scholarly journals Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 922
Author(s):  
Gaurav Sinsinbar ◽  
Sushanth Gudlur ◽  
Kevin J Metcalf ◽  
Milan Mrksich ◽  
Madhavan Nallani ◽  
...  
Keyword(s):  
Defense Mechanism ◽  
Protective Role ◽  
Binding Motif ◽  
Host Immune System ◽  
E Coli ◽  
Sds Page ◽  
Lps Binding

Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

scholarly journals Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jun-Xian Du ◽  
Yi-Hong Luo ◽  
Si-Jia Zhang ◽  
Biao Wang ◽  
Cong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
High Risk Group ◽  
Clinical Samples ◽  
Binding Motif ◽  
Ki 67 ◽  
Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

scholarly journals A Protective Role of Paeoniflorin in Fluctuant Hyperglycemia-Induced Vascular Endothelial Injuries through Antioxidative and Anti-Inflammatory Effects and Reduction of PKCβ1

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Jing-Shang Wang ◽  
Ye Huang ◽  
Shuping Zhang ◽  
Hui-Jun Yin ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Level ◽  
Umbilical Vein ◽  
Protective Role ◽  
Vascular Endothelial ◽  
Glucose Levels ◽  
Umbilical Vein Endothelial Cells

Hyperglycemia fluctuation is associated with diabetes mellitus (DM) complications when compared to persistent hyperglycemia. Previous studies have shown that paeoniflorin (PF), through its antiapoptosis, anti-inflammation, and antithrombotic properties, effectively protects against cardiovascular and cerebrovascular disease. However, the mechanism underlying the protection from PF against vascular injuries induced by hyperglycemia fluctuations remains poorly understood. Herein, we investigated the potential protective role of PF on human umbilical vein endothelial cells (HUVECs) subjected to intermittent glucose levels in vitro and in DM rats with fluctuating hyperglycemia in vivo. A remarkable increased apoptosis associated with elevated inflammation, increased oxidative stress, and high protein level of PKCβ1 was induced in HUVECs by intermittently changing glucose for 8 days, and PF recovered those detrimental changes. LY333531, a potent PKCβ1 inhibitor, and metformin manifested similar effects. Additionally, in DM rats with fluctuating hyperglycemia, PF protected against vascular damage as what has been observed in vitro. Taken together, PF attenuates the vascular injury induced by fluctuant hyperglycemia through oxidative stress inhibition, inflammatory reaction reduction, and PKCβ1 protein level repression, suggesting its perspective clinical usage.

scholarly journals HDAC2 attenuates airway inflammation by suppressing IL-17A production in HDM-challenged mice

2019 ◽  
Vol 316 (1) ◽  
pp. L269-L279 ◽  
Author(s):  
Tianwen Lai ◽  
Mindan Wu ◽  
Chao Zhang ◽  
Luanqing Che ◽  
Feng Xu ◽  
...  
Keyword(s):  
Airway Inflammation ◽  
Inflammatory Cytokines ◽  
Allergic Inflammation ◽  
Allergic Airway Inflammation ◽  
Inflammatory Cells ◽  
Protective Role ◽  
In Vivo Models

Histone deacetylase (HDAC)2 is expressed in airway epithelium and plays a pivotal role in inflammatory cells. However, the role of HDAC2 in allergic airway inflammation remains poorly understood. In the present study, we determined the role of HDAC2 in airway inflammation using in vivo models of house dust mite (HDM)-induced allergic inflammation and in vitro cultures of human bronchial epithelial (HBE) cells exposed to HDM, IL-17A, or both. We observed that HDM-challenged Hdac2+/− mice exhibited substantially enhanced infiltration of inflammatory cells. Higher levels of T helper 2 cytokines and IL-17A expression were found in lung tissues of HDM-challenged Hdac2+/− mice. Interestingly, IL-17A deletion or anti-IL-17A treatment reversed the enhanced airway inflammation induced by HDAC2 impairment. In vitro, HDM and IL-17A synergistically decreased HDAC2 expression in HBE cells. HDAC2 gene silencing further enhanced HDM- and/or IL-17A-induced inflammatory cytokines in HBE cells. HDAC2 overexpresion or blocking IL-17A gene expression restored the enhanced inflammatory cytokines. Collectively, these results support a protective role of HDAC2 in HDM-induced airway inflammation by suppressing IL-17A production and might suggest that activation of HDAC2 and/or inhibition of IL-17A production could prevent the development of allergic airway inflammation.

scholarly journals Regulatory Role of PlaR (YiaJ) for Plant Utilization in Escherichia coli K-12

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tomohiro Shimada ◽  
Yui Yokoyama ◽  
Takumi Anzai ◽  
Kaneyoshi Yamamoto ◽  
Akira Ishihama
Keyword(s):  
Escherichia Coli ◽  
Genetic System ◽  
Regulatory Role ◽  
E Coli ◽  
Warm Blooded Animal ◽  
Plant Utilization ◽  
K 12

AbstractOutside a warm-blooded animal host, the enterobacterium Escherichia coli K-12 is also able to grow and survive in stressful nature. The major organic substance in nature is plant, but the genetic system of E. coli how to utilize plant-derived materials as nutrients is poorly understood. Here we describe the set of regulatory targets for uncharacterized IclR-family transcription factor YiaJ on the E. coli genome, using gSELEX screening system. Among a total of 18 high-affinity binding targets of YiaJ, the major regulatory target was identified to be the yiaLMNOPQRS operon for utilization of ascorbate from fruits and galacturonate from plant pectin. The targets of YiaJ also include the genes involved in the utilization for other plant-derived materials as nutrients such as fructose, sorbitol, glycerol and fructoselysine. Detailed in vitro and in vivo analyses suggest that L-ascorbate and α-D-galacturonate are the effector ligands for regulation of YiaJ function. These findings altogether indicate that YiaJ plays a major regulatory role in expression of a set of the genes for the utilization of plant-derived materials as nutrients for survival. PlaR was also suggested to play protecting roles of E. coli under stressful environments in nature, including the formation of biofilm. We then propose renaming YiaJ to PlaR (regulator of plant utilization).

scholarly journals TheEscherichia coli-Derived Thymosinβ4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Xiaolei Wang ◽  
Guihua Yang ◽  
Shanshuang Li ◽  
Meifeng Gao ◽  
Pangfeng Zhao ◽  
...  
Keyword(s):  
Spleen Cells ◽  
New Approach ◽  
E Coli ◽  
Sds Page ◽  
Strain Bl21 ◽  
Healing Wound ◽  
Production Strategy ◽  
Better Than

Thymosinβ4 (Tβ4) is one of the most promising thymosins for future clinical applications, and it is anticipated that commercial demand for Tβ4 will increase. In order to develop a new approach to produce recombinant Tβ4, a 168 bp DNA (termedTβ4) was designed based on the Tβ4 protein sequence and used to express a 4 ×Tβ4 concatemer (four tandem copies of Tβ4, termed 4 ×Tβ4) together with a histidine tag (6 × His) inE. coli(strain BL21). SDS-PAGE and western blot analysis were used to confirm that a recombinant 4 × Tβ4 protein of the expected size (30.87 kDa) was produced following the induction of the bacterial cultures with isopropylβ-D-thiogalactoside (IPTG). TheE. coli-derived 4 ×Tβ4 was purified by Ni-NTA resin, and its activities were examined with regard to both stimulating proliferation of the mice spleen cellsin vitroandin vivowound healing. The results demonstrate that these activities of theE. coli-derived recombinant 4 × Tβ4 were similar or even better than existing commercially obtained Tβ4. This production strategy therefore represents a potentially valuable approach for future commercial production of recombinant Tβ4.

scholarly journals In Vivo Effect of Mutations at the PRPP Binding Site of the Bacillus subtilis Purine Repressor

2003 ◽  
Vol 185 (22) ◽  
pp. 6728-6731 ◽  
Author(s):  
Pekka Rappu ◽  
Terhi Pullinen ◽  
Pekka Mäntsälä
Keyword(s):  
Bacillus Subtilis ◽  
Binding Site ◽  
Binding Motif

ABSTRACT The Bacillus subtilis PurR mediates adenine repression and guanosine induction of purA. PRPP inhibits binding of PurR to DNA in vitro. Mutations in the PRPP binding motif of PurR caused strong repression regardless of purine exclusions or additions, establishing the role of PRPP as regulator of PurR.

scholarly journals Role of interleukin-1 and tumour necrosis factor in leukocyte recruitment to acute dermal inflammation

1992 ◽  
Vol 1 (5) ◽  
pp. 347-353 ◽  
Author(s):  
Andrew C. Issekutz ◽  
Nancy Lopes ◽  
Thomas B. Issekutz
Keyword(s):  
Monoclonal Antibody ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
E Coli ◽  
Tnf Α ◽  
Lps Activation ◽  
Necrosis Factor

The cytokines IL-1 and TNF-α are involved in inflammation and their production is stimulated by various agents, especially endotoxin (LPS). Here, using the human IL-1 receptor antagonist (IL-1RA) and a new monoclonal antibody (mAb 7F11) to rabbit TNF, the role of endogenous IL-l and TNF production in acute (3h) leukocyte (PMNL) recruitment to dermal inflammation in rabbits has been studied. IL-1RA inhibited by 27% the PMNL accumulation in reactions induced by killed Escherichia coli (p < 0.05) but not by LPS. The monoclonal antibody to TNF inhibited by 27% and 38% (p < 0.002) the PMNL accumulation in LPS and E. coli reactions respectively, but a combination of the mAb with IL-1RA was not more effective. Treatment of human umbilical vein endothelium with LPS for 3 h activated endothelium to induce PMNL transendothelial migration in vitro, which was not inhibited by IL-1RA, antibody to TNF-α, IL-1 or to IL-8. In conclusion, TNF and IL-1 may partially mediate acute PMNL infiltration in vivo to LPS and Gram negative bacteria, but there is a major IL-1/TNF independent mechanism, at least in dermal inflammation, which may be due to direct LPS activation of the microvasculature or perhaps the generation of cytokines other than IL-1 and TNF.

scholarly journals Role of Escherichia coli Nitrogen Regulatory Genes in the Nitrogen Response of the Azotobacter vinelandiiNifL-NifA Complex

2001 ◽  
Vol 183 (10) ◽  
pp. 3076-3082 ◽  
Author(s):  
Francisca Reyes-Ramirez ◽  
Richard Little ◽  
Ray Dixon
Keyword(s):  
Escherichia Coli ◽  
Signal Transduction ◽  
Nitrogen Control ◽  
Fixed Nitrogen ◽  
Nitrogen Response ◽  
E Coli ◽  
Nitrogen Excess

ABSTRACT The redox-sensing flavoprotein NifL inhibits the activity of the nitrogen fixation (nif)-specific transcriptional activator NifA in Azotobacter vinelandii in response to molecular oxygen and fixed nitrogen. Although the mechanism whereby the A. vinelandii NifL-NifA system responds to fixed nitrogen in vivo is unknown, the glnK gene, which encodes a PII-like signal transduction protein, has been implicated in nitrogen control. However, the precise function of A. vinelandii glnK in this response is difficult to establish because of the essential nature of this gene. We have shown previously that A. vinelandii NifL is able to respond to fixed nitrogen to control NifA activity when expressed inEscherichia coli. In this study, we investigated the role of the E. coli PII-like signal transduction proteins in nitrogen control of the A. vinelandii NifL-NifA regulatory system in vivo. In contrast to recent findings with Klebsiella pneumoniae NifL, our results indicate that neither the E. coli PII nor GlnK protein is required to relieve inhibition byA. vinelandii NifL under nitrogen-limiting conditions. Moreover, disruption of both the E. coli glnB andntrC genes resulted in a complete loss of nitrogen regulation of NifA activity by NifL. We observe that glnB ntrC and glnB glnK ntrC mutant strains accumulate high levels of intracellular 2-oxoglutarate under conditions of nitrogen excess. These findings are in accord with our recent in vitro observations (R. Little, F. Reyes-Ramirez, Y. Zhang, W. Van Heeswijk, and R. Dixon, EMBO J. 19:6041–6050, 2000) and suggest a model in which nitrogen control of the A. vinelandii NifL-NifA system is achieved through the response to the level of 2-oxoglutarate and an interaction with PII-like proteins under conditions of nitrogen excess.

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