Protein and Peptide Letters最新文献

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Necrotrophic phytopathogens pose a serious challenge to the productivity of several crops causing seedling damage, pre- and post-emergence damping-off and root rot thus reducing plant growth and yield. They are known to gain nutrition by secreting a diverse array of hydrolytic enzymes and thereby causing extensive host plant tissue maceration. Amongst the diverse hydrolases, proteases play a pivotal role in the necrotrophic mode of nutrition and thereby in determining pathogenic virulence. Host plants often counteract the necrotrophic proteolysis events by proteins (peptides), particularly through protease inhibitors (PIs). PIs play an important role in host innate immunity function by functioning as anti-metabolic proteins inhibiting the activity of phytopathogenic secretory proteases. Their abundance in plant storage organs explains their anti-nutritional interaction which stalls pathogenic invasion. PIs, therefore, constitute potential candidates that can be deployed as effective antimicrobials in agriculture, particularly against necrotrophic soil-borne pathogens. The present review traces the progress made in the identification of PIs from plants, and their inhibitory potential against necrotrophic phytopathogens and explores prospects of utilizing these molecules as effective anti-necrotrophic formulations for disease management.

Cold-induced RNA-binding protein (CIRP) and RNA-binding motif protein 3 (RBM3) have recently been reported to be involved in cold stress in mammals. These proteins are expressed at low levels in various normal cells, tissues, and organs but can be upregulated upon stimulation by multiple stressors. Studies have shown that CIRP and RBM3 are multifunctional RNA molecular chaperones with different biological functions in various physiological and pathophysiological processes, such as reproductive development, the inflammatory response, the immune response, nerve injury regulation, and tumorigenesis. This paper reviews recent studies on the structure, localization and correlation of CIRP and RBM3 with reproductive development and reproductive system diseases.

The high global burden of tuberculosis (TB) and the increasing emergence of the drugresistant (DR) strain of Mycobacterium tuberculosis (Mtb) emphasize the urgent need for novel antimycobacterial agents. Antimicrobial peptides (AMPs) are small peptides widely existing in a variety of organisms and usually have amphiphilic cationic structures, which have a selective affinity to the negatively charged bacterial cell wall. Besides direct bactericidal mechanisms, including interacting with the bacterial cell membrane and interfering with the biosynthesis of the cell wall, DNA, or protein, some AMPs are involved in the host's innate immunity. AMPs are promising alternative or complementary agents for the treatment of DR-TB, given their various antibacterial mechanisms and low cytotoxicity. A large number of AMPs, synthetic or natural, from human to bacteriophage sources, have displayed potent anti-mycobacterial activity in vitro and in vivo. In this review, we summarized the features, antimycobacterial activity, and mechanisms of action of the AMPs according to their sources. Although AMPs have not yet met the expectations for clinical application due to their low bioavailabilities, high cost, and difficulties in large-scale production, their potent antimycobacterial activity and action mechanisms, which are different from conventional antibiotics, make them promising antibacterial agents against DR-Mtb in the future.

Introduction: Sensitive methods are necessary to identify the residual structure in an unfolded protein, which may be similar to the functionally native structure. Signal intensity in NMR experiments is useful for analyzing the line width for a dynamic structure; however, another contribution is contained.

Methods: Here, the signal-intensity difference along the sequence was used for probability to calculate the standard deviation.

Results: The relative values of the standard deviations were 0.57, 0.57, and 0.66 for alpha-synuclein wild-type, A53T, and A30P, respectively. This revealed that the flexible region was mainly in the Cterminal region of alpha-synuclein at higher temperatures as observed by the amide-proton exchange studies.

Conclusion: In particular, the flexible structure was induced by the A30P mutation.

Background: The CRISPR-Cas system is an adaptive immune mechanism for bacteria and archaea to resist foreign invasion. Currently, Cas9 and Cpf1 have been widely studied and applied in gene editing. C2c1 is a newly discovered CRISPR-Cas system endonuclease. It has broad application prospects due to its small molecular weight and high substrate recognition specificity.

Objectives: Bacillus thermoamylovorans C2c1(BthC2c1) was expressed in E. coli C43 (DE3) competent cells, purified, and the BthC2c1-sgRNA-dsDNA complex was assembled. The effect of temperature on the cleavage ability of the BthC2c1 system was investigated.

Methods: The cDNA of BthC2c1 was cloned into the vector pGEX-6P-1. BthC2c1 was expressed in E. coli C43(DE3) cells and purified using a GST affinity column and FPLC. The sgRNAs were transcribed and purified in vitro, and the complexes were assembled by gel filtration chromatography. The enzyme cleavage activity of BthC2c1 at different temperatures was investigated using an in vitro cleavage assay. Microscale Thermophoresis detected the affinity of the BthC2c1-sgRNA complexes to substrate DNA.

Results: BthC2c1 proteins were prokaryotically expressed and purified. The complex of BthC2c1 with sgRNA and dsDNA was assembled. In vitro cleavage assay results showed that BthC2c1 cleaved the target DNA at temperatures ranging from 37°C to 67°C. The cleavage ability of BthC2c1 at 42^oC was stronger than that at 37^oC. The results of affinity detection showed that the affinity between the BthC2c1-sgRNA complex and ds36/36 at 42^oC was stronger than that at 37^oC.

Conclusion: In this study, BthC2c1 was expressed, purified, and assembled into a complex with sgRNA and dsDNA. BthC2c1 cleaved DNA within the temperature range of 37^oC to 67^oC. The affinity of BthC2c1-sgRNA to DNA at 42°C was significantly enhanced than that at 37°C. It may be related to its stringent substrate recognition pattern, which differs from Cas9 and Cpf1. The temperature-dependent affinity changes of substrate binding may be part of the reason for the stronger cleavage activity of BthC2c1 at 42^oC. This study may provide an experimental basis for optimizing and modifying the C2c1 gene editing system.

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.

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.

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.