bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural and Mechanistic Basis for Self versus Non‐self RNA discrimination by the Innate Immune Receptor RIG‐I
Authors: Devarkar Swapnil , Wang Chen , Ramanathan Anand , Jiang Fuguo , Miller Matthew , Khan Abdul , Marcotrigiano Joseph , Patel Smita 
Published: November 2023

RNAs with 5′‐triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor RIG‐I, whose activation triggers a Type I interferon response. Cellular RNAs undergo post‐transcriptional modifications which ensures that self RNAs do not carry 5′ppp and/or blunt‐ends. We carried out a thorough biochemical characterization of RIG‐I complexes with RNAs carrying various end‐modifications as well as measured the effect of these end‐modifications on their signaling activity. We find that discrimination of RNA ligands occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high katpase/Kd. Our studies reveal that the autoinhibitory interface between CARD2 (second CARD) and Hel2i (Helicase 2 insertion domain) plays a critical role in RNA selectivity, wherein this interface acts as a “gate” that prevents cellular RNAs from generating productive complexes that can signal.It is thought that self RNAs like mRNAs are not recognized by RIG‐I because 5′ppp is capped by the addition of a 7‐methyl guanosine (m7G) (Cap‐0) and the 5′‐end nucleotide ribose is 2′‐O‐methylated (Cap‐1). We provide a structural and mechanistic basis for the exact roles of capping and 2′‐O‐methylation in evading RIG‐I recognition using enzymatic assays, X‐ray crystallography and cell‐based signaling studies. Surprisingly, we find that Cap‐0 and 5′ppp dsRNA have similar binding affinities, activate RIG‐I's ATPase to the same extent, and shows the same cellular signaling response. Three new crystal structures of RIG‐I complexes with dsRNA bearing 5′OH, 5′ppp, and 5′Cap‐0 reveal that RIG‐I accommodates m7G in a cavity created through conformational changes in helicase‐motif IVa without perturbing the triphosphate interactions. In contrast, Cap‐1 modifications abrogate RIG‐I signaling through a mechanism involving the H830 residue, wherein 2′‐O‐methylation works synergistically with m7G capping to weaken RNA affinity and ATPase activity. Our work provides new structural insights into the host and viral immune evasive mechanisms from RIG‐I explaining the complexity of cap structures over evolution.Support or Funding InformationThis work was supported by NIH grant GM111959 to Joseph Marcotrigiano and Smita S Patel.
In Vivo Studies of Function of RRF (Ribosome Recycling Factor) with Translationally Coupled Two ORFs Revealed Its Major Function: Release of mRNA from the Post‐Termination Complex
Authors: Kaji Akira , Inokuchi Yoshio , Hirashima Akikazu , Kaji Hideko 
Published: November 2023

ObjectiveStudy the RRF reaction in vivo to correct wrong prevalent concept that RRF's major function is to split ribosomes of post‐termination complexes.MethodsUse translationally coupled two ORFs and determine how much of the upstream ribosomes is reading the downstream ORF using the reporter gene. RRF is known to be essential for the translational coupling. We control RRF activity using tsRRF.ResultsIn the translationally coupled 2 ORFs, the downstream ORF is exclusively read by the ribosomes reading the upstream ORF. No free ribosomes participate in the reading of the downstream ORF. In this system, the border region of the two ORFs is such that termination codon of the upstream ORF and the initiation codon of the downstream ORF overlap as in UAAUG. At this junction, ribosomes just completed reading the upstream are released by RRF and about 25% of the released ribosomes re‐bind to the AUG and start translating the downstream gene. For this reason, the system offers unique opportunity to study the fate of ribosome finishing one round of translation which is under the control of ribosome recycling Factor (RRF). We placed this system in a strain (LJ14) which contains the temperature sensitive RRF so that one can study what happens in the absence and presence of RRF. Using this system, we showed 1) The major function of RRF is to release the ribosomes from mRNA but not splitting the ribosome. 2) When Ribosomes are not released due to the inactivation of RRF, ribosomes at UAA undergoes thermal frame shift and read all three frames. 3) For the ribosome released by RRF from UAA, AUG but not other triplet functions to read the downstream ORF. 4) This binding of released ribosomes occurs only if the AUG is present near UAA. 5) Introduction of short sequence hybridizable with ribosomal RNA helps ribosomes to rebind to the AUG. 6) When the upstream sequence is extremely short (4 codon or less), the ribosomes released from UAA by RRF rebinds to the upstream AUG with Shine Dalgarno (SD) sequence and the reading of the downstream ORF does not occur.ConclusionsOur results show that the early workers were led to a wrong concept that RRF does not release mRNA from the ribosome because they studied the action of RRF using short ORF with SD. Therefore, our original concept for which RRF was discovered as “ribosome releasing factor” was correct. The current wrong concept of the RRF action or ribosome recycling factor in general including the eukaryotes stems from the use of short ORF in in vitro studies in the early days.Support or Funding InformationFunded by Creative Bio‐medical Research Institute, Phildelphia Pa 19147
Structural Characterization of a Mutant form of the Chlamydial Protein Pgp3
Authors: Trinh Timothy , Waninger‐Saroni Jessica , Galaleldeen Ahmad 
Published: November 2023

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide. Untreated infection of the human ocular or urogenital tract can lead to blindness, ectopic pregnancy, pelvic inflammation, and infertility. The C. trachomatis protein Pgp3 is a homotrimer believed to play a prominent role in chlamydial pathogenesis. Pgp3 is one of the most immunodominant antigens found in infected people, and it stimulates macrophages into releasing inflammatory cytokines.We have recently determined the structure of Pgp3. The protein is comprised of three domains: a C‐terminal domain that resembles the TNF family of cytokines, an N‐terminal domain with structural motifs similar to those found in trimeric viral proteins, and a central triple‐helical coiled‐coil (THCC) that forms a rare right‐handed superhelical twist.The structure determination of Pgp3 was challenging due to the strong diffuse scattering observed in the diffraction pattern of the crystals. The diffuse scatter is believed to be caused by the flexibility of the THCC domain as a result of a sharp turn at glycine 85.To improve the model and produce better diffracting crystals, we mutated glycine 85 into alanine to remove the sharp turn, thus producing better diffracting crystals to build a better model of the THCC domain.The G85A protein was expressed, purified, and, crystallized, and the structure was determined. The crystal shares the same space group with WT Pgp3 and the diffuse scattering persisted. The G85A mutation did not alter the twist in the THCC domain.
Blocking the Protein‐Protein Interaction between Gαh and PLCδ1 Inhibits Metastatic Progression in Triple‐Negative Breast Cancer
Authors: Lin Yuan‐Feng , Liu Pei‐Yao , Hsiao Michael 
Published: November 2023

Breast cancer is thought to be a leading cause of poor survival rate in woman diseases. Especially triple‐negative breast cancer (TNBC) severely risks patient's life, usually owing to its metastatic progression. However, the molecular mechanism for cancer metastasis is still unclear in TNBC.Here find that Gαh which is originally identified as tissue transglutaminase acts as a poor prognostic marker in clinical cohort with breast cancer and exerts its G protein function to promote the invasiveness of TNBC cells in vitro and in vivo. Our data showed that the expression of Gαh causally associates with the in vitro invasion ability of TNBC cell lines. Moreover, the knockdown of Gαh obviously suppressed the in vitro invasion ability and the in vivo lung colonization capacity of highly metastatic TNBC cell lines MDA‐MB231 and HCC38 that express Gαh protein abundantly. Conversely, the enforced expression of exogenous Gah markedly fostered the metastatic potentials in vitro and in vivo of TNBC cell lines MDA‐MB157 and HCC70 that express Gαh protein poorly. Certainly, the GTP‐binding activity, but not transamidating activity, of Gαh is crucial for promoting the metastatic evolution in TNBC cells. Furthermore, interrupting the protein‐protein interaction (PPI) of phospholipase C‐δ1 (PLCδ1) with Gαh by a synthetic peptide corresponding to PLCδ1 amino acid sequence TIPLNSLKQGYRHVHLM robustly suppressed the metastatic abilities of MDA‐MB231 cells in vitro and in vivo.These findings not only elucidate the critical role of Gαh/PLCδ1 signaling pathway in promoting the metastatic progression of TNBC but also provide a new strategy to combat TNBC metastasis via targeting the PPI of Gαh/PLCδ1 complex.
Serum Proteins Binding to Tamm‐Horsfall Protein
Authors: Araujo Hugo C. , Rhodes Diana C.J. , Rhodes James A. 
Published: November 2023

Tamm‐Horsfall protein (THP), also called uromodulin, is an approximately 86kD, heavily glycosylated, single‐chain acidic protein (estimated pI=3). THP is produced in the kidney within the thick ascending limb of the loop of Henle and is excreted in milligram quantities daily into the urine. Despite its abundance, the normal physiologic function of THP remains enigmatic. One theory is that THP is an immunomodulatory molecule, due to its ability to bind various immune proteins such as IL‐1, TNF‐α, IgG, complement 1q (C1q), and factor H (fH). The purpose of the present study was to determine if other serum proteins could be shown to bind to immobilized THP. Multiple attempts to couple THP (purified from the urine of one healthy female) to a commercially available N‐hydroxysuccinimide (NHS)‐activated agarose resin (Thermo ScientificTM Pierce®) failed, at least partially because of solubility issues with THP, a protein that readily forms large aggregates in solution. As an alternative approach, purified THP was coated to microtiter plate wells and normal human serum diluted 1:5 with 20 mM Tris/20 mM NaCl/0.05% Tween 20, pH 7.5 was incubated in THP‐coated or non‐THP‐coated wells. After rinsing to remove unbound protein, the proteins bound to the wells were extracted by addition of 1X sample buffer containing 5% β‐mercaptoethanol, and the recovered samples were analyzed by SDS‐PAGE. IgG in serum did not appear to bind THP, even though binding of purified IgG to THP could be detected by this method. Among the proteins that specifically bound to the THP‐coated wells was a prominent protein with an estimated molecular weight of about 78 kD. Based on its molecular weight and abundance, transferrin seemed to be a likely candidate for this protein. Purified human transferrin (Sigma) and the protein recovered from THP‐coated microtiter plates had identical mobilities on SDS‐PAGE gels. Direct binding of THP to purified transferrin was analyzed by ELISA, incubating human transferrin (from 4 mg/ml to 100 μg/ml) in THP‐coated and non‐THP‐coated microtiter plate wells and then detecting bound transferrin with goat anti‐transferrin antiserum and rabbit anti‐goat IgG‐alkaline phosphatase. This ELISA demonstrated that transferrin bound to immobilized THP in a dose‐dependent manner. In summary, these studies indicated that immobilized THP does not bind to IgG within diluted serum, even though THP binds to purified IgG (in the absence of diluted serum), suggesting that a component of serum inhibits the THP/IgG interaction. These studies also provided evidence for an interaction between THP and transferrin, the significance of which remains to be elucidated.Support or Funding InformationFunded by the PNWU Research Seed Program.
Binding of Tamm‐Horsfall Protein to Properdin in Various Ionic Environments
Authors: Morgan Jarrad , Rhodes Diana 
Published: November 2023

Tamm‐Horsfall protein (THP), also known as uromodulin, is produced in the kidney and is the most abundant protein excreted into urine. It is an ~86kD acidic glycoprotein (pI≈3) with complex N‐linked carbohydrates often capped with sialic acid. THP may act as an immunomodulatory protein since it binds to IgG, complement 1q (C1q), complement 3b (C3b), and factor H (fH); inhibits the classical complement pathway; and acts as a cofactor for factor I in C3b degradation. THP also binds properdin which is a basic protein (pI > 9.5) consisting of multiple, identical 53 kD subunits. Properdin is the only known positive regulator of complement, stabilizing C3b. The purpose of this study was to investigate the effect of different ionic conditions on the interaction between THP and properdin, which are oppositely charged proteins. Previous studies of the interaction between THP and properdin had utilized Falcon Pro Bind microtiter plates that are no longer available. Hence, the first studies compared the remaining Falcon plates to three other microtiter plates. For these and all subsequent ELISAs, wells were coated with THP purified from the urine of one healthy male. Multiple concentrations of properdin (Complement Technologies) were added to THP‐coated wells, and the bound properdin was detected with goat anti‐properdin serum and rabbit anti‐goat IgG‐alkaline phosphatase. Of the plates tested, the Corning Costar plates acted in a manner most similar to Falcon plates and were used in the remaining studies. The binding buffer used in all studies was 20 mM Tris/1 mM MgCl2/1 mM CaCl2/0.05% Tween 20 (pH ~7.6) with concentrations of NaCl varying from 20 mM to 154 mM. These studies showed that the optimal NaCl concentration was dependent on the properdin concentration, with maximal binding of properdin to THP occurring at 70–80 mM NaCl for low properdin concentrations (0.25 and 0.5 μg/ml) and maximal binding of properdin to THP occurring at 20 mM NaCl with high properdin concentrations (5 μg/ml). The lowest amount of THP/properdin binding consistently was seen in the 154 mM NaCl buffer. These results differ somewhat from our very early, much more limited experiments with the Falcon microtiter plates, where THP binding to properdin was higher at all concentrations of properdin in a 90 mM ionic strength buffer. It is noteworthy that if the interaction between THP and properdin was purely electrostatic in nature, binding would be consistently higher in low ionic strength buffers, which was not what the present study showed. Further studies, not only on the effect of ionic strength but also on the effect of pH on the THP/properdin interaction are warranted since urine, which is where THP and properdin most likely could interact, varies considerably in these parameters.Support or Funding InformationSupported by the PNWU Research Funds.
A group of novel serum diagnostic biomarkers for multidrug‐resistant tuberculosis by iTRAQ‐2D LC‐MS/MS and Solexa sequencing
Authors: WANG CHONG , LIU CHANG‐MING , WEI LI‐LIANG , SHI LI‐YING , PAN ZHI‐FEN , MAO LIAN‐GEN , WAN XIAO‐CHEN , PING ZE‐PENG , JIANG TING‐TING , CHEN ZHONG‐LIANG , LI ZHONG‐JIE , LI JI‐CHENG 
Published: November 2023

The epidemic of pulmonary tuberculosis (TB), especially multidrug‐resistance tuberculosis (MDR‐TB) presented a major challenge for TB treatment today. We performed iTRAQ labeling coupled with two‐dimensional liquid chromatography‐tandem mass spectrometry (2D LC‐MS/MS) and Solexa sequencing among MDR‐TB patients, drug‐sensitive tuberculosis (DS‐TB) patients, and healthy controls. A total of 50 differentially expressed proteins and 43 differentially expressed miRNAs (fold change >1.50 or <0.60, P<0.05) were identified in the MDR‐TB patients compared to both DS‐TB patients and healthy controls. We found that 22.00% of differentially expressed proteins and 32.56% of differentially expressed miRNAs were related, and could construct a network mainly in complement and coagulation cascades. Significant differences in CD44 antigen (CD44), coagulation factor XI (F11), kininogen‐1 (KNG1), miR‐4433b‐5p, miR‐424‐5p, and miR‐199b‐5p were found among MDR‐TB patients, DS‐TB patients and healthy controls (P<0.05) by enzyme‐linked immunosorbent assay (ELISA) and SYBR green qRT‐PCR validation. A strong negative correlation, consistent with the target gene prediction, was found between KNG1 and miR‐199b‐5p (r=−0.232, P=0.017). Moreover, we established a diagnostic model for MDR‐TB patients and healthy controls with a sensitivity of 100.00% and a specificity of 88.33% (overall accuracy 93.14%), and also established a diagnostic model for MDR‐TB patients and DS‐TB patients with a sensitivity of 83.33% and a specificity of 88.33% (overall accuracy 86.27%). Our study proposes potential biomarkers for MDR‐TB diagnosis, and also provides a new experimental basis to understand the pathogenesis of MDR‐TB.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China (No.81573709, No.81273882), the National Basic Research Program of China (No.2014CB543002) and the National Special Sci‐Tech Projects (No.2012ZX10005001‐006).
Metal affinity chromatography and protein refolding techniques: some observations and strategies
Authors: Odunuga Odutayo O , Weihe Jamie M , Tovar Carmel N 
Published: November 2023

Immobilized metal affinity chromatography (IMAC) is a popular technique used for large‐scale purification of proteins because of its high binding specificity, capacity and relative ease of operation. Despite recent advances in improving the robustness of the technique, several factors still affect column binding capacity, yield and purity of proteins during IMAC. Using (His)6‐tagged proteins with comparably high level of expression, we investigated the effect of protein size on the binding capacities of commercial Ni2+ and Co2+ IMAC columns. Our data suggest that small and medium‐sized proteins (< 50 kDa) bind more tightly to the columns (Ni‐NTA and Co‐TALON) than bigger proteins, resulting in higher yields during purification. In another study, we employed a modified gel filtration technique, gradient gel filtration to successfully refold a spectrum of highly aggregation‐prone enzymatic proteins back to their native states during denaturing purification. Some of the proteins chosen are well‐known to precipitate significantly during refolding. However, using this technique, precipitation was either so minimal or non‐existent. Enzymatic assays and dynamic molecular mass determination confirmed that the proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification step.Support or Funding InformationWelch Foundation Grant # AN‐0008
Synthesis of Artificial Bacterial Cell Wall Fragments as Tools to Study the Innate Immune System
Authors: Gregorzak Gabriel , Melnyk James , DeMeester Kristen , Grimes Catherine 
Published: November 2023

During a bacterial infection, the body's first line of defense is provided by the innate immune system. This system is triggered by pathogen‐associated molecular patterns (PAMPs) that are recognized by different members of the body's innate immune system called pattern recognition receptors (PRRs). One such PRR of interest is nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2). Mammalian Nod2 is a cytosolic protein that binds to and signals in response to carbohydrate containing bacterial cell wall fragments; the smallest of which is muramyl dipeptide (MDP). Mutations in Nod2 increase the likelihood of the development of Crohn's Disease and other chronic inflammatory diseases. Previously, our lab has shown that Nod2 binds directly to MDP, but currently the molecular mechanisms of activation and binding are still unknown1. Currently, we are addressing this issue using synthetic chemistry. This project focuses on the synthesis of an assortment of MDP dimers, which vary in the length and functionality of their connecting linkers. To avoid troublesome late‐stage de‐protection issues, the synthesis diverges in its early stages to result in two differing MDP‐like fragments. These two fragments can then be coupled together and de‐protected, resulting in the MDP dimer. Once synthesized, these dimers will be used in in vitro SPR binding assays, as well as in cell‐based assays to elucidate the binding and activation mechanisms used by Nod2. Ultimately, these synthetic tools will aid in the advancement of our understanding of how are body's sense and respond to bacteria.Support or Funding InformationHoward Hughes Medical Institute
GRP78 is Not Expressed on the Surface of Triple Negative Human Breast Cancer Cells
Authors: Serrano‐Negron Jesus E , Romero Eva C , Sanchez Neysharie , Banerjee Aditi , Baksi Krishna , Banerjee Dipak K 
Published: November 2023

GRP78, an Mr78 kDa glucose responsive protein is located in the lumen of the endoplasmic reticulum (ER). It functions as ER chaperone in translocating protein across the ER membrane that needs to be glycosylated at the asparagine residue present in the sequeon Asn‐X‐Ser/Thr (N‐linked glycosylation). This is an important biochemical event in protein folding. When N‐glycosylation is impaired with an N‐glycosylation inhibitor tunicamycin, angiogenesis, a hallmark for tumor progression and metastasis is inhibited. The result is regression of breast tumor. Reduced N‐glycan expression correlates with thinning and narrowing of the tumor microvessels. On the other hand, GRP78 expression in microvasculature and in tumor tissue is upregulated, concluding that GRP78 is a master regulator in ER stress induced unfolded protein response (upr)‐mediated apoptosis in tumor microvasculature (J. Biol. Chem. 286, 29127–29138, 2011; Pure Appl. Chem. 84, 1907–1918, 2012). This contradicts the current dogma that GRP78 expresses on the cell surface, interferes with the therapeutic approach(es) and becomes a tumor promoter. To evaluate the GRP78 localization in our study, we have used a triple negative (ER−/PR−/HER2−) human breast cancer cell line MDA‐MB‐231 cultured normally or without serum as well as before after inducing ER stress, and immunofluorescence microscopy. Unfixed cells were stained with Concanavalin A (Con A; Specificity = α‐D‐Mannose, α‐D‐Glucose, Branched mannose), Wheat germ agglutinin (WGA; Specificity = (GlcNAc‐β‐(1,4)‐GlcNAc)1‐4>β‐GlcNAc‐NeuAc) as well as with anti‐GRP78 antibody followed by their detection either with Rhodamin or Alexa–conjugated secondary antibody. The fluorescence images were captured in a Zeiss microscope with Axiocam camera. Con A and WGA staining have provided images of N‐glycans on the cell surface and supported the intactness of the cell membrane. On the other hand, GRP78 fluorescence was absent from the surface of the cancer cell. The results were the same whether the cells were cultured in the absence of serum and/or in the presence of 1μg/mL of tunicamycin. GRP78 fluorescence however was detectable in cells after either fixing them with ice‐cold methanol or permeabilizing them with digitonin. We, therefore, conclude that GRP78 is not expressed on the outer‐leaflet of the cell surface of the triple negative human breast cancer cells MDA‐MB‐231. But, its intracellular expression is anti‐tumorigenic.Support or Funding InformationSupported in part by grants from NIH U54‐CA096297 and Susan G. Komen for Cure BCRT58206 (DKB) and NIH/NIMHD 2G12MD007583 (KB).