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1.
J Adv Res ; 37: 255-266, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35499043

RESUMO

Introduction: The striking imbalance between the ever-increasing amount of nanomedicines and low clinical translation of products has become the focus of intense debate. For clinical translation, the critical issue is to select the appropriate agents and combination regimen for targeted diseases, not to prepare increasingly complex nanoplatforms. Objectives: A safe and efficient platform, α-tocopheryl succinate (α-TOS) married 2D molybdenum disulfide, was devised by a facile method and applied for cooperative imaging-guided photothermal-selective chemotherapy of ovarian carcinoma. Methods: A novel platform of PEGylated α-TOS and folic acid (FA) conjugated 2D MoS2 nanoflakes was fabricated  for the cooperative multimode computed tomography (CT)/photoacoustic (PA)/thermal imaging-guided photothermal-selective chemotherapy of ovarian carcinoma. Results: The photothermal efficiency (65.3%) of the platform under safe near-infrared irradiation is much higher than that of other photothermal materials reported elsewhere. Moreover, the covalently linked α-TOS renders platform with selective chemotherapy for cancer cells. Remarkably, with these excellent properties, the platform can be used to completely eliminate the solid tumor by safe photothermal therapy, and then kill the residual cancer cells by selective chemotherapy to prevent tumor recurrence. More significantly, barely side effects occur in the whole treatment process. The excellent efficacy and safety benefits in vivo lead to the prominent survival rate of 100% over 91 days. Conclusion: The safe and efficient platform might be a candidate of clinical nanomedicines for multimode theranostics. This study demonstrates an innovative thought in precise nanomedicine regarding the design of next generation of cancer theranostic protocol for potential clinical practice.


Assuntos
Molibdênio , Neoplasias Ovarianas , Dissulfetos , Feminino , Humanos , Neoplasias Ovarianas/diagnóstico por imagem , Neoplasias Ovarianas/tratamento farmacológico , Fototerapia , Nanomedicina Teranóstica/métodos
2.
Sci Rep ; 12(1): 7262, 2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508689

RESUMO

Next-generation site-specific cysteine-based antibody-drug-conjugates (ADCs) broaden therapeutic index by precise drug-antibody attachments. However, manufacturing such ADCs for clinical validation requires complex full reduction and reoxidation processes, impacting product quality. To overcome this technical challenge, we developed a novel antibody manufacturing process through cysteine (Cys) metabolic engineering in Chinese hamster ovary cells implementing a unique cysteine-capping technology. This development enabled a direct conjugation of drugs after chemoselective-reduction with mild reductant tris(3-sulfonatophenyl)phosphine. This innovative platform produces clinical ADC products with superior quality through a simplified manufacturing process. This technology also has the potential to integrate Cys-based site-specific conjugation with other site-specific conjugation methodologies to develop multi-drug ADCs and exploit multi-mechanisms of action for effective cancer treatments.


Assuntos
Antineoplásicos , Imunoconjugados , Animais , Anticorpos , Antineoplásicos/uso terapêutico , Células CHO , Cricetinae , Cricetulus , Cisteína , Dissulfetos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Engenharia Metabólica
3.
J Nanobiotechnology ; 20(1): 210, 2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35524268

RESUMO

A sort of composite hydrogel with good biocompatibility, suppleness, high conductivity, and anti-inflammatory activity based on polyvinyl alcohol (PVA) and molybdenum sulfide/graphene oxide (MoS2/GO) nanomaterial has been developed for spinal cord injury (SCI) restoration. The developed (MoS2/GO/PVA) hydrogel exhibits excellent mechanical properties, outstanding electronic conductivity, and inflammation attenuation activity. It can promote neural stem cells into neurons differentiation as well as inhibit the astrocytes development in vitro. In addition, the composite hydrogel shows a high anti-inflammatory effect. After implantation of the composite hydrogel in mice, it could activate the endogenous regeneration of the spinal cord and inhibit the activation of glial cells in the injured area, thus resulting in the recovery of locomotor function. Overall, our work provides a new sort of hydrogels for SCI reparation, which shows great promise for improving the dilemma in SCI therapy.


Assuntos
Álcool de Polivinil , Traumatismos da Medula Espinal , Animais , Anti-Inflamatórios/uso terapêutico , Dissulfetos , Grafite , Hidrogéis , Camundongos , Molibdênio/uso terapêutico , Nanogéis , Traumatismos da Medula Espinal/tratamento farmacológico
4.
Biochemistry (Mosc) ; 87(4): 319-330, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35527370

RESUMO

Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein-protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Dissulfetos , Epitopos , Humanos , Imunização , Camundongos , Proteínas Recombinantes/genética , SARS-CoV-2
5.
BMC Plant Biol ; 22(1): 172, 2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35379184

RESUMO

PURPOSE: Verticillium wilt is a destructive vascular disease in eggplants. The complex defensive mechanisms of eggplant against this disease are very limited. METHODS: Our work examined the bioactive properties of garlic allelochemical diallyl disulfide (DADS) as potential biostimulants for defense against V. dahliae in eggplant seedlings. We, therefore, foliar sprayed DADS on eggplants to study the defense response during the early biotrophic phase of V. dahliae (a hemibiotroph). RESULTS: DADS application significantly increased root peroxidase (POD), phenylalanine-ammonia lyase (PAL) enzyme activity, and reduced H2O2 levels after 24 h of fungal inoculation. Salicylic acid (SA) in leaves and roots was significantly increased while, the jasmonic acid (JA), indole acetic acid (IAA), and abscisic acid (ABA) levels were decreased. The microscopic examinations of V. dahliae infection in roots displayed that the progression of infection was restricted in DADS-treated plants. Depositions of lignin and phenolic compounds such as ferulic acid, p-coumaric acid, and caffeic acid content were significantly higher in DADS-treated plants at 48 h post-inoculation. Similarly, the DADS application up-regulated pathogenesis-related (PR1, PR2, and PR5), mitogen-activated protein kinase (MPK1), and lipoxygenase (LOX) genes. Furthermore, DADS-treated plants exhibited a lower disease severity index (23.3% vs. 57.0% in controls), indicating successful defense against V. dahliae. CONCLUSIONS: Our findings concluded that the biological function of garlic allelochemical DADS has a prominent role in the higher defense resistance of eggplants during the early infection of V. dahliae.


Assuntos
Solanum melongena , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/análogos & derivados , Compostos Alílicos , Dissulfetos , Peróxido de Hidrogênio , Verticillium
6.
Int J Nanomedicine ; 17: 1567-1575, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35401000

RESUMO

Background: Multifunctional nanocarriers based on tumor targeting and intracellular monitoring have received much attention and been a subject of intensive study by researchers in recent years. In this study, we report multifunctional glyconanoparticles with activatable near-infrared probes for tumor imaging and targeted drug delivery. Methods: Disulfide-functionalized dicyanomethylene-4H-pyran (DCM-SS-NH2) and amino-functionalized lactose were modified and loaded onto the surfaces of polydopamine nanoparticles (NPs) by Michael addition or Schiff-base reaction as GSH stimulation-responsive fluorescent probes and tumor-targeting moieties, respectively. Doxorubicin (DOX), a model anticancer drug, was loaded onto polydopamine through π-π interactions directly to prepare multifunctional PLDD (PDA@Lac/DCM/DOX) NPs. Results: Experimental results showed that PLDD NPs had been successfully prepared. DCM, the fluorescence of which was quenched in PLDD NPs, was able to restore red fluorescence in a solution with a GSH concentration of 5 mM. The amount of DOX released from PLDD NPs was 44% over 72 hours in a weak-acid environment (pH 5). The results of CLSM and flow cytometry indicated that the PLDD NPs had good HepG2-targeting ability due to the special recognition between lactose derivative of NPs and overexpressed asialoglycoprotein receptors on HepG2 cell membrane. More importantly, the disulfide bond of DCM-SS-NH2 was broken by the high concentration of GSH inside cancer cells, activating the near-infrared fluorescence probe DCM for cancer-cell imaging. MTT assays indicated that PLDD NPs exhibited higher anticancer efficiency for HepG2 cells and had reduced side effects on normal cells compared with free DOX. Conclusion: The fluorescence of modified DCM loaded onto PLDD NPs is able to be restored in the high-concentration GSH environment within cancer cells, while improving the effectiveness of chemotherapy with reduced side effects. It provides a good example of integration of tumor imaging and targeted drug delivery.


Assuntos
Lactose , Nanopartículas , Dissulfetos , Doxorrubicina , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química
7.
Int J Mol Sci ; 23(7)2022 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-35409101

RESUMO

N-Glycosylation (NG) and disulfide bonds (DBs) are two prevalent co/post-translational modifications (PTMs) that are often conserved and coexist in membrane and secreted proteins involved in a large number of diseases. Both in the past and in recent times, the enzymes and chaperones regulating these PTMs have been constantly discovered to directly interact with each other or colocalize in the ER. However, beyond a few model proteins, how such cooperation affects N-glycan modification and disulfide bonding at selective sites in individual proteins is largely unknown. Here, we reviewed the literature to discover the current status in understanding the relationships between NG and DBs in individual proteins. Our results showed that more than 2700 human proteins carry both PTMs, and fewer than 2% of them have been investigated in the associations between NG and DBs. We summarized both these proteins with the reported relationships in the two PTMs and the tools used to discover the relationships. We hope that, by exposing this largely understudied field, more investigations can be encouraged to unveil the hidden relationships of NG and DBs in the majority of membranes and secreted proteins for pathophysiological understanding and biotherapeutic development.


Assuntos
Chaperonas Moleculares , Processamento de Proteína Pós-Traducional , Dissulfetos/química , Glicosilação , Humanos , Chaperonas Moleculares/metabolismo , Domínios Proteicos
8.
Int J Mol Sci ; 23(7)2022 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-35409419

RESUMO

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design.


Assuntos
Receptores de Antígenos Quiméricos , Animais , Linhagem Celular Tumoral , Cisteína/metabolismo , Dissulfetos/metabolismo , Imunoterapia Adotiva , Camundongos , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
J Agric Food Chem ; 70(14): 4391-4406, 2022 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-35380828

RESUMO

Thermal treatment is often employed in food processing to tailor product properties by manipulating the ingredient functionality, but these elevated temperatures may accelerate oxidation and nutrient loss. Here, oxidation of different whey protein systems [α-lactalbumin (α-LA), ß-lactoglobulin (ß-LG), a mix of α-LA and ß-LG (whey model), and a commercial whey protein isolate (WPI)] was investigated during heat treatment at 60-90 °C and a UHT-like treatment by LC-MS-based proteomic analysis. The relative modification levels of each oxidation site were calculated and compared among different heat treatments and sample systems. Oxidation increased significantly in protein systems after heating at ≥90 °C but decreased in systems with higher complexity [pure protein (α-LA > ß-LG) > whey model > WPI]. In α-LA, Cys, Met, and Trp residues were found to be most prone to oxidation. In ß-LG-containing protein systems, Cys residues were suggested to scavenge most of the reactive oxidants and undergo an oxidation-mediated disulfide rearrangement. The rearranged disulfide bonds contributed to protein aggregation, which was suggested to provide physical protection against oxidation. Overall, limited loss of amino acid residues was detected after acidic hydrolysis followed by UHPLC analysis, which showed only a minor effect of heat treatment on protein oxidation in these protein systems.


Assuntos
Proteínas do Leite , Proteômica , Cromatografia Líquida , Dissulfetos , Temperatura Alta , Lactalbumina/química , Lactoglobulinas/química , Proteínas do Leite/química , Espectrometria de Massas em Tandem , Proteínas do Soro do Leite/análise
10.
Commun Biol ; 5(1): 331, 2022 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-35393494

RESUMO

Cerebral small vessel disease (SVD) is a prevalent disease of aging and a major contributor to stroke and dementia. The most commonly inherited SVD, CADASIL, is caused by dominantly acting cysteine-altering mutations in NOTCH3. These mutations change the number of cysteines from an even to an odd number, but the impact of these alterations on NOTCH3 protein structure remain unclear. Here, we prepared wildtype and four mutant recombinant NOTCH3 protein fragments to analyze the impact of CADASIL mutations on oligomerization, thiol status, and protein stability. Using gel electrophoresis, tandem MS/MS, and collision-induced unfolding, we find that NOTCH3 mutant proteins feature increased amounts of inappropriate disulfide bridges, reduced cysteines, and structural instability. Presence of a second protein factor, an N-terminal fragment of NOTCH3 (NTF), is capable of further altering disulfide statuses of both wildtype and mutant proteins, leading to increased numbers of reduced cysteines and further destabilization of NOTCH3 structure. In sum, these studies identify specific cysteine residues alterations and quaternary structure induced by CADASIL mutations in NOTCH3; further, we validate that reductive factors alter the structure and stability of this small vessel disease protein.


Assuntos
CADASIL , Demência Vascular , Receptor Notch3 , CADASIL/genética , CADASIL/metabolismo , Cisteína/genética , Dissulfetos , Humanos , Proteínas Mutantes , Receptor Notch3/genética , Receptores Notch/metabolismo , Espectrometria de Massas em Tandem
11.
Toxins (Basel) ; 14(4)2022 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-35448841

RESUMO

Disintegrin-like/cysteine-rich (DC) proteins have long been regarded just as products of proteolysis of P-III snake venom metalloproteinases (SVMPs). However, here we demonstrate that a DC protein from the venom of Vipera ammodytes (Vaa; nose-horned viper), VaaMPIII-3, is encoded per se by a P-III SVMP-like gene that has a deletion in the region of the catalytic metalloproteinase domain and in part of the non-catalytic disintegrin-like domain. In this way, we justify the proposal of the introduction of a new subclass P-IIIe of SVMP-derived DC proteins. We purified VaaMPIII-3 from the venom of Vaa in a series of chromatographic steps. A covalent chromatography step based on thiol-disulphide exchange revealed that VaaMPIII-3 contains an unpaired Cys residue. This was demonstrated to be Cys6 in about 90% and Cys19 in about 10% of the VaaMPIII-3 molecules. We further constructed a three-dimensional homology model of VaaMPIII-3. From this model, it is evident that both Cys6 and Cys19 can pair with Cys26, which suggests that the intramolecular thiol-disulphide exchange has a regulatory function. VaaMPIII-3 is an acidic 21-kDa monomeric glycoprotein that exists in at least six N-glycoforms, with isoelectric points ranging from pH 4.5 to 5.1. Consistent with the presence of an integrin-binding motif in its sequence, SECD, VaaMPIII-3 inhibited collagen-induced platelet aggregation. It also inhibited ADP- and arachidonic-acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination and the blood coagulation cascade.


Assuntos
Venenos de Crotalídeos , Desintegrinas , Sequência de Aminoácidos , Cisteína , Desintegrinas/farmacologia , Dissulfetos , Metaloendopeptidases/química , Metaloproteases/química , Venenos de Serpentes/química
12.
In Vivo ; 36(3): 1195-1202, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35478119

RESUMO

BACKGROUND/AIM: Allicin has been known to improve wound healing via antimicrobial and anti-inflammatory properties. The aim of this study was to evaluate whether an allicin-coated tracheal tube can prevent tracheal stenosis through improving wound healing after tracheal injury. MATERIALS AND METHODS: Allicin-coated silicone tracheal tube (t-tube) was prepared by the polydopamine-mediated coating method. Tracheal mucosa was injured, and an allicin-coated t-tube was placed into the trachea to evaluate mucosal changes until designated time point. Anti-inflammatory, anti-bacterial and cytotoxic effects of allicin were also investigated in in vitro. RESULTS: Allicin- coated silicone was not cytotoxic, and it showed anti-inflammatory and anti-bacterial effects in in vitro analysis. The use of allicin-coated t-tube in a rabbit model showed favorable mucosal healing with significant decrease of proinflammatory cytokines compared to the non-coated tube group. The allicin-coated tube showed obvious decreased number of cocci-shaped bacterial attached to the tube surface. From the histological point of view, the allicin- coated tube showed faster regeneration of the normal respiratory epithelial structure compared to the non-coated group. CONCLUSION: Allicin-coated t-tube showed anti-inflammatory and anti-bacterial effects on injured tracheal mucosa. We suggest that allicin-coated t-tube can be used for promoting physiological wound healing to prevent laryngotracheal stenosis.


Assuntos
Traqueia , Estenose Traqueal , Animais , Anti-Inflamatórios/farmacologia , Bactérias , Dissulfetos , Membrana Mucosa , Coelhos , Ácidos Sulfínicos/farmacologia , Estenose Traqueal/prevenção & controle
13.
Carbohydr Polym ; 288: 119407, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35450658

RESUMO

The piezoelectric effect is one of the most promising electromechanical coupling processes for mechanical energy conversion and energy harvesting. However, natural polymer based piezoelectric materials are of poor piezoelectric performance. we developed flexible porous piezoelectric aerogel films based on TEMPO-oxidized cellulose nanofibrils (TOCN) and MoS2 nanosheets. Those aerogel films possessed large specific surface areas and abundant mesopores. Moreover, they exhibited very good piezoelectric properties when a field strength of 20 MV/m was used to polarize MoS2 nanosheets and air in the mesopores. When assembled to piezoelectric nanogenerators (PENGs), a TOCN/MoS2 aerogel film PENG containing 6 wt% of MoS2 exhibited the best output performance. It generated an open circuit voltage of 42 V and a short-circuit current of 1.1 µA, a maximum area power density of 1.29 µW/cm2 and a maximum volume power density of 0.143 µW/cm3. These features enable them to be promising piezoelectric materials for energy harvesting.


Assuntos
Celulose Oxidada , Celulose , Dissulfetos/química , Molibdênio/química , Porosidade
14.
J Mol Model ; 28(5): 129, 2022 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-35469101

RESUMO

Zinc (II), the second most abundant transition metal in blood, binds to the initiator of the contact pathway, factor XII (FXII). This binding induces conformational changes in the structure of FXII eventually leading to its activation. Despite many in vitro and in vivo studies on zinc-mediated activation of FXII, its molecular mechanism remains elusive mainly due to absence of a full-length structural model of FXII. To this end, this study investigated the role of zinc in the structure and dynamics of the full-length structure FXII that was obtained through molecular modeling. We have used four structural templates covering more than 70% of the FXII sequence and the remaining interconnecting regions were built by loop modeling. The resulting full-length structure of FXII contained disordered regions, but in comparison to the AlphaFold (AF) prediction, our full-length model represented a more realistic structure because of the disordered regions which were modeled to yield a more compact full-length structure in our model than the AF structure. Other than the disordered regions, our model and AF prediction were highly similar. The resulting full-length FXII structure was used to generate different systems representing the zinc-bound form (holo). Further to assess the contribution of the disulfide bridges, we also analyzed the apo and holo FXII structures with oxidized or reduced cysteine side-chains. Simulations suggested zinc binding conferred rigidity to the structure, particularly to the light chain of FXII. Zinc binding alone was sufficient to limit the backbone flexibility while 15 disulfide bonds, which were scattered throughout the structure, made a less significant contribution to the backbone rigidity. Altogether our results provide insights into the first realistic full-length structure of FXII focusing on the impact of structural zinc and disulfide bridges in the dynamics of this structure.


Assuntos
Fator XII , Zinco , Dissulfetos , Fator XII/química , Fator XII/metabolismo , Ligação Proteica , Domínios Proteicos
15.
Chem Commun (Camb) ; 58(35): 5359-5362, 2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35394478

RESUMO

Herein we report a fundamental discovery on the use of tris(dialkylamino)phosphine reagents for peptide and protein modification. We discovered that C-terminal thiophosphonium species, which are uniquely stable, could be selectively and rapidly generated from their disulfide counterparts. In sharp and direct contrast, internal thiophosphonium species rapidly degrade to dehydroalanine. We demonstrate this remarkable chemoselectivity on a bis-cysteine model peptide, and the formation of a stable C-terminal-thiophosphonium adduct on an antibody fragment, as well as characterise the species in various small molecule/peptide studies.


Assuntos
Cisteína , Proteínas , Dissulfetos , Peptídeos , Processamento de Proteína Pós-Traducional
16.
Ulus Travma Acil Cerrahi Derg ; 28(4): 403-410, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35485506

RESUMO

BACKGROUND: Acute mesenteric ischemia (AMI) is a rarely observed acute abdominal disease that may be mortal and is difficult to diagnose early. The aim of our study is to assess the role of Thiol-Disulphide Haemostasis (TDH), a new method for AMI which still has no specific biochemical markers for early diagnosis, and to assess it together with Ischemia-Modified Albumin (IMA) which has previously proven reliability for AMI. METHODS: The study included 32 Wistar albino rats in four groups. The 1st group (n=8) was the control group, 2nd group (n=8) was the sham group, 3rd group (n=8) had 3 h of arterial mesentery ischemia and the 4th group (n=8) had 6 h of arterial mesentery ischemia. TDH, IMA, and serum lactate values were measured at h 0, 1, 3, and 6. RESULTS: In the 3rd and 6th h, serum total thiol and native thiol values significantly reduced (p<0.001), while serum disulfide, IMA, and lactate values clearly increased (p<0.001). Serum thiol values were observed to reduce from the 1st h. CONCLUSION: TDH changes in the early period of AMI. The TDH parameters can be used with IMA as diagnostic parameters for patients with suspected AMI in the early period.


Assuntos
Dissulfetos , Isquemia Mesentérica , Biomarcadores , Diagnóstico Precoce , Hemostasia , Humanos , Lactatos , Isquemia Mesentérica/diagnóstico , Mesentério , Estresse Oxidativo , Reprodutibilidade dos Testes , Albumina Sérica , Compostos de Sulfidrila
17.
Nat Commun ; 13(1): 1754, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35365603

RESUMO

The cyclic five-membered disulfide 1,2-dithiolane has been widely used in chemical biology and in redox probes. Contradictory reports have described it either as nonspecifically reduced in cells, or else as a highly specific substrate for thioredoxin reductase (TrxR). Here we show that 1,2-dithiolane probes, such as "TRFS" probes, are nonspecifically reduced by thiol reductants and redox-active proteins, and their cellular performance is barely affected by TrxR inhibition or knockout. Therefore, results of cellular imaging or inhibitor screening using 1,2-dithiolanes should not be interpreted as reflecting TrxR activity, and previous studies may need re-evaluation. To understand 1,2-dithiolanes' complex behaviour, probe localisation, environment-dependent fluorescence, reduction-independent ring-opening polymerisation, and thiol-dependent cellular uptake must all be considered; particular caution is needed when co-applying thiophilic inhibitors. We present a general approach controlling against assay misinterpretation with reducible probes, to ensure future TrxR-targeted designs are robustly evaluated for selectivity, and to better orient future research.


Assuntos
Dissulfetos , Tiorredoxina Dissulfeto Redutase , Dissulfetos/metabolismo , Fluorescência , Oxirredução , Tiorredoxina Dissulfeto Redutase/metabolismo
18.
Protein Expr Purif ; 195-196: 106096, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35460871

RESUMO

Plasmodium vivax ookinete surface protein, Pvs25, is a candidate for a transmission-blocking vaccine (TBV) for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds, a structural feature that makes its recombinant protein expression difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoforms with inappropriate disulfide bonds were found, requiring no further purification step, which is necessary in the case of Pichia pastoris-based expression systems. The Pvs25 from silkworm was confirmed to be molecularly uniform by sodium dodecyl sulfate gel electrophoresis and size-exclusion chromatography. To examine the immunogenicity, the Pvs25 from B. mori was administered to BALB/c mice subcutaneously with oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune responses, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a candidate for a malaria TBV. To the best of our knowledge, this is the first study to construct a system for mass-producing malaria TBV antigens using silkworm.


Assuntos
Bombyx , Vacinas Antimaláricas , Malária Vivax , Animais , Antígenos de Protozoários/genética , Antígenos de Superfície , Bombyx/genética , Dissulfetos , Vacinas Antimaláricas/genética , Malária Vivax/prevenção & controle , Camundongos , Plasmodium vivax/genética
19.
Appl Microbiol Biotechnol ; 106(8): 3093-3102, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35471617

RESUMO

Chaetomium fungi produce a diversity of bioactive compounds. Chaetomium cochliodes SD-280 possesses 91 secondary metabolite gene clusters and exhibits strong antibacterial activity. One of the active compounds responsible for that activity, chetomin, has a minimum inhibitory concentration (MIC) for anti-methicillin-resistant Staphylococcus aureus (MRSA) of 0.05 µg/mL (vancomycin: 0.625 µg/mL). This study demonstrated that the addition of glutathione (GSH) can enhance chetomin yield dramatically, increasing its production 15.43-fold. Following genome sequencing, cluster prediction, and transcriptome and proteome analyses of the fungus were carried out. Furthermore, a relatively complete chetomin biosynthetic gene cluster was proposed, and the coding sequences were acquired. In the cluster of GSH-treated cells, proteome analysis revealed two up-regulated proteins that are critical enzymes for chetomin biosynthesis. One of these enzymes, a nonribosomal peptide synthetase (NRPS), was heterologously expressed in Aspergillus nidulans, and one of its metabolites was determined to be an intermediate in the chetomin biosynthetic pathway. We present here, to our knowledge, the first experimental evidence that chetomin exhibits strong bioactivity against MRSA. Our work also provides extensive insights into the biosynthetic pathway of chetomin, in particular identifying two key enzymes (glutathione S-transferase (CheG) and NRPS (CheP)) that substantially up-regulate chetomin. These mechanistic insights into chetomin biosynthesis will provide the foundation for further investigation into the anti-pathogenic properties and applications of chetomin. KEY POINTS: • Chetomin exhibits strong anti-MRSA activity with MIC of 0.05 µg/mL. • Addition of glutathione improved the yield of chetomin by 15.43-fold. • CheG and CheP involved in the chetomin biosynthesis were revealed for the first time.


Assuntos
Vias Biossintéticas , Staphylococcus aureus Resistente à Meticilina , Antibacterianos/química , Antibacterianos/farmacologia , Vias Biossintéticas/genética , Dissulfetos , Glutationa , Alcaloides Indólicos , Chumbo , Proteoma
20.
Protein Sci ; 31(5): e4289, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35481637

RESUMO

The Plasmodium ookinete uses chitinase activity to penetrate the acellular, chitin-containing peritrophic matrix to invade the mosquito vector. Plasmodium ookinetes from different parasite clades secrete two structurally distinct forms of chitinase, one, a short form lacking a C-terminal putative chitin-binding domain (CBD), the other, a long form with both proenzyme and C-terminal putative chitin-binding domains. Here, we structurally and functionally characterize the three cysteines in the short chitinase of the human-infecting malaria parasite, P. falciparum testing the hypothesis that one unpaired cysteine would not contribute to chitinase-specific enzymatic activity which would identify this residue as potentially involved in intermolecular disulfide bonding and heteromultimeric invasion complex formation as previously described. To test this hypothesis, we produced and characterized recombinant wild-type and cysteine-mutation PfCHT1 proteins in E. coli and used biophysical and enzymatic approaches to examine their enzymatic activities and chitin-binding affinities. The cysteine-203 PfCHT1 mutation had no effect on chitinolytic and chitin-binding functions. The cysteine-220 and cysteine-230 mutants were enzymatically inactive and did not bind to chitin. The artificial intelligence-based protein prediction algorithm, AlphaFold, correctly identified the involvement of cys-220 and cys-230 in the intramolecular disulfide linkages key to maintaining properly folded chitinase structural integrity. AlphaFold predicted that cys-203 cysteine is surface exposed and thus involved in intermolecular protein-protein interaction. Production of the cys-to-ser 203 PfCHT1 mutant facilitated recombinant protein production. Future cellular and biochemical studies are needed to further understand details of Plasmodium ookinete mosquito midgut invasion.


Assuntos
Quitinases , Plasmodium falciparum , Animais , Inteligência Artificial , Quitina/metabolismo , Quitinases/química , Cisteína/genética , Dissulfetos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Mosquitos Vetores , Plasmodium falciparum/genética , Proteínas de Protozoários
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