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1.
Materials (Basel) ; 13(11)2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32532009

RESUMO

This paper describes the investigations on the possibilities of treatment of wastewater generated in an industrial laundry with application of a combined biological-photooxidation- membrane system aimed at water recycle and reuse. The two treatment schemes were compared: 1) scheme A consisting of a treatment in a moving bed biological reactor (MBBR) followed by microfiltration (MF) and nanofiltration (NF), and 2) scheme B comprising MBBR followed by oxidation by photolysis enhanced with in situ generated O3 (UV/O3) after which MF and NF were applied. The removal efficiency in MBBR reached 95-97% for the biochemical oxygen demand; 90-93% for the chemical oxygen demand and 89-99% for an anionic and a nonionic surfactants. The application of UV/O3 system allowed to decrease the content of the total organic carbon by 68% after 36 h of operation with a mineralization rate of 0.36 mg/L·h. Due to UV/O3 pretreatment, a significant mitigation of membrane fouling in the case of both MF and NF processes was achieved. The MF permeate flux in the system B was over two times higher compared to that in the system A. Based on the obtained results it was concluded that the laundry wastewater pretreated in the MBBR-UV/O3-MF-NF system could be recycled to any stage of the laundry process.

2.
Plant Physiol ; 182(2): 1142-1160, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31699848

RESUMO

SNF1-related protein kinases 2 (SnRK2s) are key signaling elements regulating abscisic acid-dependent plant development and responses to environmental stresses. Our previous data showed that the SnRK2-interacting Calcium Sensor (SCS) inhibits SnRK2 activity. Use of alternative transcription start sites located within the Arabidopsis (Arabidopsis thaliana) AtSCS gene results in two in-frame transcripts and subsequently two proteins, that differ only by the sequence position of the N terminus. We previously described the longer AtSCS-A, and now describe the shorter AtSCS-B and compare the two isoforms. The two isoforms differ substantially in their expression profiles in plant organs and in response to environmental stresses, in their calcium binding properties, and in their conformational dynamics in the presence and absence of Ca2+ Only AtSCS-A has the features of a calcium sensor. Both forms inhibit SnRK2 activity, but while AtSCS-A requires calcium for inhibition, AtSCS-B does not. Analysis of Arabidopsis plants stably expressing 35S::AtSCS-A-c-myc or 35S::AtSCS-B-c-myc in the scs-1 knockout mutant background revealed that, in planta, both forms are negative regulators of abscisic acid-induced SnRK2 activity and regulate plant resistance against water deficit. Moreover, the data highlight biochemical, biophysical, and functional properties of EF-hand-like motifs in plant proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cálcio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Salino/genética , Estresse Fisiológico/genética , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Algoritmos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Dicroísmo Circular , Simulação por Computador , Desidratação/genética , Desidratação/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Inativação de Genes , Espectrometria de Massa com Troca Hidrogênio-Deutério , Modelos Químicos , Plantas Geneticamente Modificadas , Conformação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes , Estresse Fisiológico/efeitos dos fármacos
3.
Sci Total Environ ; 627: 1638-1643, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29467086

RESUMO

Large, laboratory scale biological treatment tests of real industrial wastewater, generated in a large industrial laundry facility, was conducted from October 2014 to January 2015. This research sought to develop laundry wastewater treatment technology which included tests of a two-stage Moving Bed Bio Reactor (MBBR); this had two reactors, was filled with carriers Kaldnes K5 (specific area - 800 m2/m3) and were realized in aerobic condition. Operating on site, in the laundry, reactors were fed actual wastewater from the laundry retention tank. The laundry wastewater contained mainly surfactants and impurities originating from washed fabrics; a solution of urea to supplement nitrogen content and a solution of acid to correct pH were added. The daily flow of raw wastewater Qd varied from 0.6-1.0 m3/d. Wastewater quality indicators showed that the reduction of pollutants was obtained: BOD5 by 95-98%, COD by 89-94%, the sum of anionic and nonionic surfactants by 85-96%. The quality of the purified wastewater after the start-up period met legal requirements regarding the standards for wastewater discharged into the environment.

4.
J Am Soc Mass Spectrom ; 29(1): 158-173, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29080206

RESUMO

The human GW182 protein plays an essential role in micro(mi)RNA-dependent gene silencing. miRNA silencing is mediated, in part, by a GW182 C-terminal region called the silencing domain, which interacts with the poly(A) binding protein and the CCR4-NOT deadenylase complex to repress protein synthesis. Structural studies of this GW182 fragment are challenging due to its predicted intrinsically disordered character, except for its RRM domain. However, detailed insights into the properties of proteins containing disordered regions can be provided by hydrogen-deuterium exchange mass spectrometry (HDX/MS). In this work, we applied HDX/MS to define the structural state of the GW182 silencing domain. HDX/MS analysis revealed that this domain is clearly divided into a natively unstructured part, including the CCR4-NOT interacting motif 1, and a distinct RRM domain. The GW182 RRM has a very dynamic structure, since water molecules can penetrate the whole domain in 2 h. The finding of this high structural dynamics sheds new light on the RRM structure. Though this domain is one of the most frequently occurring canonical protein domains in eukaryotes, these results are - to our knowledge - the first HDX/MS characteristics of an RRM. The HDX/MS studies show also that the α2 helix of the RRM can display EX1 behavior after a freezing-thawing cycle. This means that the RRM structure is sensitive to environmental conditions and can change its conformation, which suggests that the state of the RRM containing proteins should be checked by HDX/MS in regard of the conformational uniformity. Graphical Abstract.


Assuntos
Autoantígenos/química , Espectrometria de Massas/métodos , Proteínas de Ligação a RNA/química , Autoantígenos/metabolismo , Medição da Troca de Deutério/métodos , Cinética , Conformação Proteica , Domínios Proteicos , Motivo de Reconhecimento de RNA , Proteínas de Ligação a RNA/metabolismo
5.
Environ Sci Pollut Res Int ; 23(19): 19145-55, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27351873

RESUMO

The possibilities of application of a three-step system combining hybrid biological treatment followed by advanced UV/O3 oxidation with in situ generated O3 and membrane separation (ultrafiltration (UF) and nanofiltration (NF)) to treat and reuse the wastewater from an industrial laundry are presented. By the application of a hybrid moving bed biofilm reactor (HMBBR), the total organic carbon concentration was reduced for about 90 %. However, since the HMBBR effluent still contained organic contaminants as well as high concentrations of inorganic ions and exhibited significant turbidity (8.2 NTU), its further treatment before a possible reuse in the laundry was necessary. The UV/O3 pretreatment prior to UF was found to be an efficient method of the membrane fouling alleviation. During UF, the turbidity of wastewater was reduced below 0.3 NTU. To remove the inorganic salts, the UF permeate was further treated during NF. The NF permeate exhibited very low conductivity (27-75 µS/cm) and contained only small amounts of Ca(2+) and Mg(2+); thus ,it could be reused at any stage of the laundry process.


Assuntos
Águas Residuárias/química , Purificação da Água/métodos , Reatores Biológicos , Resíduos Industriais , Indústrias , Membranas Artificiais , Ozônio/química , Reciclagem/métodos , Ultrafiltração , Raios Ultravioleta , Eliminação de Resíduos Líquidos , Águas Residuárias/estatística & dados numéricos
6.
J Mol Biol ; 428(6): 1180-1196, 2016 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-26860583

RESUMO

Histone pre-mRNAs are cleaved at the 3' end by a complex that contains U7 snRNP, the FLICE-associated huge protein (FLASH) and histone pre-mRNA cleavage complex (HCC) consisting of several polyadenylation factors. Within the complex, the N terminus of FLASH interacts with the N terminus of the U7 snRNP protein Lsm11, and together they recruit the HCC. FLASH through its distant C terminus independently interacts with the C-terminal SANT/Myb-like domain of nuclear protein, ataxia-telangiectasia locus (NPAT), a transcriptional co-activator required for expression of histone genes in S phase. To gain structural information on these interactions, we used mass spectrometry to monitor hydrogen/deuterium exchange in various regions of FLASH, Lsm11 and NPAT alone or in the presence of their respective binding partners. Our results indicate that the FLASH-interacting domain in Lsm11 is highly dynamic, while the more downstream region required for recruiting the HCC exchanges deuterium slowly and likely folds into a stable structure. In FLASH, a stable structure is adopted by the domain that interacts with Lsm11 and this domain is further stabilized by binding Lsm11. Notably, both hydrogen/deuterium exchange experiments and in vitro binding assays demonstrate that Lsm11, in addition to interacting with the N-terminal region of FLASH, also contacts the C-terminal SANT/Myb-like domain of FLASH, the same region that binds NPAT. However, while NPAT stabilizes this domain, Lsm11 causes its partial relaxation. These competing reactions may play a role in regulating histone gene expression in vivo.


Assuntos
Regulação da Expressão Gênica , Histonas/genética , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Animais , Drosophila , Espectrometria de Massas , Modelos Biológicos , Vertebrados
7.
J Am Soc Mass Spectrom ; 27(1): 73-82, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26337516

RESUMO

The biologically active form of purine nucleoside phosphorylase (PNP) from Escherichia coli (EC 2.4.2.1) is a homohexamer unit, assembled as a trimer of dimers. Upon binding of phosphate, neighboring monomers adopt different active site conformations, described as open and closed. To get insight into the functions of the two distinctive active site conformations, virtually inactive Arg24Ala mutant is complexed with phosphate; all active sites are found to be in the open conformation. To understand how the sites of neighboring monomers communicate with each other, we have combined H/D exchange (H/DX) experiments with molecular dynamics (MD) simulations. Both methods point to the mobility of the enzyme, associated with a few flexible regions situated at the surface and within the dimer interface. Although H/DX provides an average extent of deuterium uptake for all six hexamer active sites, it was able to indicate the dynamic mechanism of cross-talk between monomers, allostery. Using this technique, it was found that phosphate binding to the wild type (WT) causes arrest of the molecular motion in backbone fragments that are flexible in a ligand-free state. This was not the case for the Arg24Ala mutant. Upon nucleoside substrate/inhibitor binding, some release of the phosphate-induced arrest is observed for the WT, whereas the opposite effects occur for the Arg24Ala mutant. MD simulations confirmed that phosphate is bound tightly in the closed active sites of the WT; conversely, in the open conformation of the active site of the WT phosphate is bound loosely moving towards the exit of the active site. In Arg24Ala mutant binary complex Pi is bound loosely, too.


Assuntos
Proteínas de Bactérias/química , Domínio Catalítico , Medição da Troca de Deutério/métodos , Escherichia coli/enzimologia , Simulação de Dinâmica Molecular , Purina-Núcleosídeo Fosforilase/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Fosfatos/química , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica , Purina-Núcleosídeo Fosforilase/metabolismo
8.
J Struct Biol ; 192(3): 426-440, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26434626

RESUMO

Keratins are intermediate filament (IF) proteins that form complex filament systems in epithelial cells, thus serving as scaffolding elements and mechanical stress absorbers. The building blocks of keratin IFs are parallel coiled-coil dimers of two distinct sequence-related proteins distinguished as type I and type II keratins. To gain more insight into their structural dynamics, we resorted to hydrogen-deuterium exchange mass spectrometry of keratins K8 and K18, which are characteristic for simple epithelial cells. Using this powerful technique not employed with IFs before, we mapped patterns of protected versus unprotected regions in keratin complexes at various assembly levels. In particular, we localized protein segments exhibiting different hydrogen exchange patterns in tetramers versus filaments. We observed a general pattern of precisely positioned regions of stability intertwining with flexible regions, mostly represented by the non-α-helical segments. Notably, some regions within the coiled-coil domains are significantly more dynamic than others, while the IF-consensus motifs at the end domains of the central α-helical "rod" segment, which mediate the "head-to-tail" dimer-dimer interaction in the filament elongation process, become distinctly more protected upon formation of filaments. Moreover, to gain more insight into the dynamics of the individual keratins, we investigated the properties of homomeric preparations of K8 and K18. The physiological importance of keratins without a partner is encountered in both pathological and experimental situations when one of the two species is present in robust excess or completely absent, such as in gene-targeted mice.


Assuntos
Medição da Troca de Deutério , Células Epiteliais/metabolismo , Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Sequência de Aminoácidos , Citoesqueleto/metabolismo , Estrutura Terciária de Proteína
9.
Protein Sci ; 23(5): 639-51, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24591271

RESUMO

RACK1 is a member of the WD repeat family of proteins and is involved in multiple fundamental cellular processes. An intriguing feature of RACK1 is its ability to interact with at least 80 different protein partners. Thus, the structural features enabling such interactomic flexibility are of great interest. Several previous studies of the crystal structures of RACK1 orthologs described its detailed architecture and confirmed predictions that RACK1 adopts a seven-bladed ß-propeller fold. However, this did not explain its ability to bind to multiple partners. We performed hydrogen-deuterium (H-D) exchange mass spectrometry on three orthologs of RACK1 (human, yeast, and plant) to obtain insights into the dynamic properties of RACK1 in solution. All three variants retained similar patterns of deuterium uptake, with some pronounced differences that can be attributed to RACK1's divergent biological functions. In all cases, the most rigid structural elements were confined to B-C turns and, to some extent, strands B and C, while the remaining regions retained much flexibility. We also compared the average rate constants for H-D exchange in different regions of RACK1 and found that amide protons in some regions exchanged at least 1000-fold faster than in others. We conclude that its evolutionarily retained structural architecture might have allowed RACK1 to accommodate multiple molecular partners. This was exemplified by our additional analysis of yeast RACK1 dimer, which showed stabilization, as well as destabilization, of several interface regions upon dimer formation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas de Ligação ao GTP/química , Proteínas de Neoplasias/química , Receptores de Superfície Celular/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Medição da Troca de Deutério , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Multimerização Proteica , Receptores de Quinase C Ativada , Alinhamento de Sequência
10.
PLoS One ; 8(10): e76353, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098480

RESUMO

Activation of the receptor for advanced glycation end products (RAGE) leads to a chronic proinflammatory signal, affecting patients with a variety of diseases. Potentially beneficial modification of RAGE activity requires understanding the signal transduction mechanism at the molecular level. The ligand binding domain is structurally uncoupled from the cytoplasmic domain, suggesting receptor oligomerization is a requirement for receptor activation. In this study, we used hydrogen-deuterium exchange and mass spectrometry to map structural differences between the monomeric and oligomeric forms of RAGE. Our results indicated the presence of a region shielded from exchange in the oligomeric form of RAGE and led to the identification of a new oligomerization interface localized at the linker region between domains C1 and C2. Based on this finding, a model of a RAGE dimer and higher oligomeric state was constructed.


Assuntos
Deutério/química , Hidrogênio/química , Espectrometria de Massas , Multimerização Proteica , Receptores Imunológicos/química , Espaço Extracelular , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo
11.
EMBO Mol Med ; 5(10): 1484-501, 2013 10.
Artigo em Inglês | MEDLINE | ID: mdl-23982976

RESUMO

The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508-CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508-NBD1 and housekeeping proteins prevents ΔF508-CFTR delivery to the plasma membrane. Based on this assumption we applied structure-based virtual screening to identify new low-molecular-weight compounds that should bind to ΔF508-NBD1 and act as protein-protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico-selected compounds induced functional expression of ΔF508-CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508-CFTR mice. The proposed compounds disrupt keratin8-ΔF508-CFTR interaction in ΔF508-CFTR HeLa cells. Structural analysis of ΔF508-NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508-CFTR trafficking defect known to date.


Assuntos
Brônquios/citologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Animais , Sítios de Ligação , Brônquios/efeitos dos fármacos , Brônquios/fisiologia , Células Cultivadas , Canais de Cloreto/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Células HeLa , Homozigoto , Humanos , Queratina-8/química , Queratina-8/metabolismo , Camundongos , Técnicas de Patch-Clamp , Ligação Proteica , Mapas de Interação de Proteínas/efeitos dos fármacos , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
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