Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme's regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature.

Middle East respiratory syndrome coronavirus (MERS-CoV) has evolved to navigate through the sophisticated network of a host's immune system. The immune evasion mechanism including type 1 interferon and protein kinase R-mediated antiviral stress responses has been recently attributed to the involvement of MERS-CoV protein 4a (p4a) that masks the viral dsRNA. However, the structural mechanism of how p4a recognizes and establishes contacts with dsRNA is not well explained. In this study, we report a dynamic mechanism deployed by p4a to engage the viral dsRNA and make it unavailable to the host immune system. Multiple variants of p4a-dsRNA were created and investigated through extensive molecular dynamics procedures to highlight crucial interfacial residues that may be used as potential pharmacophores for future drug development. The structural analysis revealed that p4a exhibits a typical αβββα fold structure, as found in other dsRNA-binding proteins. The α1 helix and the β1-β2 loop play a crucial role in recognizing and establishing contacts with the minor grooves of dsRNA. Further, mutational and binding free energy analyses suggested that in addition to K63 and K67, two other residues, K27 and W45, might also be crucial for p4a-dsRNA stability.

Direct inhibition of tumor necrosis factor-alpha (TNF-α) action is considered a promising way to prevent or treat TNF-α-associated diseases. The trimeric form of TNF-α binds to its receptor (TNFR) and activates the downstream signaling pathway. The interaction of TNF-α with molecular-grade dimethyl sulfoxide (DMSO) in an equal volumetric ratio renders TNF-α inert, in this state, TNF-α fails to activate TNFR. Here, we aimed to examine the inhibition of TNF-α function by various concentrations of DMSO. Its higher concentration led to stronger attenuation of TNF-α-induced cytokine secretion by fibroblasts, and of their death. We found that this inhibition was mediated by a perturbation in the formation of the functional TNF-α trimer. Molecular dynamics simulations revealed a transient interaction between DMSO molecules and the central hydrophobic cavity of the TNF-α homodimer, indicating that a brief interaction of DMSO with the TNF-α homodimer may disrupt the formation of the functional homotrimer. We also found that the sensitizing effect of actinomycin D on TNF-α-induced cell death depends upon the timing of these treatments and on the cell type. This study will help to select an appropriate concentration of DMSO as a working solvent for the screening of water-insoluble TNF-α inhibitors.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo--a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.

Toll-like receptors (TLRs) play a fundamental role in the inflammatory response against invading pathogens. However, the dysregulation of TLR-signaling pathways is implicated in several autoimmune/inflammatory diseases. Here, we show that a novel small molecule TLR-inhibitor (TAC5) and its derivatives TAC5-a, TAC5-c, TAC5-d, and TAC5-e predominantly antagonized poly(I:C) (TLR3)-, imiquimod (TLR7)-, TL8-506 (TLR8)-, and CpG-oligodeoxynucleotide (TLR9)-induced signaling pathways. TAC5 and TAC5-a significantly hindered the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), reduced the phosphorylation of mitogen-activated protein kinases, and inhibited the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6. Besides, TAC5-a prevented the progression of psoriasis and systemic lupus erythematosus (SLE) in mice. Interestingly, TAC5 and TAC5-a did not affect Pam3CSK4 (TLR1/2)-, FSL-1 (TLR2/6)-, or lipopolysaccharide (TLR4)-induced TNF-α secretion, indicating their specificity towards endosomal TLRs (TLR3/7/8/9). Collectively, our data suggest that the TAC5 series of compounds are potential candidates for treating autoimmune diseases such as psoriasis or SLE.

Cathelicidins are an ancient class of antimicrobial peptides (AMPs) with broad spectrum bactericidal activities. In this study, we investigated the diversity and biological activity of cathelicidins of buffalo, a species known for its disease resistance. A series of new homologs of cathelicidin4 (CATHL4), which were structurally diverse in their antimicrobial domain, was identified in buffalo. AMPs of newly identified buffalo CATHL4s (buCATHL4s) displayed potent antimicrobial activity against selected Gram positive (G+) and Gram negative (G-) bacteria. These peptides were prompt to disrupt the membrane integrity of bacteria and induced specific changes such as blebing, budding, and pore like structure formation on bacterial membrane. The peptides assumed different secondary structure conformations in aqueous and membrane-mimicking environments. Simulation studies suggested that the amphipathic design of buCATHL4 was crucial for water permeation following membrane disruption. A great diversity, broad-spectrum antimicrobial action, and ability to induce an inflammatory response indicated the pleiotropic role of cathelicidins in innate immunity of buffalo. This study suggests short buffalo cathelicidin peptides with potent bactericidal properties and low cytotoxicity have potential translational applications for the development of novel antibiotics and antimicrobial peptidomimetics.

The NADPH-dependent HC-toxin reductases (HCTR1 and 2) encoded by enzymatic class of disease resistance homologous genes (Hm1 and Hm2) protect maize by detoxifying a cyclic tetrapeptide, HC-toxin, secreted by the fungus Cochliobolus carbonum race 1(CCR1). Unlike the other classes' resistance (R) genes, HCTR-mediated disease resistance is an inimitable mechanism where the avirulence (Avr) component from CCR1 is not involved in toxin degradation. In this study, we attempted to decipher cofactor (NADPH) recognition and mode of HC-toxin binding to HCTRs through molecular docking, molecular dynamics (MD) simulations and binding free energy calculation methods. The rationality and the stability of docked complexes were validated by 30-ns MD simulation. The binding free energy decomposition of enzyme-cofactor complex was calculated to find the driving force behind cofactor recognition. The overall binding free energies of HCTR1-NADPH and HCTR2-NADPH were found to be -616.989 and -16.9749 kJ mol-1 respectively. The binding free energy decomposition revealed that the binding of NADPH to the HCTR1 is mainly governed by van der Waals and nonpolar interactions, whereas electrostatic terms play dominant role in stabilizing the binding mode between HCTR2 and NADPH. Further, docking analysis of HC-toxin with HCTR-NADPH complexes showed a distinct mode of binding and the complexes were stabilized by a strong network of hydrogen bond and hydrophobic interactions. This study is the first in silico attempt to unravel the biophysical and biochemical basis of cofactor recognition in enzymatic class of R genes in cereal crop maize.

The innate immune system is activated upon virus invasion of a host cell by recognizing viral component, such as dsRNA through specific receptors, resulting in the production of type- I IFNs, which confer an antiviral state within the invaded as well as surrounding cells. In the present study, fibroblast, monocyte and macrophage cells derived from water Buffalo (Bubalus bubalis) were exposed to a synthetic dsRNA analogue, poly I:C to mimic viral invasion in each cell type. Recognition of poly I:C through cytosolic helicase receptors RIG-I and MDA5 molecule lead to the activation of the RLR pathway, subsequently activating the MAVS-IRF3/7 cascade and the production of antiviral effector molecule like IFNβ and ISGs. Within the different cell types, we identified variability in RLR receptor and IFNβ expression after poly I:C administration. Fibroblasts responded quickly and strongly with IFNβ production, followed by macrophages and monocytes. Despite absolute expression variability among different cell types the expression trend of RLRs pathway genes were similar. Length of poly I:C molecule also influence IFNβ expression in response of RLR pathway. Short (LMW) poly I:C induce stronger IFN-β expression in myeloid (macrophage and monocyte) cells. In contrast long (HMW) poly I:C preferably elicit higher IFNβ expression in non-myeloid (fibroblast) cell. Therefore, MDA5 and RIG-1 plays an indispensable role in eliciting antiviral response in non- immune (fibroblast) host cell. Thus, stimulation of RLR pathway with suitable and potentially cell-type specific agonist molecules successfully elicit antiviral state in the host animal, with fibroblasts conferring a stronger antiviral state compared with the monocytes and macrophages.

The nucleotide-binding and oligomerization domain (NOD)-containing protein 1 (NOD1) plays the pivotal role in host-pathogen interface of innate immunity and triggers immune signalling pathways for the maturation and release of pro-inflammatory cytokines. Upon the recognition of iE-DAP, NOD1 self-oligomerizes in an ATP-dependent fashion and interacts with adaptor molecule receptor-interacting protein 2 (RIP2) for the propagation of innate immune signalling and initiation of pro-inflammatory immune responses. This interaction (mediated by NOD1 and RIP2) helps in transmitting the downstream signals for the activation of NF-κB signalling pathway, and has been arbitrated by respective caspase-recruitment domains (CARDs). The so-called CARD-CARD interaction still remained contradictory due to inconsistent results. Henceforth, to understand the mode and the nature of the interaction, structural bioinformatics approaches were employed. MD simulation of modelled 1:1 heterodimeric complexes revealed that the type-Ia interface of NOD1CARD and the type-Ib interface of RIP2CARD might be the suitable interfaces for the said interaction. Moreover, we perceived three dynamically stable heterotrimeric complexes with an NOD1:RIP2 ratio of 1:2 (two numbers) and 2:1. Out of which, in the first trimeric complex, a type-I NOD1-RIP2 heterodimer was found interacting with an RIP2CARD using their type-IIa and IIIa interfaces. However, in the second and third heterotrimer, we observed type-I homodimers of NOD1 and RIP2 CARDs were interacting individually with RIP2CARD and NOD1CARD (in type-II and type-III interface), respectively. Overall, this study provides structural and dynamic insights into the NOD1-RIP2 oligomer formation, which will be crucial in understanding the molecular basis of NOD1-mediated CARD-CARD interaction in higher and lower eukaryotes.

In response to double stranded RNA (dsRNA) viruses, toll-like receptor 3 (TLR3) in fish activates signaling like human, and induces innate immunity. This suggested the existence of dsRNA binding domains in fish TLR3 as reported in higher vertebrates. In in silico analysis, leucine rich repeat (LRR) regions (4-6, 13-14, 20-22), and LRR (8-15, 17-24) were identified as key domains in rohu TLR3 as poly I:C and dsRNA of fish reovirus (AGCRV,VHSV and IHNV) binding regions. 3D-models of rohu TLR3-TIR and zebrafish TRIF were generated by homology and ab initio modeling respectively, and their interacting domains were predicted. This is the first report of TLR3 modeling in fish.

RIG1 and MDA5 have emerged as important intracellular innate pattern recognition receptors that recognize viral RNA and mediate cellular signals controlling Type I interferon (IFN-I) response. Buffalo RIG1 and MDA5 genes were investigated to understand the mechanism of receptor induced antiviral response. Sequence analysis revealed that RIG1 and MDA5 maintain a domain arrangement that is common in mammals. Critical binding site residues of the receptors are evolutionary conserved among mammals. Molecular dynamics simulations suggested that RIG1 and MDA5 follow a similar, if not identical, dsRNA binding pattern that has been previously reported in human. Moreover, binding free energy calculation revealed that MDA5 had a greater affinity towards dsRNA compared to RIG1. Constitutive expressions of RLR genes were ubiquitous in different tissues without being specific to immune organs. Poly I:C stimulation induced elevated expressions of IFN-β and IFN-stimulated genes (ISGs) through interferon regulatory factors (IRFs) mediated pathway in buffalo foetal fibroblast cells. The present study provides crucial insights into the structure and function of RIG1 and MDA5 receptors in buffalo.

Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), the first line of defense, are the cytosolic pattern recognition receptors (PRRs) that regulate the inflammatory activity in response to invading pathogens. NLRs are the members of AAA+ ATPase superfamily that comprises of N-terminal EBD(s), a centrally positioned NOD/NACHT and varying range of LRRs towards the C-terminal end. Due to the lack of structural data, the functional aspects of NLRP-signaling mechanism, which includes pathogen recognition, nucleotide-binding, and sensor-adaptor-effector interactions, are not fully understood. In this study, we implemented structural bioinformatics approaches including protein modeling, docking, and molecular dynamics simulations to explore the structural-dynamic features of ADP-/ATP-Mg2+ binding in NLRPNACHT models. Our results indicate a similar mode of ATP-Mg2+ binding in all NLRPNACHT models and the interacting residues are found consistent with reported mutagenesis data. Accompanied by the key amino acids (proposed to be crucial for ATP-Mg2+ coordination), we further have noticed that some additional conserved residues (including 'Trp' of the PhhCW motif, and 'Phe' and 'Tyr' of the GFxxxxRxxYF motif) are potentially interacting with ATP during dynamics; which require further experimentation for legitimacy. Overall, this study will help in understanding the ADP-/ATP-Mg2+ binding mechanisms in NLRPs in a broader perspective and the proposed ATP-binding pocket will aid in designing novel inhibitors for the regulation of inflammasome activity.

TLRs are some of the actively pursued drug-targets in immune disorders. Owing to a recent surge in the cognizance of TLR structural biology and signalling pathways, numerous therapeutic modulators, ranging from low-molecular-weight organic compounds to polypeptides and nucleic acid agents have been developed.

Excessive signaling by the proinflammatory cytokine TNF is involved in several autoimmune diseases, including rheumatoid arthritis (RA). However, unlike the approved biologics currently used to treat this and other conditions, commercially available small-molecule inhibitors of TNF trimerization are cytotoxic or exhibit low potency. Here, we report a TNF-inhibitory molecule (TIM) that reduced TNF signaling in vitro and was an effective treatment in a mouse model of RA. The initial lead compound, TIM1, attenuated TNF-induced apoptosis of human and mouse cells by delaying the induction of proinflammatory NF-κB and MAPK signaling and caspase 3- and caspase 8-dependent apoptosis. TIM1 inhibited the secretion of the proinflammatory cytokines IL-6 and IL-8 by disrupting TNF homotrimerization, thereby preventing its association with the TNF receptor. In a mouse model of collagen-induced polyarthritis, the more potent TIM1 analog TIM1c was orally bioavailable and reduced paw swelling, histological indicators of knee joint pathology, inflammatory infiltration of the joint, and the overall arthritis index. Orally delivered TIM1c showed immunological effects similar to those elicited by intraperitoneal injection of the FDA-approved TNF receptor decoy etanercept. Thus, TIM1c is a promising lead compound for the development of small-molecule therapies for the treatment of RA and other TNF-dependent systemic inflammation disorders.

Recent global health concern motivated the exploration of natural medicinal plant resources as an alternative target for treating COVID-19 infection and associated inflammation. In the current study, a phytochemical, 6-shogaol [1-(4-hydroxy-3-methoxyphenyl)dec-4-en-3-one; 6-SHO] was investigated as a potential anti-inflammatory and anti-COVID-19 agent. In virus release assay, 6-SHO efficiently (94.5%) inhibited SARS-CoV2 replication. When tested in the inflammasome activation model, 6-SHO displayed mechanistic action by regulating the expression of the inflammasome pathway molecules. In comparison to the existing drugs, remdesivir and hydroxy-chloroquine, 6-SHO was not only found to be as effective as the standard anti-viral drugs but also much superior and safe in terms of predicted physicochemical properties and clinical toxicity. Comparative molecular dynamics simulation demonstrated a stable interaction of 6-SHO with NLRP3 (the key inflammasome regulator) in the explicit water environment. Overall, this study provides important cues for further development of 6-SHO as potential anti-inflammatory and anti-viral therapeutic agents.

Toll-like receptor 3 (TLR3) provides the host with antiviral defense by initiating an immune signaling cascade for the production of type I interferons. The X-ray structures of isolated TLR3 ectodomain (ECD) and transmembrane (TM) domains have been reported; however, the structure of a membrane-solvated, full-length receptor remains elusive. We investigated an all-residue TLR3 model embedded inside a phospholipid bilayer using molecular dynamics simulations. The TLR3-ECD exhibited a ~30°-35° tilt on the membrane due to the electrostatic interaction between the N-terminal subdomain and phospholipid headgroups. Although the movement of dsRNA did not affect the dimer integrity of TLR3, its sugar-phosphate backbone was slightly distorted with the orientation of the ECD. TM helices exhibited a noticeable tilt and curvature but maintained a consistent crossing angle, avoiding the hydrophobic mismatch with the bilayer. Residues from the αD helix and the CD and DE loops of the Toll/interleukin-1 receptor (TIR) domains were partially absorbed into the lower leaflet of the bilayer. We found that the previously unknown TLR3-TIR dimerization interface could be stabilized by the reciprocal contact between αC and αD helices of one subunit and the αC helix and the BB loop of the other. Overall, the present study can be helpful to understand the signaling-competent form of TLR3 in physiological environments.

The toll/interleukin 1 receptor (TIR) domain-containing adaptor protein (TIRAP) plays an important role in the toll-like receptor (TLR) 2, TLR4, TLR7, and TLR9 signaling pathways. TIRAP anchors to phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) on the plasma membrane and PI (3,4,5)-trisphosphate (PIP3) on the endosomal membrane and assists in recruitment of the myeloid differentiation primary response 88 protein to activated TLRs. To date, the structure and mechanism of TIRAP's membrane association are only partially understood. Here, we modeled an all-residue TIRAP dimer using homology modeling, threading, and protein-protein docking strategies. Molecular dynamics simulations revealed that PIP2 creates a stable microdomain in a dipalmitoylphosphatidylcholine bilayer, providing TIRAP with its physiologically relevant orientation. Computed binding free energy values suggest that the affinity of PI-binding domain (PBD) for PIP2 is stronger than that of TIRAP as a whole for PIP2 and that the short PI-binding motif (PBM) contributes to the affinity between PBD and PIP2. Four PIP2 molecules can be accommodated by distinct lysine-rich surfaces on the dimeric PBM. Along with the known PI-binding residues (K15, K16, K31, and K32), additional positively charged residues (K34, K35, and R36) showed strong affinity toward PIP2. Lysine-to-alanine mutations at the PI-binding residues abolished TIRAP's affinity for PIP2; however, K34, K35, and R36 consistently interacted with PIP2 headgroups through hydrogen bond (H-bond) and electrostatic interactions. TIRAP exhibited a PIP2-analogous intermolecular contact and binding affinity toward PIP3, aided by an H-bond network involving K34, K35, and R36. The present study extends our understanding of TIRAP's membrane association, which could be helpful in designing peptide decoys to block TLR2-, TLR4-, TLR7-, and TLR9-mediated autoimmune diseases.

Toll-like receptors (TLRs) are a unique category of pattern recognition receptors that recognize distinct pathogenic components, often utilizing the same set of downstream adaptors. Specific molecular features of extracellular, transmembrane (TM), and cytoplasmic domains of TLRs are crucial for coordinating the complex, innate immune signaling pathway. Here, we constructed a full-length structural model of TLR4-a widely studied member of the interleukin-1 receptor/TLR superfamily-using homology modeling, protein-protein docking, and molecular dynamics simulations to understand the differential domain organization of TLR4 in a membrane-aqueous environment. Results showed that each functional domain of the membrane-bound TLR4 displayed several structural transitions that are biophysically essential for plasma membrane integration. Specifically, the extracellular and cytoplasmic domains were partially immersed in the upper and lower leaflets of the membrane bilayer. Meanwhile, TM domains tilted considerably to overcome the hydrophobic mismatch with the bilayer core. Our analysis indicates an alternate dimerization or a potential oligomerization interface of TLR4-TM. Moreover, the helical properties of an isolated TM dimer partly agree with that of the full-length receptor. Furthermore, membrane-absorbed or solvent-exposed surfaces of the toll/interleukin-1 receptor domain are consistent with previous X-ray crystallography and biochemical studies. Collectively, we provided a complete structural model of membrane-bound TLR4 that strengthens our current understanding of the complex mechanism of receptor activation and adaptor recruitment in the innate immune signaling pathway.

Toll-like receptors (TLRs) recognize pathogen/damage-associated molecular patterns and initiate inflammatory signaling cascades. Occasionally, overexpression of TLRs leads to the onset of numerous inflammatory diseases, necessitating the development of selective inhibitors to allow a protective yet balanced immune response. Here, we demonstrate that a novel peptide (TIP1) derived from Toll/interleukin-1 receptor (TIR) domain-containing adapter protein inhibited multiple TLR signaling pathways (MyD88-dependent and MyD88-independent) in murine and human cell lines. TIP1 also inhibited NLRP3-mediated IL-1β secretion, as we validated at both the protein and mRNA levels. Biophysical experiments confirmed that TIP1 specifically binds to the BB loop of the TLR4-TIR domain. Animal studies revealed that TIP1 inhibited the secretion of lipopolysaccharide (LPS)-induced proinflammatory cytokines in collagen-induced arthritis (CIA) and kaolin/carrageenan-induced arthritis (K/C) rodent models. TIP1 also rescued animals from sepsis and from LPS-induced kidney/liver damage. Importantly, TIP1 ameliorated the symptoms of rheumatoid arthritis in CIA and K/C rodent models, suggesting that TIP1 has therapeutic potential for the treatment of TLR-mediated autoimmune/inflammatory diseases.