Protein and Peptide Letters最新文献

Background: Sevoflurane (Sev) is a type of volatile anesthetic commonly used in clinic practices and can initiate long-term neurotoxicity, while dexmedetomidine (Dex) possesses a neuroprotective function in multiple neurological disorders.

Objective: This work expounded on the function of Dex pretreatment in Sev-initiated neurotoxicity.

Methods: At first, human neuroblastoma cells (SK-N-SH cells) were treated with different concentrations of Sev or Dex, followed by the cell counting kit (CCK)-8 assay to decide the appropriate concentrations of Sev or Dex. Cell viability, lactate dehydrogenase (LDH) productions, and apoptotic rate of SK-N-SH cells were examined by the CCK-8 assay, LDH cytotoxicity kit, and flow cytometry assay in sequence. Further, reactive oxygen species (ROS) levels and proinflammatory cytokine contents were examined by the ROS assay kit and the enzyme-linked immunosorbent assay kits. The expression patterns of microRNA (miR)-204-5p and SRY-box transcription factor 4 (SOX4) in SK-N-SH cells were measured by real-time quantitative polymerase chain reaction or Western blotting. The binding relationship between miR-204-5p and SOX4 was confirmed by the dual-luciferase assay. After transfection of miR-204-5p mimics or SOX4 siRNA, the role of the miR-204-5p/SOX4 axis in Sev-initiated neurotoxicity was detected.

Results: Sev treatment reduced SK-N-SH cell viability in a concentration-dependent manner, and Dex pretreatment diminished Sev-initiated neurotoxicity. Mechanically, Dex pretreatment limited Sevinduced upregulation of miR-204-5p and further increased SOX4 expression levels. miR-204-5p upregulation or SOX4 knockdown averted the neuroprotection function of Dex pretreatment in Sevinitiated neurotoxicity.

Conclusion: Dex pretreatment decreased miR-204-5p expression levels and upregulated SOX4 expression levels, palliating Sev-initiated neurotoxicity.

Background: Glutathionylation is a protein post-translational modification triggered by oxidative stress. The susceptible proteins are modified by the addition of glutathione to specific cysteine residues. Virus infection also induces oxidative stress in the cell, which affects cellular homeostasis. It is not just the cellular proteins but the viral proteins that can also be modified by glutathionylation events, thereby impacting the function of the viral proteins.

Objectives: This study was conducted to identify the effects of modification by glutathionylation on the guanylyltransferase activity of NS5 and identify the cysteine residues modified for the three flavivirus NS5 proteins.

Methods: The capping domain of NS5 proteins from 3 flaviviruses was cloned and expressed as recombinant proteins. A gel-based assay for guanylyltransferase activity was performed using a GTP analog labeled with the fluorescent dye Cy5 as substrate. The protein modification by glutathionylation was induced by GSSG and evaluated by western blot. The reactive cysteine residues were identified by mass spectrometry.

Results: It was found that the three flavivirus proteins behaved in a similar fashion with increasing glutathionylation yielding decreased guanylyltransferase activity. The three proteins also possessed conserved cysteines and they appeared to be modified for all three proteins.

Conclusion: The glutathionylation appeared to induce conformational changes that affect enzyme activity. The conformational changes might also create binding sites for host cell protein interactions at later stages of viral propagation with the glutathionylation event, thereby serving as a switch for function change.

Introduction: Effective T-cell-mediated immunity has emerged as an essential component of human immunodeficiency virus-1 (HIV-1) vaccination. Thus, inducing an immune response against HIV proteins such as Nef and Vif, two major accessory proteins with critical roles in HIV pathogenesis and immune evasion, may lead to an effective approach.

Aim: Our goal is to evaluate and compare Montanide ISA-720 and heat shock protein 27 in increasing immunostimulatory properties of HIV-1 Nef-Vif fusion protein as a vaccine candidate.

Methods: In this study, the nef-vif fusion gene with and without the heat shock protein 27 (hsp27) gene was cloned in the prokaryotic pET24a (+) vector. Then, the recombinant Nef-Vif and Hsp27-Nef- Vif proteins were generated in the E. coli system. Finally, their immunostimulatory properties were evaluated in mice. Indeed, the potency of Hsp27 as an endogenous natural adjuvant was investigated to enhance HIV-1 Nef-Vif antigen-specific immunity compared to Montanide ISA-720 as a commercial adjuvant in protein-based immunization strategy.

Results: Our results approved the role of Hsp27 as an effective adjuvant in the stimulation of B- and T-cell immunity. The linkage of Hsp27 to antigen could elicit higher levels of IgG1, IgG2a, IFN-γ, IL- 5 and Granzyme B than antigen mixed with Montanide ISA-720. Moreover, the ratios of IFN-γ/IL-5 and IgG2a/IgG1 were significantly increased in groups receiving Nef-Vif protein + Montanide ISA- 720 and Hsp27-Nef-Vif protein indicating the direction of the immune response pathway toward strong Th1 response. These ratios were higher in the group receiving Hsp27-Nef-Vif protein than in the group receiving Nef-Vif protein + Montanide ISA-720.

Conclusion: Our findings suggest that Hsp27 can be used as an effective adjuvant to enhance antigenspecific immune responses in HIV-1 infectious models for therapeutic vaccine development.

Background: It has been reported that activation of glutamate kainate receptor subunit 2 (GluK2) subunit-containing glutamate receptors and the following Fas ligand(FasL) up-regulation, caspase-3 activation, result in delayed apoptosis-like neuronal death in hippocampus CA1 subfield after cerebral ischemia and reperfusion. Nitric oxide-mediated S-nitrosylation might inhibit the procaspase activation, whereas denitrosylation might contribute to cleavage and activation of procaspases.

Objectives: The study aimed to elucidate the molecular mechanisms underlying procaspase-3 denitrosylation and activation following kainic acid (KA)-induced excitotoxicity in rat hippocampus.

Methods: S-nitrosylation of procaspase-3 was detected by biotin-switch method. Activation of procaspase-3 was shown as cleavage of procaspase-3 detected by immunoblotting. FasL expression was detected by immunoblotting. Cresyl violets and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were used to detect apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields.

Results: KA led to the activation of procaspase-3 in a dose- and time-dependent manner, and the activation was inhibited by KA receptor antagonist NS102. Procaspase-3 was denitrosylated at 3 h after kainic acid administration, and the denitrosylation was reversed by SNP and GSNO. FasL ASODNs inhibited the procaspase-3 denitrosylation and activation. Moreover, thioredoxin reductase (TrxR) inhibitor auranofin prevented the denitrosylation and activation of procaspase-3 in rat hippocampal CA1 and CA3 subfields. NS102, FasL AS-ODNs, and auranofin reversed the KAinduced apoptosis and cell death in hippocampal CA1 and CA3 subfields.

Conclusions: KA led to denitrosylation and activation of procaspase-3 via FasL and TrxR. Inhibition of procaspase-3 denitrosylation by auranofin, SNP, and GSNO played protective effects against KA-induced apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. These investigations revealed that the procaspase-3 undergoes an initial denitrosylation process before becoming activated, providing valuable insights into the underlying mechanisms and possible treatment of excitotoxicity.

Background: UDP-glucuronosyltransferases (UGTs) play a crucial role in maintaining endobiotic homeostasis and metabolizing xenobiotic compounds, particularly clinical drugs. However, the detailed catalytic mechanism of UGTs has not been fully elucidated due to the limited availability of reliable protein structures. Determining the catalytic domain of human UGTs has proven to be a significant challenge, primarily due to the difficulty in purifying and crystallizing the full-length protein.

Objectives: This study focused on the human UGT2B10 C-terminal cofactor binding domain, aiming to provide structural insights into the fundamental catalytic mechanisms.

Methods: In this study, the C-terminal sugar-donor binding domain of human UGT2B10 was purified and crystallized using the vapor-diffusion method. The resulting UGT2B10 CTD crystals displayed high-quality diffraction patterns, allowing for data collection at an impressive resolution of 1.53 Å using synchrotron radiation. Subsequently, the structure of the UGT2B10 CTD was determined using the molecule replacement method with a homologous structure.

Results: The crystals were monoclinic, belonging to the space C2 with unit-cell parameters a = 85.90 Å, b = 58.39 Å, c = 68.87 Å, α = γ = 90°, and β = 98.138°. The Matthews coefficient V_M was determined to be 2.24 ų Da^-1 (solvent content 46.43%) with two molecules in the asymmetric unit.

Conclusion: The crystal structure of UGT2B10 CTD was solved at a high resolution of 1.53 Å, revealing a conserved cofactor binding pocket. This is the first study determining the C-terminal cofactor binding domain of human UGT2B10, which plays a key role in additive drug metabolism.

Background: STAM-binding protein-like 1 (STAMBPL1) functions as a deubiquitinase to cleave Lys63 ubiquitin linkage, and is associated with cancer dissemination and progression. The role of STAMBPL1 in colorectal cancer (CRC) remains unclear.

Methods: STAMBPL1 expression was determined by western blot and qRT-PCR. Cell proliferation was detected by colony formation and MTT assays, and apoptosis was assessed by flow cytometry. The metastasis was evaluated by transwell and wound healing assays. An animal xenograft experiment was used to investigate the effect of STAMBPL1 on tumor growth.

Results: The expression of STAMBPL1 was elevated in CRC cells. Knockdown of STAMBPL1 reduced cell viability of CRC and suppressed the proliferation, invasion, and migration. Apoptosis of CRC was induced by silence of STAMBPL1. Tumor growth of CRC was also suppressed by the silence of STAMBPL1. Knockdown of STAMBPL1 increased IκB and decreased phosphorylation of IκB to reduce p65 phosphorylation.

Conclusion: Knockdown of STAMBPL1 inhibited cell growth and metastasis of CRC through inactivation of the NF-κB pathway.

Objectives: The present study investigated the anti-depressive-like (anti-immobility) effect of a lectin from Moringa oleifera seeds (WSMoL) in mice.

Methods: To evaluate an acute effect, the animals were treated with WSMoL (1, 2, and 4 mg/kg, i.p.) 30 min before the tail suspension test (TST). To investigate the involvement of monoaminergic and nitrergic signaling, the mice were pre-treated with selective antagonists. The role of the WSMoL carbohydrate-recognizing domain (CRD) was verified using previous blockage with casein (0.5 mg/mL). The subacute anti-immobility effect was also evaluated by administering WSMoL (1, 2, and 4 mg/kg, i.p.) once a day for 7 d. Finally, an open field test (OFT) was performed to identify possible interferences of WSMoL on animal locomotory behavior.

Results: WSMoL reduced the immobility time of mice in the TST at all doses, and combined treatment with fluoxetine (5 mg/kg, i.p.) and WSMoL (1 mg/kg) was also effective. The CRD appeared to be involved in the anti-immobility effect since the solution of WSMoL (4 mg/kg) pre-incubated with casein showed no activity. The lectin effect was prevented by the pre-treatment of mice with ketanserin, yohimbine, and SCH 23390, thereby demonstrating the involvement of monoaminergic pathways. In contrast, pre-treatment with L-NAME, aminoguanidine, and L-arginine did not interfere with lectin action. WSMoL exhibited a subacute effect in the TST, thereby reducing immobility time and increasing agitation time even on the seventh day. OFT data revealed that the anti-immobility effect was not caused by interference with locomotor behavior.

Conclusion: WSMoL elicits an anti-depressant-like effect that is dependent on monoaminergic signaling.

Background: Gastric cancer (GC) is a malignant tumor with seriously poor outcomes. Studies have shown that microRNAs (miRNAs) play an omnifarious regulatory effect in GC. However, the role of miR-3650 in the progression of GC is not well known.

Methods: In this study, miR-3650 expression and its clinical significance were determined using clinical specimens. The biological functions of miR-3650 were determined in gastric cancer cell lines through CCK-8, cell scratch, and transwell experiments. Bioinformatics predictions, combined with Western blot experiments, were employed to explore its downstream molecular targets.

Results: We observed that miR-3650 was overexpressed in GC specimens and most cell lines, i.e., 77.8% (MKN28, SNU1, AGS, MKN45, N87, BGC823 and SGC7901). The overexpression correlated with advanced T-stage, N-stage, M-stage, and TNM-stage. Furthermore, miR-3650 promoted the proliferation and migration of gastric cancer cells, and its overexpression promoted the PI3K-AKT-mTOR pathway and inhibited the PTEN and hippo pathways. The potassium ion signaling pathway was also involved in the biological process of miR-3650 promoting cancer.

Conclusion: Therefore, we concluded that miR-3650/PTEN/PI3K-AKT-mTOR and miR-3650/hippo pathways are vital in the progression of GC and serve as novel targets for GC therapy.

Background: α-Synuclein, a natively disordered protein, is a key component of Lewy bodies, the ubiquitinated protein aggregates which are the pathological hallmark of Parkinson's disease (PD). Meclofenoxate (centrophenoxine) is a nootropic drug which has shown beneficial therapeutic effects in various neuronal diseases. Administration of meclofenoxate enhanced levels of dopamine and improved motor function in animal models of Parkinson's disease (PD). Evidence suggested that dopamine interacts with and modulates α-synuclein aggregation.

Objective: The aim of this work was to investigate whether the observed positive effect of addition of meclofenoxate, a nootropic agent, on dopamine level, could be correlated with its effect on aggregation of α-synuclein.

Methods: Purification of recombinant human α-synuclein was performed by anion exchange chromatography. The purified protein was incubated in the absence and presence of meclofenoxate and was analyzed for aggregation by Thioflavin T fluorescence spectroscopy. Conformational changes in α-synuclein were monitored by fluorescence spectroscopy and fluorescence quenching studies using a neutral quencher. Secondary structure analysis of α-synuclein was monitored by circular dichroism spectroscopy.

Results: Recombinant human α-synuclein was expressed and purified by anion-exchange chromatography. Incubation of α-synuclein with meclofenoxate led to lowering aggregation in a concentration-dependent manner. Reduction in formation of oligomers was seen which suggested the formation of an off-pathway species which did not give rise to an aggregation-competent entity. Fluorescence quenching studies revealed that the additive distorted the native conformation of α- synuclein, leading to the formation of lower amounts of aggregation-prone species.

Conclusion: In the presence of higher concentrations of meclofenoxate, α-synuclein undergoes a change in its conformation. This change is not dependent on the concentration of the additive. This non-native conformer promotes the formation of a species which does not undergo further aggregation. Our study provides a mechanistic explanation of the earlier observation that meclofenoxate has a beneficial effect on progression of PD in animal models.

Background: Bromelain is a complex mixture of protease enzyme extract from the fruit or stem of the pineapple plant and it has a history of folk medicine use. It is known to have a wide range of biological actions and it is most commonly used as an anti-inflammatory agent, though scientists have also discovered its potential as an anticancer and antimicrobial agent, it has been reported to have positive effects on the respiratory, digestive, circulatory systems and potentially on the immune system.

Objective: This study was designed to investigate the antidepressant potential of Bromelain in the chronic unpredictable stress (CUS) model of depression.

Methods: We studied the antioxidant activity, and neuroprotective effect of Bromelain by analyzing the fear and anxiety behavior, antioxidants, and neurotransmitter levels, and also by analyzing the histopathological changes. Adult male Wistar albino rats were divided into 5 groups, Control; Bromelain; CUS; CUS + Bromelain, CUS + fluoxetine. Animals of the CUS group, CUS + Bromelain group, and CUS + Fluoxetine group were exposed to CUS for 30 days. Animals of the Bromelain group and CUS + Bromelain group were treated orally with 40 mg/kg Bromelain throughout the period of CUS whereas, the positive control group was treated with fluoxetine.

Results: Results showed a significant decrease in oxidative stress marker (lipid peroxidation), and the stress hormone cortisol, in Bromelain-treated CUS-induced depression. Bromelain treatment in CUS has also resulted in a significant increase in neurotransmitter levels, which indicates the efficacy of Bromelain to counteract the monamine neurotransmitter changes in depression by increasing their synthesis and reducing their metabolism. In addition, the antioxidant activity of Bromelain prevented oxidative stress in depressed rats. Also, hematoxylin and eosin staining of hippocampus sections has revealed that Bromelain treatment has protected the degeneration of nerve cells by chronic unpredictable stress exposure.

Conclusion: This data provides evidence for the antidepressant-like action of Bromelain by preventing neurobehavioral, biochemical, and monoamine alterations.