Most folding studies on proteins and nucleic acids have been addressed to the transition between the folded and unfolded states of an intact molecule, where an entire residue sequence is present during the folding event. However, since these polymers are synthesized sequentially from one terminus to the other in vivo, their folding pathways may be influenced greatly by the sequential appearance of the residues as a function of time. The three-dimensional structure of yeast tRNAPhe in the crystalline state is correlated with 360 MHz proton nuclear magnetic resonances from three fragments plus an intact molecule of the tRNA that share a common 5’ end and are in a solution condition similar to that of the crystal structure. This has allowed identification of folded structures present in the fragments and presumably present in the growing tRNA molecule as it is being synthesized from the 5’ end. The experiments show that only the correct stems are formed in the fragments; no additional or competing helical region is produced. This suggests that in the biosynthesis of this tRNA, correct folding of helical stems occurs before the entire molecule is formed. Further, some of the tertiary interactions (hydrogen bonds) found in the crystal structure are also probably present before the synthesis is completed. These findings are generalized to consider the precursor of the tRNA as well as other tRNAs.

Sequential Folding of Transfer RNA. A Nuclear Magnetic Resonance Study of Successively Longer tRNA Fragments with a Common 5’ End

Cerebral ischemia/reperfusion (I/R) injury is a clinical event associated with high morbidity and mortality. Neuroinflammation plays a crucial role in the pathogenesis of I/R-induced brain injury and cognitive decline. Low-density lipoprotein receptor-related protein-1 (LRP1) can exert strong neuroprotection in experimental intracerebral hemorrhage. However, whether LRP1 can confer neuroprotective effects after cerebral I/R is yet to be elucidated. The present study is aimed at investigating the effects of LRP1 activation on cerebral I/R injury and deducing the underlying mechanism involving TXNIP/NLRP3 signaling pathway. Cerebral I/R injury was induced in mice by bilateral common carotid artery occlusion. LPR1 ligand, apoE-mimic peptide COG1410, was administered intraperitoneally. To elucidate the underlying mechanism, overexpression of TXNIP was achieved via the hippocampal injection of AAV-TXNIP before COG1410 treatment. Neurobehavioral tests, brain water content, immunofluorescence, Western blot, enzyme-linked immunosorbent assay, HE, and terminal deoxynucleotidyl transferase dUTP nick end labeling staining were performed. Our results showed that the expressions of endogenous LRP1, TXNIP, NLRP3, procaspase-1, and cleaved caspase-1 were increased after cerebral I/R. COG1410 significantly ameliorated cerebral I/R-induced neurobehavioral deficits, brain edema, histopathological damage, and poor survival rate. Interestingly, COG1410 inhibited microglia proinflammatory polarization and promoted anti-inflammatory polarization, decreased oxidative stress, attenuated apoptosis, and inhibited the expression of the TXNIP/NLRP3 signaling pathway. However, the benefits of COG1410 were abolished by TXNIP overexpression. Thus, our study suggested that LRP1 activation with COG1410 attenuated cerebral I/R injury at least partially related to modulating microglial polarization through TXNIP/NLRP3 signaling pathway in mice. Thus, COG1410 treatment might serve as a promising therapeutic approach in the management of cerebral I/R patients.

https://downloads.hindawi.com/journals/omcl/2022/8729398.pdf

Activation of LRP1 Ameliorates Cerebral Ischemia/Reperfusion Injury and Cognitive Decline by Suppressing Neuroinflammation and Oxidative Stress through TXNIP/NLRP3 Signaling Pathway in Mice

S-nitrosylation of proteins occurs as a consequence of the derivatization of cysteine thiols with nitric oxide (NO) and is often associated with diseases and protein malfunction. Aberrant S-nitrosylation, in addition to other genetic and epigenetic factors, has gained rapid importance as a prime cause of various metabolic, respiratory, and cardiac disorders, with a major emphasis on cancer and neurodegeneration. The S-nitrosoproteome, a term used to collectively refer to the diverse and dynamic repertoire of S-nitrosylated proteins, is relatively less explored in the field of redox biochemistry, in contrast to other covalently modified versions of the same set of proteins. Advancing research is gradually unveiling the enormous clinical importance of S-nitrosylation in the etiology of diseases and is opening up new avenues of prompt diagnosis that harness this phenomenon. Ever since the discovery of the two robust and highly conserved S-nitrosoglutathione reductase and thioredoxin systems as candidate denitrosylases, years of rampant speculation centered around the identification of specific substrates and other candidate denitrosylases, subcellular localization of both substrates and denitrosylases, the position of susceptible thiols, mechanisms of S-denitrosylation under basal and stimulus-dependent conditions, impact on protein conformation and function, and extrapolating these findings towards the understanding of diseases, aging and the development of novel therapeutic strategies. However, newer insights in the ever-expanding field of redox biology reveal distinct gaps in exploring the crucial crosstalk between the redoxins/major denitrosylase systems. Clarifying the importance of the functional overlap of the glutaredoxin, glutathione, and thioredoxin systems and examining their complementary functions as denitrosylases and antioxidant enzymatic defense systems are essential prerequisites for devising a rationale that could aid in predicting the extent of cell survival under high oxidative/nitrosative stress while taking into account the existence of the alternative and compensatory regulatory mechanisms. This review thus attempts to highlight major gaps in our understanding of the robust cellular redox regulation system, which is upheld by the concerted efforts of various denitrosylases and antioxidants.

/pdf/s-denitrosylation-a-crosstalk-between-glutathione-and-imwprsdo.pdf

S-Denitrosylation: A Crosstalk between Glutathione and Redoxin Systems

Cancer, which killed ten million people in 2020, is expected to become the world’s leading health problem and financial burden. Despite the development of effective therapeutic approaches, cancer-related deaths have increased by 25.4% in the last ten years. Current therapies promote apoptosis and oxidative stress DNA damage and inhibit inflammatory mediators and angiogenesis from providing temporary relief. Thioredoxin-binding protein (TXNIP) causes oxidative stress by inhibiting the function of the thioredoxin system. It is an important regulator of many redox-related signal transduction pathways in cells. In cancer cells, it functions as a tumor suppressor protein that inhibits cell proliferation. In addition, TXNIP levels in hemocytes increased after immune stimulation, suggesting that TXNIP plays an important role in immunity. Several studies have provided experimental evidence for the immune modulatory role of TXNIP in cancer impediments. TXNIP also has the potential to act against immune cells in cancer by mediating the JAK-STAT, MAPK, and PI3K/Akt pathways. To date, therapies targeting TXNIP in cancer are still under investigation. This review highlights the role of TXNIP in preventing cancer, as well as recent reports describing its functions in various immune cells, signaling pathways, and promoting action against cancer.

/pdf/immunomodulatory-role-of-thioredoxin-interacting-protein-in-eh2i1y4s.pdf

Immunomodulatory Role of Thioredoxin Interacting Protein in Cancer’s Impediments: Current Understanding and Therapeutic Implications

Simple Summary Neuroblastoma (NB) is the most common extracranial solid tumor in children, and the majority of patients with high-risk disease are unable to be cured. There is an urgent need to design novel therapeutics for NB patients. In NB, the RAS-MAPK pathway plays a crucial role in essential processes such as cell proliferation, survival, and chemoresistance. In the present study, we determined the therapeutic potential of the ERK inhibitor ulixertinib in NB using a panel of NB cell lines, patient-derived xenograft (PDX) cell lines, and NB xenograft mouse models. Ulixertinib significantly and potently inhibited NB cell proliferation and tumor growth, as well as prolonged survival in the treated mice. Additionally, ulixertinib synergistically sensitized NB cells to the conventional chemotherapeutic drug doxorubicin. This study provides proof-of-concept pre-clinical evidence for exploring ulixertinib as a novel therapeutic approach for NB. Abstract Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Approximately 80% of relapsed NB show RAS-MAPK pathway mutations that activate ERK, resulting in the promotion of cell proliferation and drug resistance. Ulixertinib, a first-in-class ERK-specific inhibitor, has shown promising antitumor activity in phase 1 clinical trials for advanced solid tumors. Here, we show that ulixertinib significantly and dose-dependently inhibits cell proliferation and colony formation in different NB cell lines, including PDX cells. Transcriptomic analysis revealed that ulixertinib extensively inhibits different oncogenic and neuronal developmental pathways, including EGFR, VEGF, WNT, MAPK, NGF, and NTRK1. The proteomic analysis further revealed that ulixertinib inhibits the cell cycle and promotes apoptosis in NB cells. Additionally, ulixertinib treatment significantly sensitized NB cells to the conventional chemotherapeutic agent doxorubicin. Furthermore, ulixertinib potently inhibited NB tumor growth and prolonged the overall survival of the treated mice in two different NB mice models. Our preclinical study demonstrates that ulixertinib, either as a single agent or in combination with current therapies, is a novel and practical therapeutic approach for NB.

/pdf/erk-inhibitor-ulixertinib-inhibits-high-risk-neuroblastoma-1uekvbzv.pdf

ERK Inhibitor Ulixertinib Inhibits High-Risk Neuroblastoma Growth In Vitro and In Vivo

Thioredoxin-interacting protein (TXNIP) was originally named vitamin D3 upregulated protein-1 (VDUP1) because of its ability to bind to thioredoxin (TRX) and inhibit TRX function and expression. TXNIP is an alpha-arrestin protein that is essential for redox homeostasis in the human body. TXNIP may act as a double-edged sword in the cell. The balance of TXNIP is crucial. A study has shown that TXNIP can travel between diverse intracellular locations and bind to different proteins to play different roles under oxidative stress. The primary function of TXNIP is to induce apoptosis or pyroptosis under oxidative stress. TXNIP also inhibits proliferation and migration in cancer cells, although TXNIP levels decrease, and function diminishes in various cancers. In this review, we summarized the main structure, binding proteins, pathways, and the role of TXNIP in diseases, aiming to explore the double-edged sword role of TXNIP, and expect it to be helpful for future treatment using TXNIP as a therapeutic target.

/pdf/txnip-a-double-edged-sword-in-disease-and-therapeutic-3c7upvbk.pdf

TXNIP: A Double-Edged Sword in Disease and Therapeutic Outlook

Nowadays, RNA is extensively acknowledged an important role in the functions of information transfer, structural components, gene regulation and etc. The secondary structure of RNA becomes a key to understand structure-function relationship. Computational prediction of RNA secondary structure does not only provide possible structures, but also elucidates the mechanism of RNA folding. Conventional prediction programs are either derived from evolutionary perspective, or aimed to achieve minimum free energy. In vivo, RNA folds during transcription, which indicates that native RNA structure is a result from both thermodynamics and kinetics. 
In this thesis, I first reviewed the current leading kinetic folding programs and demonstrate that these programs are not able to predict secondary structure accurately. Upon that, I proposed a new sequential folding program called GTkinetics. Given an RNA sequence, GTkinetics predicts a secondary structure and a series of RNA folding trajectories. It treats the RNA as a growing chain, and adds stable local structures sequentially. It is featured with a Z-score to evaluate stability of local structures, which is able to locate native local structures with high confidence. Since all stable local structures are captured in GTkinetics, it results in some false positives, which prevents the native structure to form as the chain grows. This suggests a refolding model to melt the false positive hairpins, probable intermediate structures, and to fold the RNA into a new structure with reliable long-range helices. By analyzing suboptimal ensemble along the folding pathway, I suggested a refolding mechanism, with which refolding can be evaluated whether or not to take place. 
Another way to favor local structures over long-distance structures, we introduced a distance penalty function into the free energy calculation. I used a sigmoidal function to compute the energy penalty according to the distance in the primary sequence between two nucleotides of a base pair. For both the training dataset and the test dataset, the distance function improves the prediction to some extent. 
In order to characterize the differences between local and long-range helices, I carried out analysis of standardized local nucleotide composition and base pair composition according to the two groups. The results show that adenine accumulates on the 5' side of local structure, but not on that of long-range helices. GU base pairs occur significantly more frequent in the local helices than that in the long-range helices. These indicate that the mechanisms to form local and long range helices are different, which is encoded in the sequence itself. 
Based on all the results, I will draw conclusions and suggest future directions to enhance the current sequential folding program.

RNA secondary sturcture prediction using a combined method of thermodynamics and kinetics

Bacillus pumilus is widely used as a biocontrol agent. To further develop the biological control potential of B. pumilus LYMC-3 against pine blight, a statistical experimental design was used to optimize a liquid medium using low-cost substrates to improve its antagonistic activity against Sphaeropsis sapinea. Through the plate antagonism test and greenhouse control effect test, this study determines the antifungal effect of strain LYMC-3 against S. sapinea and pine blight. Moreover, response surface optimization methodologies were used to systematically optimize the medium composition and culture conditions of the LYMC-3 strain. The plate antagonism test showed that the inhibition rate of LYMC-3 fermentation filtrate (diluted 5-fold) was 66.09%. The greenhouse control effect test showed that the control effect of its fermentation filtrate on shoot blight reached 89.99%. The response surface optimization test ultimately determined that a higher inhibition rate can be achieved under these conditions: the optimal medium components were 7.2 g/L glucose, 15 g/L peptone, and 7.1 g/L magnesium sulfate; the optimal culture conditions were 52% liquid volume, 28 °C culture temperature, an initial pH of 7, and 1% bacterial inoculation volume. Under the optimized system, the five-fold diluted LYMC-3 fermentation filtrate inhibition rate against S. sapinea was 81.23%, which was 15.84% higher than that before optimization. Meanwhile, optimize the selection of lower-cost and more commonly used glucose instead of beef paste as the carbon source for the culture medium, and choose cheaper magnesium sulfate instead of sodium chloride as the nitrogen source.

/pdf/optimization-of-fermentation-conditions-for-bacillus-pumilus-1hzefgjs.pdf

Minmin Pan

Papers

TXNIP: A Double-Edged Sword in Disease and Therapeutic Outlook

RNA secondary sturcture prediction using a combined method of thermodynamics and kinetics

Optimization of Fermentation Conditions for Bacillus pumilus LYMC-3 to Antagonize Sphaeropsis sapinea