin interact with each other collectively suggest that the limited proteolytic cleavage of talin is one of the possible mechanisms through which CalpB regulates cell migration.
Assuntos
Calpaína/metabolismo , Movimento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Animais , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Adesões Focais/metabolismo , Talina/metabolismoRESUMO
Coenzymeâ B(12) can assist radical enzymes that accomplish the vicinal interchange of a hydrogen atom with a functional group. It has been proposed that the Co-C bond homolysis of coenzymeâ B(12) to cob(II)alamin and the 5'-deoxyadenosyl radical is aided by hydrogen bonding of the corrin C19-H to the 3'-O of the ribose moiety of the incipient 5'-deoxyadenosyl radical, which is stabilized by 30â kJ mol(-1) (B. Durbeej etâ al., Chem. Eur. J. 2009, 15, 8578-8585). The diastereoisomers (R)- and (S)-2,3-dihydroxypropylcobalamin were used as models for coenzymeâ B(12). A downfield shift of the NMR signal for the C19-H proton was observed for the (R)-isomer (δ=4.45 versus 4.01â ppm for the (S)-isomer) and can be ascribed to an intramolecular hydrogen bond between the C19-H and the oxygen of CHOH. Crystal structures of (R)- and (S)-2,3-dihydroxypropylcobalamin showed C19-Hâ â â O distances of 3.214(7)â Å (R-isomer) and 3.281(11)â Å (S-isomer), which suggest weak hydrogen-bond interactions (-ΔG<6â kJ mol(-1)) between the CHOH of the dihydroxypropyl ligand and the C19-H. Exchange of the C19-H, which is dependent on the cobalt redox state, was investigated with cob(I)alamin, cob(II)alamin, and cob(III)alamin by using NMR spectroscopy to monitor the uptake of deuterium from deuterated water in the pH range 3-11. No exchange was found for any of the cobalt oxidation states. 3',5'-Dideoxyadenosylcobalamin, but not the 2',5'-isomer, was found to act as a coenzyme for glutamate mutase, with a 15-fold lower k(cat)/K(M) than 5'-deoxyadenosylcobalamin. This indicates that stabilization of the 5'-deoxyadenosyl radical by a hydrogen bond that involves the C19-H and the 3'-OH group of the cofactor is, at most, 7â kJ mol(-1) (-ΔG). Examination of the crystal structure of glutamate mutase revealed additional stabilizing factors: hydrogen bonds between both the 2'-OH and 3'-OH groups and glutamate 330. The actual strength of a hydrogen bond between the C19-H and the 3'-O of the ribose moiety of the 5'-deoxyadenosyl group is concluded not to exceed 6â kJ mol(-1) (-ΔG).
Assuntos
Cobamidas/química , Vitamina B 12/análogos & derivados , Vitamina B 12/química , Ligação de Hidrogênio , Cinética , Espectroscopia de Ressonância Magnética , Especificidade por SubstratoRESUMO
In the genome of Drosophila melanogaster, there are 19 phosphoprotein phosphatase (PPP) catalytic subunit coding genes. Seven of the novel members of the gene family turned out to be Drosophila-specific. The expression and evolution of these genes was investigated in the present study. CG11597 is a recently evolved gene that is expressed during all stages of morphogenesis in D. melanogaster. In contrast, the transcription of PpD5, PpD6, Pp1-Y1, and Pp1-Y2 genes is restricted to the pupa and imago developmental stages and to the testis of the males, just as that of the previously characterized PpY-55A and PpN58A. With the exception of the Y-localized Pp1-Y1 and Pp1-Y2, the testis-specific phosphatase genes are expressed in X/0 males, while none of them are expressed in XX/Y females. The mRNA of PpD5, Pp1-Y1, and PpY-55A were detected in the developing cysts by in situ hybridization, in contrast with the PpD6 transcript that was found in the distal ends of elongating spermatids. The latter localization suggests post-meiotic expression. The comparison of PPP genes in five Drosophila species revealed that the sequence of the six testis-specific phosphatases changed more rapidly than that of the housekeeping phosphatases. Our results support the "faster male" hypothesis. On the other hand, the male-biased expression of the six genes remained conserved during evolution despite the fact that Pp1-Y1, Pp1-Y2, and PpD6 moved from autosomes to the Y chromosome. Interestingly, the PpD6 gene was found to be Y-linked only in Drosophila ananassae.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Perfilação da Expressão Gênica , Fosfoproteínas Fosfatases/genética , Animais , Cromossomos de Insetos/genética , Drosophila/classificação , Drosophila/crescimento & desenvolvimento , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Evolução Molecular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ , Isoenzimas/genética , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores Sexuais , Especificidade da Espécie , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Cromossomo Y/genéticaRESUMO
Calpains are cysteine proteases involved in a number of physiological and pathological processes, yet our knowledge of substrates cleaved in vivo, in intact cells, is scarce. In this work we made an attempt to develop a technique for finding calpain substrates in intact Drosophila Schneider S2 cells. The procedure consists in comparative 2D gelelectrophoresis: three identical samples were treated in different ways: A (control, no addition), B, activated (Ca(2+) and ionomycin added), C, inactivated (additions as in B+specific calpain inhibitor). 2D gel pattern were analyzed by densitometry. Spots showing density relation A>B<Assuntos
Calpaína/metabolismo
, Proteínas de Drosophila/metabolismo
, Drosophila/metabolismo
, Animais
, Cálcio/farmacologia
, Calpaína/antagonistas & inibidores
, Calpaína/genética
, Linhagem Celular
, Primers do DNA
, Proteínas de Drosophila/genética
, Ionomicina/farmacologia
, Espectrometria de Massas
, Reação em Cadeia da Polimerase
, Proteínas Recombinantes/metabolismo
, Especificidade por Substrato
RESUMO
Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca(2+) sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an alpha-helical conformation in 50% CD(3)OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca(2+) induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain.
Assuntos
Calpaína/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Peptídeos/química , Peptídeos/farmacologia , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/química , Ativação Enzimática/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Earlier we have shown that an equimolar mixture of calpastatin subdomains A and C (19 amino acids each) strongly activates m-calpain in vitro. In the present work we developed a membrane-permeable activator system, by conjugating an oligo-arginine tail to both peptides. We tested calpain activation as well as synaptic excitability on rat brain slices ex vivo. In hippocampal slices both basic excitability and long-term synaptic efficacy were significantly increased upon treatment with the activator. We propose that the activator peptide conjugates can be used with any mammalian cell, to specifically challenge the calpain system apparently without raising cytoplasmic Ca2+. Such an effector may be a useful tool in dissecting intracellular mechanisms involving the calpain system.
Assuntos
Cálcio/metabolismo , Proteínas de Membrana/síntese química , Proteínas de Membrana/fisiologia , Neurônios/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Cálcio/fisiologia , Chlorocebus aethiops , Masculino , Dados de Sequência Molecular , Ratos , Ratos Wistar , Potenciais Sinápticos/efeitos dos fármacos , Potenciais Sinápticos/fisiologiaRESUMO
Missing, ineligible or delayed reference data to establish conventional dental or DNA identification are common scenarios in forensic practice. Therefore, it is worthwhile to explore new avenues that facilitate human identification. Due to the recent remarkable evolution in the prosthetic dental restorations based on dental implants and the emergence of novel DNA technologies utilized to infer the biological profile, the identification process has become easier than ever before. We report on a characteristic case, which highlights the particular importance of dental implants and DNA approaches in the prospective investigations for human identification. The aim of this publication is to focus on the possibility of identifying the batch numbers, even if they were not engraved in dental implants, making antemortem dental records of dental implants more easily accessible to establish a comparative dental identification. In addition, the reported case presents the supplementary data yielded through estimating the epigenetic age using DNA methylation as well as the biogeographical origin using Y-Haplotype and mitochondrial DNA analyses. Our results demonstrate that expanded oral implant investigations that also include implants extraction and comprehensive microscopic measurements can lead to identifying their batch numbers despite the numerous number of implants systems manufactured and distributed worldwide. Data saved by dental implant manufacturers can be very supportive and represent additional reference data for dental identification, when antemortem dental records are still missing. Furthermore, DNA methylation and mitochondrial DNA analyses can support the progress of investigation.
Assuntos
Impressões Digitais de DNA , Implantes Dentários , Repetições de Microssatélites , Idoso , Idoso de 80 Anos ou mais , Ilhas de CpG/genética , Metilação de DNA , Dente Suporte , Odontologia Legal , Genética Forense , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Radiografia PanorâmicaRESUMO
The activity of calpain is controlled by the free intracellular calcium level and by the protein's intrinsically disordered endogenous inhibitor, calpastatin, mediated by short conserved segments: subdomains A-C. The exact binding mode of calpastatin to the enzyme has until now been unclear. Our NMR data of the 141 amino acid long inhibitor, with and without calcium and calpain, have revealed structural changes and a tripartite binding mode, in which the disordered inhibitor wraps around, and contacts, the enzyme at three points, facilitated by flexible linkers. This unprecedented binding mode permits a unique combination of specificity, speed and binding strength in regulation.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Calpaína/antagonistas & inibidores , Calpaína/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
The calpain enzymes play important roles in numerous processes in the cell. In vivo analysis of calpain activity might be useful for clarification of their role in different diseases. Our early results suggested that a peptide substrate, Dabcyl-TPLKSPPPSPR- EDANS, based on the calpain cleavage sequences is suitable for developing a new cell-penetrating calpain substrate. This conjugate with the Dabcyl and EDANS fluorophores as a FRET pair is specific for calpain even in cell lysate, but unfortunately has poor cell uptake. Therefore, we have modified this sequence by C-terminal elongation with heptaarginine unit possessing cell-penetrating activity. In order to preserve the necessary distance between the two FRET partners, we inserted a Glu residue between the substrate and heptaarginine parts of the peptide. Thus, the cell-penetrating substrate Dabcyl-TPLKSPPPSPRE( EDANS)R 7 was synthesized. This peptide not only retained the substrate property, but was a better substrate of Calpain B enzyme. The cell uptake of the substrate conjugate was studied by fluorescence microscopy and flow cytometry. The results showed that the conjugate enters COS-7 cells more efficiently than the peptide substrate without heptaarginine. The uptake occurs already at low concentration and the compound is distributed homogeneously inside cells. These observations might indicate that this new cell-penetrating substrate could be useful for determining calpain activity in cell lysate or in intact cells of various origins.
Assuntos
Calpaína/metabolismo , Células/metabolismo , Peptídeos/síntese química , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Transferência Ressonante de Energia de Fluorescência , Espaço Intracelular/metabolismo , Cinética , Microscopia de Fluorescência , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Peptídeos/química , PermeabilidadeRESUMO
Typical calpains in mammals become activated on binding of 8-12 Ca2+ ions per enzyme molecule, giving an example of integrated, manifold regulation by calcium. Besides two identified Ca2+ sites in catalytic domain II and several EF-hand motifs in domains IV and VI, an acidic loop in the centrally positioned domain III seems to harbour Ca2+. The mediator of distant Ca2+-induced structural transitions is an elongated structural element, the 'transducer'. By site-directed mutagenesis along the transducer, we have generated various forms of rat m-calpain in which critical intramolecular interactions, as judged from the X-ray structure, would be abolished or modified. The kinetic parameters of these mutant enzymes support a model featuring shrinkage of transducer as a contributor to structural changes involved in calpain activation.
Assuntos
Cálcio/farmacologia , Calpaína/química , Calpaína/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Sinalização do Cálcio/fisiologia , Calpaína/genética , Domínio Catalítico , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , RatosRESUMO
Intrinsically unstructured proteins (IUPs) are devoid of extensive structural order but often display signs of local and limited residual structure. To explain their effective functioning, we reasoned that such residual structure can be crucial in their interactions with their structured partner(s) in a way that preformed structural elements presage their final conformational state. To check this assumption, a database of 24 IUPs with known 3D structures in the bound state has been assembled and the distribution of secondary structure elements and backbone torsion angles have been analysed. The high proportion of residues in coil conformation and with phi, psi angles in the disallowed regions of the Ramachandran map compared to the reference set of globular proteins shows that IUPs are not fully ordered even in their bound form. To probe the effect of partner proteins on IUP folding, inherent conformational preferences of IUP sequences have been assessed by secondary structure predictions using the GOR, ALB and PROF algorithms. The accuracy of predicting secondary structure elements of IUPs is similar to that of their partner proteins and is significantly higher than the corresponding values for random sequences. We propose that strong conformational preferences mark regions in IUPs (mostly helices), which correspond to their final structural state, while regions with weak conformational preferences represent flexible linkers between them. In our interpretation, preformed elements could serve as initial contact points, the binding of which facilitates the reeling of the flexible regions onto the template. This finding implies that IUPs draw a functional advantage from preformed structural elements, as they enable their facile, kinetically and energetically less demanding, interaction with their physiological partner.
Assuntos
Proteínas/química , Proteínas/metabolismo , Bases de Dados de Proteínas , Ligação Proteica/fisiologia , Estrutura Secundária de ProteínaRESUMO
UK114, the goat liver tumour antigen, is a member of a widely distributed family of conserved low-molecular-mass proteins (YER057c/YjgF/UK114), the function of which is ill understood. To the various orthologues diverse functions have been ascribed, such as translation inhibition, regulation of purine repressor or calpain activation. Owing to a limited sequence similarity to Hsp90 (heat-shock protein 90), they have also been proposed to be molecular chaperones; however, this has never been tested. In the present paper, we report the cloning and characterization of the Drosophila orthologue, DUK114. In brief, DUK114 had no effect that would have qualified it as a calpain activator. In contrast, it proved to be a very potent molecular chaperone in in vitro assays. In a heat-aggregation test, it significantly decelerated the formation of citrate synthase aggregates. In a reverse assay, the recovery of the enzyme from urea- and heat-induced denatured states was accelerated almost 3-fold. On a molar basis, the chaperone activity of the 15-kDa DUK114 is comparable with that of Hsp90, the almost 6-times-larger archetypal molecular chaperone. In similar assays, DUK114 was ineffective with Drosophila calpain A or calpain B. To test for its chaperone activity in vivo, DUK114 was transfected into Schneider (S2) cells; after heat shock, the number of viable non-transfected cells started to increase after a lag time; in the presence of DUK114, cell proliferation started at once. Our work is the first experimental evidence that DUK114, and possibly other members of this family, are molecular chaperones.
Assuntos
Chaperonas Moleculares/fisiologia , Sequência de Aminoácidos , Animais , Calpaína/química , Calpaína/fisiologia , Bovinos , Linhagem Celular , Citrato (si)-Sintase/metabolismo , Clonagem Molecular , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Temperatura Alta , Proteínas de Membrana/química , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/química , Dados de Sequência Molecular , Conformação Proteica , Desnaturação Proteica , Renaturação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência de AminoácidosRESUMO
Calpain B is one of the two calpain homologues in Drosophila melanogaster that are proteolytically active. We studied its activation by Ca2+ both in vitro and in vivo, in Schneider (S2) cells. Activation involves the autolytic cleavage, at two major sites, of the N-terminal segment, the length of which was earlier underestimated. Site-directed mutagenesis at the autolytic sites did not prevent autolysis, but only shifted its sites. Calpain B mRNA was detectable in all developmental stages of the fly. In situ hybridization and immunostaining showed expression in ovaries, embryo and larvae, with high abundance in larval salivary glands. In S2 cells, calpain B was mainly in the cytoplasm and upon a rise in Ca2+ the enzyme adhered to intracellular membranes.
Assuntos
Calpaína/análise , Calpaína/metabolismo , Proteínas de Drosophila/análise , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Sequência de Aminoácidos , Animais , Calpaína/genética , Linhagem Celular , Citoplasma/enzimologia , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Ativação Enzimática , Membranas Intracelulares/enzimologia , Dados de Sequência Molecular , RNA Mensageiro/metabolismoRESUMO
Calpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca(2+)-dependent manner. The peptides are disordered in water but show a high propensity for alpha-helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca(2+), and is clearly seen upon binding of the peptides to the enzyme. Secondary-structure prediction of all calpastatin sequences shows that the helix-forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding-induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.