NBD Domain Research Articles

Three cases of autoinflammatory disease with novel NLRC4 mutations, and the first mutation reported in the CARD domain of NLRC4 associated with autoinflammatory infantile enterocolitis (AIFEC)

BackgroundGain of function (GOF) mutations in NOD-like receptor family CARD-containing 4 protein (NLRC4) gene induce a wide spectrum of autoinflammatory phenotypes. Currently, we categorize them into four groups: familial cold autoinflammatory syndrome (FCAS)4, autoinflammatory infantile enterocolitis (AIFEC), NLRC4-macrophage associated syndrome (MAS), and neonatal-onset multisystem inflammatory disease (NOMID). The rarity and complexity of the disease necessitate the description of new cases and a reexamination of our understanding of the condition.Case presentationsWe present three patients with NLRC4-GOF mutations and AIFEC phenotypes. The first patient is an infant girl with periodic fever, seizure, high inflammatory markers, and an episode of macrophage associated syndrome (MAS). History of recurrent fever episodes since childhood was reported in mother and maternal grandmother. A heterozygous mutation was found in CARD domain of NLRC4: c.A91C: p.Asn31His. The second patient is an adolescent boy with periodic fever, diarrhea, aphthous stomatitis, seizure, and central nervous system (CNS) vasculitis. A heterozygous mutation was found in NLRC4 gene: c.1202T > C. p. Val401Ala. The third patient is a child with chronic diarrhea and elevated inflammatory markers. We found a heterozygous mutation in NLRC4 gene: c.390delG: p.S132Afs*21. All mutations have been reported for the first time as NLRC4 mutations associated with autoinflammation. We introduced novel mutations in the CARD domain and between CARD and NBD domain in the first and third cases, respectively. All three children are under remission following treatment.ConclusionsNLRC4-GOF mutations can be associated with autoinflammation with diverse symptoms. Given the rarity of the disease and the possibility of new mutations being identified, the existence of a phenotype/genotype correlation has yet to be thoroughly investigated. The variety in manifestations and severity spectrum mandates a variety of treatments. Adalimumab has shown favorable outcomes in our AIFEC cases.

HSPA8 inhibitors augment cancer chemotherapeutic effectiveness via potentiating necroptosis.

Our recent work has uncovered a novel function of HSPA8 as an amyloidase, capable of dismantling the RHIM-containing protein fibrils to suppress necroptosis. However, the impact of HSPA8 inhibitors on cancer regression via necroptosis remains unexplored. In this study, we conducted a comprehensive investigation to assess the potential of HSPA8 inhibitors in enhancing necroptosis both in vitro and in vivo. Our findings indicate that pharmacologic inhibition of HSPA8, achieved either through VER (VER-155008) targeting the nucleotide binding domain or pifithrin-μ targeting the substrate binding domain of HSPA8, significantly potentiates necroptosis induced by diverse treatments in cellular assays. These inhibitors effectively disrupt the binding of HSPA8 to the RHIM protein, impeding its regulatory function on RHIM amyloid formation. Importantly, HSPA8 inhibitors significantly enhanced cancer cell sensitivity to microtubule-targeting agents (MTAs) in vitro, while reversing chemoresistance and facilitating tumor regression by augmenting necroptosis in vivo. Our findings suggest a promising therapeutic approach to cancer through necroptosis modulation via HSPA8 targeting, particularly in combination with MTA drugs for enhanced treatment efficacy.

Structural Insight into the Binding Pattern and Interaction Mechanism of Antagonist MCC950 and Agonist BMS986299 with NLRP3 by Molecular Dynamics Simulation.

The NLRP3 inflammasome mediates a range of inflammatory responses that are associated with an increasing number of pathological mechanisms. Over-activation of NLRP3 can exacerbate many diseases. However, NLRP3 antagonists have significant therapeutic potential. Moreover, NLRP3 plays an important role in limiting the growth and spread of some tumors, and NLRP3 agonists also have clinical value. MCC950 and BMS986299 are an antagonist and agonist of NLRP3, respectively. In light of the important clinical applications of NLRP3, especially for NLRP3 inhibitors, a computational method was used to investigate the interaction modes of MCC950 and BMS986299 with NLRP3 in order to design and develop more potent NLRP3 regulators. In this study, the conformational behaviors of NLRP3 bound to the antagonist MCC950 in an inactive state and the agonist BMS986299 in an active state were investigated using 200 ns equilibrium all-atom molecular dynamics (MD) simulations, and then the analyses of the MD trajectories (RMSD, Rg, RMSF, SASA, PCA, and DCCM) were carried out to explore the mechanism of the antagonist and agonist on NLRP3 in the two different states. The RMSD, RMSF, Rg, SASA, and PCA analyses indicated that NLRP3 was more dispersive and less energetically stable in the active state than in the inactive state and that MCC950 significantly reduced the fluctuations of the interactive residues while BMS986299 did not. The antagonist MCC950 interacted with residues mainly in the NBD, HD1, WHD, and HD2 domains of NLRP3, whereas the agonist BMS986299 mainly in the NBD and WHD of NLRP3. Additionally, both compounds did not interact with residues located in the FISNA domain. The conformation of the FISNA domain appeared to change significantly when NLRP3 was translated from an inactive state to an active state. The antagonist may interact with residues mainly in the NBD, HD1, WHD, and HD2 domains, and the agonist may interact in the NBD and WHD domains. Our study provided new insights into the development of NLRP3 regulators.

HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation.

Ultra-stable fibrous structure is a hallmark of amyloids. In contrast to canonical disease-related amyloids, emerging research indicates that a significant number of cellular amyloids, termed 'functional amyloids', contribute to signal transduction as temporal signaling hubs in humans. However, it is unclear how these functional amyloids are effectively disassembled to terminate signal transduction. RHIM motif-containing amyloids, the largest functional amyloid family discovered thus far, play an important role in mediating necroptosis signal transduction in mammalian cells. Here, we identify heat shock protein family A member 8(HSPA8) as a new type of enzyme-which we name as 'amyloidase'-that directly disassembles RHIM-amyloids to inhibit necroptosis signaling in cells and mice. Different from its role in chaperone-mediated autophagy where it selects substrates containing a KFERQ-like motif, HSPA8 specifically recognizes RHIM-containing proteins through a hydrophobic hexapeptide motif N(X1)φ(X3). The SBD domain of HSPA8 interacts with RHIM-containing proteins, preventing proximate RHIM monomers from stacking into functional fibrils; furthermore, with the NBD domain supplying energy via ATP hydrolysis, HSPA8 breaks down pre-formed RHIM-amyloids into non-functional monomers. Notably, HSPA8's amyloidase activity in disassembling functional RHIM-amyloids does not require its co-chaperone system. Using this amyloidase activity, HSPA8 reverses the initiator RHIM-amyloids (formed by RIP1, ZBP1, and TRIF) to prevent necroptosis initiation, and reverses RIP3-amyloid to prevent necroptosis execution, thus eliminating multi-level RHIM-amyloids to effectively prevent spontaneous necroptosis activation. The discovery that HSPA8 acts as an amyloidase dismantling functional amyloids provides a fundamental understanding of the reversibility nature of functional amyloids, a property distinguishing them from disease-related amyloids that are unbreakable in vivo.

HSPA8 regulates anti-bacterial autophagy through liquid-liquid phase separation

ABSTRACT HSPA8 (heat shock protein family A (Hsp70) member 8) plays a significant role in the autophagic degradation of proteins, however, its effect on protein stabilization and anti-bacterial autophagy remains unknown. Here, it is discovered that HSPA8, as a binding partner of RHOB and BECN1, induce autophagy for intracellular bacteria clearance. Using its NBD and LID domains, HSPA8 physically binds to RHOB residues 1–42 and 89–118 as well as to BECN1 ECD domain, preventing RHOB and BECN1 degradation. Intriguingly, HSPA8 contains predicted intrinsically disordered regions (IDRs), and drives liquid-liquid phase separation (LLPS) to concentrate RHOB and BECN1 into HSPA8-formed liquid-phase droplets, resulting in improved RHOB and BECN1 interactions. Our study reveals a novel role and mechanism of HSPA8 in modulating anti-bacterial autophagy, and highlights the effect of LLPS-related HSPA8-RHOB-BECN1 complex on enhancing protein interaction and stabilization, which improves the understanding of autophagy-mediated defense against bacteria.

Unravelling the Interaction of Piperlongumine with the Nucleotide-Binding Domain of HSP70: A Spectroscopic and In Silico Study

Piperlongumine (PPL) is an alkaloid extracted from several pepper species that exhibits anti-inflammatory and anti-carcinogenic properties. Nevertheless, the molecular mode of action of PPL that confers such powerful pharmacological properties remains unknown. From this perspective, spectroscopic methods aided by computational modeling were employed to characterize the interaction between PPL and nucleotide-binding domain of heat shock protein 70 (NBD/HSP70), which is involved in the pathogenesis of several diseases. Steady-state fluorescence spectroscopy along with time-resolved fluorescence revealed the complex formation based on a static quenching mechanism. Van’t Hoff analyses showed that the binding of PPL toward NBD is driven by equivalent contributions of entropic and enthalpic factors. Furthermore, IDF and Scatchard methods applied to fluorescence intensities determined two cooperative binding sites with Kb of (6.3 ± 0.2) × 104 M−1. Circular dichroism determined the thermal stability of the NBD domain and showed that PPL caused minor changes in the protein secondary structure. Computational simulations elucidated the microenvironment of these interactions, showing that the binding sites are composed mainly of polar amino acids and the predominant interaction of PPL with NBD is Van der Waals in nature.

Identification of molecular switch regulating tyrosine phosphorylation of Intestinal breast cancer resistance protein (BCRP) mediated mucosal wound repair and barrier functions in obesity

Breast cancer resistance protein (BCRP) is a member of ATP-binding cassette (ABC) transporter proteins whose primary function is to efflux substrates bound to the plasma membrane. In our previous study, we reported that obese individuals have compromised intestinal BCRP functions and that diet-induced obese mice recapitulate these outcomes. Furthermore we demonstrated that the compromised BCRP functions during obesity are because of loss of Janus kinase 3 (JAK3)-mediated tyrosine phosphorylation of BCRP. Janus kinase 3 (Jak3) is a non receptor tyrosine kinase expressed in both hematopoietic and nonhematopoietic cells. Although mutations that abrogate Jak3 functions cause different immunological disorders, its constitutive activation leads to various types of inflammatory diseases. Mechanistically, JAK3-mediated phosphorylation of BCRP promoted its interactions with membrane-localized β-catenin essential not only for BCRP expression and surface localization, but also for the maintenance of BCRP-mediated intestinal drug efflux and barrier functions. In the present study, we demonstrate that, BCRP interacted with Jak3, thereby facilitating intestinal barrier function and drug efflux function. Furthermore, we characterized the structural determinants that regulate the interactions between BCRP and Jak3 and pBCRP and β-catenin. Functional reconstitution of kinase activity by recombinant full-length (wt) Jak3 using Jak3-wt or BCRP-wt as substrate showed that Jak3 autophosphorylation was the rate-limiting step during interactions between Jak3 and drug transporter protein. Determination of kinetic parameters showed that phosphorylated (P) Jak3-wt binds to p-BCRP-wt with a dissociation constant K(d)) of 15 nM and a Hill's coefficient of 2.5. Pairwise binding between Jak3 mutants and P-BCRP-wt showed that the FERM domain of Jak3 was sufficient for binding to p-BCRP with a K(d) of 20.0 nM whereas NBD domain of BCRP was contributing more towards it's binding to pJak3 with a K(d) value of 30 nM. Furthermore, Phosphorylated form of F2-subdomain of FERM was responsible for binding of FERM with BCRP. On the other hand NBD domain of BCRP was sufficient to bind to pβ-catenin with K(d)of 22nM. Physiologically, Jak3-mediated phosphorylation of BCRP increased IL2-mediated mucosal wound repair and also facilitated epithelial barrier functions. Our results also show that both BCRP and β-catenin bind to FERM domain of Jak3 non-competitively. We demonstrate that mutation (Y-F) in the tyrosine residue at 123 position of NBD domain of BCRP prevented Jak3 and β-catenin significantly from binding to BCRP. Results from cell culture: Thus, we demonstrate the molecular mechanism of interactions between Jak3 and BCRP and pBCRP and further we are investigating to determine the molecular mechanism involved in BCRP phosphorylation mediated mucosal wound repair.

Synthesis, antibacterial activities and molecular docking study of thiouracil derivatives containing oxadiazole moiety

A series of novel thiouracil derivatives 9 containing an oxadiazole moiety have been synthesized by structural modification of a lead SecA inhibitor, 2. These compounds have been evaluated for their antibacterial activities against Bacillus amyloliquefaciens, Staphylococcus aureus and Bacillus subtilis. Among them, compounds 9g and 9n exhibited promising antibacterial activities against the tested strains. Compound 9g was also tested for its inhibitory activities against SecA ATPase, and the IC50 value of compound 9g was 19.9 μg/mL, lower than that of compound 2 (20.8 µg/mL). Molecular docking work indicates that compound 9g likely occupies the pocket formed by SecA IRA2 and NBD domain.

Domain interactions reveal auto-inhibition of the deubiquitinating enzyme USP19 and its activation by HSP90 in the modulation of huntingtin aggregation.

Ubiquitin-specific protease 19 (USP19) is a member of the deubiquitinating (DUB) enzymes that catalyze removing the ubiquitin signals from target proteins. Our previous research has demonstrated that USP19 up-regulates the protein level and aggregation of polyQ-expanded huntingtin through the involvement of heat shock protein 90 (HSP90). Here, we present solution structures of the CS1, CS2 and UbL domains of USP19 and structural insights into their domain interactions. We found that the tandem CS domains fold back to interact with the C-terminal USP domain (USPD) intra-molecularly that leads to inhibition of the catalytic core of USP19, especially CS1 interacts with the embedded UbL domain and CS2 does with the CH2 catalytic core. Moreover, CS2 specifically interacts with the NBD domain of HSP90, which can activate the DUB enzyme. A mechanism of auto-inhibition of USP19 and activation by HSP90 is proposed, on which USP19 modulates the protein level of polyQ-expanded huntingtin in cells. This study provides structural and mechanistic insights into the modulation of protein level and aggregation by USP19 with the assistance of HSP90.

Hematopoietic Cell Kinase (HCK) Is Essential for NLRP3 Inflammasome Activation and Lipopolysaccharide-Induced Inflammatory Response In Vivo.

Activation of the NLRP3 inflammasome results in caspase 1 cleavage, which subsequently leads to IL-1β and IL-18 secretion, as well as pyroptosis, and aberrant activation of the inflammasome is involved in several diseases such as type 2 diabetes, atherosclerosis, multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease. NLRP3 activity is regulated by various kinases. Genetic and pharmacological inhibition of the hematopoietic cell kinase (HCK), a member of the Src family of non-receptor tyrosine kinases (NRTKs) primarily expressed in myeloid cells, has previously been shown to ameliorate inflammation, indicating that it may be involved in the regulation of microglia function. However, the underlying mechanism is not known. Hence, in this study, we aimed to investigate the role of HCK in NLRP3 inflammasome activation. We demonstrated that HCK silencing inhibited NLRP3 inflammasome activation. Furthermore, the HCK-specific inhibitor, A419259, attenuated the release of IL-1β and caspase 1(P20) from the macrophages and microglia and reduced the formation of the apoptosis-associated speck-like protein with a CARD domain (ASC) oligomer. We also observed that HCK binds to full length NLRP3 and its NBD(NACHT) and LRR domains, but not to the PYD domain. In vivo, the HCK inhibitor attenuated the LPS-induced inflammatory response in the liver of LPS-challenged mice. Collectively, these results suggested that HCK plays a critical role in NLRP3 inflammasome activation. Our results will enhance current understanding regarding the effectiveness of HCK inhibitors for treating acute inflammatory diseases.

The Endogenous Metabolite Sphingosine-1-Phosphate (S1P) Activates NOD1/2-Dependent Inflammation Triggered by Cellular Stress

Cellular stress has been associated with inflammation, yet precise underlying mechanisms remain elusive. In this study, various unrelated stress inducers were employed to screen for sensors linking altered cellular homeostasis and inflammation. We identified the intracellular pattern recognition receptors NOD1/2, known receptors of bacterial peptidoglycans, as general stress sensors detecting perturbations of cellular homeostasis. NOD1/2 activation upon such perturbations required generation of the endogenous metabolite sphingosine-1-phosphate (S1P). Unlike peptidoglycan sensing via the LRR domain, cytosolic S1P directly bound to the NBD domain of NOD1/2, triggering NF-κB activation and inflammatory responses. S1P-NOD1/2 axis was essential for Bleomycin induced inflammation in vivo. In sum, we unveiled a hitherto unknown role of NOD1/2 in surveillance of cellular homeostasis through sensing of cytosolic metabolite S1P. We propose S1P as a novel endogenous stress-associated molecular pattern (SAMP) and NOD1/2 as molecular hubs integrating bacterial and metabolic cues.

Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence.

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the ΔclpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.

Abstract 2754: Affinity-based probe reveal Bim negatively regulates the chaperone functions of Hsp70, a non-Bcl-2 BH3 receptor

Abstract The identification of novel non-Bcl-2 BH3 receptors is still challenge owing to frequently weak, transient and reversible binding character of the PPIs mediated by them. Herein, we designed and synthesized an affinity-based probe (AfBP), S1b-probe, by introducing a photo-crosslinker to an artificial BH3 mimetic. Hsp70 proteins were identified as novel BH3 receptors in situ. The PPIs between Bcl-2 proteins and Hsp70 proteins were further validated by fluorescent polarization (FP), isothermal titration calorimetry (ITC) and Co-immunoprecipitation (Co-IP) experiments. Structural and functional analysis by photo-crosslinking/LC-MS/MS sequencing, 1H-15N transverse relaxation optimized spectroscopy (TROSY-HSQC), trypsin proteolysis, single-turnover ATPase rates and denatured rhodanese aggregation measurement demonstrated that BimBH3 bind in the NBD domain of Hsp70 proteins to act as a co-chaperone that negatively regulates the ATPase activity and chaperone functions. S1g, derived from BH3 mimetics, was screened as a specific Hsp70 inhibitor in native physiological context by using S1b-probe. Citation Format: Ziqian Wang, Zhichao Zhang, Ting Song, Zongwei Guo. Affinity-based probe reveal Bim negatively regulates the chaperone functions of Hsp70, a non-Bcl-2 BH3 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2754.

The nucleotide-bound/substrate-bound conformation of the Mycoplasma genitalium DnaK chaperone.

Hsp70 chaperones keep protein homeostasis facilitating the response of organisms to changes in external and internal conditions. Hsp70s have two domains-nucleotide binding domain (NBD) and substrate binding domain (SBD)-connected by a conserved hydrophobic linker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allosterically coupled, often together with cochaperones, to the binding/release of peptide substrates. Here we describe the crystal structure of the Mycoplasma genitalium DnaK (MgDnaK) protein, an Hsp70 homolog, in the noncompact, nucleotide-bound/substrate-bound conformation. The MgDnaK structure resembles the one from the thermophilic eubacteria DnaK trapped in the same state. However, in MgDnaK the NBD and SBD domains remain close to each other despite the lack of direct interaction between them and with the linker contacting the two subdomains of SBD. These observations suggest that the structures might represent an intermediate of the protein where the conserved linker binds to the SBD to favor the noncompact state of the protein by stabilizing the SBDβ-SBDα subdomains interaction, promoting the capacity of the protein to sample different conformations, which is critical for proper functioning of the molecular chaperone allosteric mechanism. Comparison of the solved structures indicates that the NBD remains essentially invariant in presence or absence of nucleotide.

Genomic analysis of NF-κB signaling pathway reveals its complexity in Crassostrea gigas

NF-κB signaling pathway is an evolutionarily conserved pathway that plays highly important roles in several developmental, cellular and immune response processes. With the recent release of the draft Pacific oyster (Crassostra gigas) genome sequence, we have sought to identify the various components of the NF-κB signaling pathway in these mollusks and investigate their gene structure. We further constructed phylogenetic trees to establish the evolutionary relationship of the oyster proteins with their homologues in vertebrates and invertebrates using BLASTX and neighbor-joining method. We report the presence of two classic NF-κB/Rel homologues in the pacific oyster namely Cgp100 and CgRel, which possess characteristic RHD domain and a consensus nuclear localization signal, similar to mammalian homologues and an additional CgRel-like protein, unique to C. gigas. Further, in addition to two classical IκB homologues, CgIκB1 and CgIκB2, we have identified three atypical IκB family members namely CgIκB3, CgIκB4 and CgBCL3 which lack the IκB degradation motif and consist of only one exon that might have arisen by retrotransposition from CgIκB1. Finally, we report the presence of three IKKs and one NEMO genes in oyster genome, named CgIKK1, CgIKK2, CgIKK3 and CgNEMO, respectively. While CgIKK1 and CgIKK3 domain structure is similar to their mammalian homologues, CgIKK2 was found to lack the HLH and NBD domains. Overall, the high conservation of the NF-κB/Rel, IκB and IKK family components in the pacific oyster and their structural similarity to the vertebrate and invertebrate homologues underline the functional importance of this pathway in regulation of critical cellular processes across species.

New ABCG2 homology model reveals importance of C‐terminus in protein trafficking

Recent genetic studies have implicated ABCG transporters in human disease and identified potential causal variants. However interpretation of the resulting molecular defect is often limited by the lack of high‐resolution crystal structures. This has been the case for ABCG2. Here we present a new homology model of ABCG2 using a recent ABCG5/G8 crystal structure (PDB:5do7) as a template in an effort to better understanding the implications of human ABCG2 gout causing mutations. Our homology model is composed of the ABCG2 NBD domains (residues 29–288) and TMD domains (residues 289–655) and was built using the Swiss‐Model server. To avoid forcing the ABCG2 into a heterodimer, the ABCG5 served as template for both ABCG2 copies because its NBD structure is closer to the canonical NBD structure. The loop regions of the model were further refined by GalaxyLoop. The final model complied with the modular multi‐domain architecture of ABC proteins, and had RMSD values with ABCG5 NBD, ABCG5 TMD, ABCG8 NBD, ABCG8 TMD of 0.2Å, 0.5Å, 1.7Å, and 2.4Å, respectively. The new ABCG2 homology model allows for the visualization of parts of the protein previously undefined. One intriguing finding was that the loop between the first and second β‐strands of the NBD (residues 42–63) is in close proximity with the TMD C‐terminus S655. Notably, the corresponding loop (residues 47–65) and the C‐terminus R651 were not resolved in the ABCG5/G8 crystal structure, suggesting a dynamic interaction in this region. We explored the significance of the C‐terminal tail (resides 652–655; KKYS) on protein abundance and trafficking. The addition of a C‐terminal Flag‐tag or the deletion of the last 4 residues (651X) led to a profound decrease in protein abundance. The 651X protein is not glycosylated and localizes to the ER only. Interestingly, a YS653AA (KKAA) substitution is less deleterious and results in protein that reaches the Golgi compartment, highlighting the dilysine motif, a potential COPI target, as key in trafficking ABCG2. Finally, we found the S655A substitution (KKYA) results is significantly greater protein abundance than wild type and partially rescues the abundance of the Q141K human gout mutation, implicating the Y654 as a key trafficking residue. In conclusion, our new ABCG2 homology model suggests the C‐terminus is in close proximity to the NBD and may be a critical feature in regulating the trafficking of the ABCG2 protein.Support or Funding InformationAmerican Heart Association 14SDG18060004

Evidence of Phospho‐degron regulating expression of urate secretory transporter ABCG2

ABCG2 is a high capacity urate secretory transporter of the renal proximal tubule. Mutations in ABCG2 cause gout in humans. The ABCG2 patient mutation ABCG2Q141K leads to instability of the ABCG2 nucleotide‐binding domain, which can be rescued by enhancing nucleotide‐binding domain sandwich formation with phosphomimetic amino acid substitutions (G188E, S187D); raising the possibility that phosphorylation of residues in the NBD domain may regulate expression and trafficking of ABCG2. An in silico analysis of ABCG2 revealed a limited number of predicted phosphorylation sites including S195, a serine conserved in the mammalian lineage. The upstream RXRXS represents a target motif for both AKT1 and PKA, and we find both co‐immunoprecipitate with ABCG2. Specifically, endogenous AKT1 can pull down both over expressed ABCG2 in HEK293 cells as well as endogenous ABCG2 in mouse kidney lysate. AKT1 and ABCG2 transcript co‐localize in the proximal S2 segment of the mammalian nephron (Mark Knepper RNA‐seq database) and inhibiting the AKT1 kinase cascade with PI3K inhibitor LY294002 dramatically up‐regulated ABCG2 expression. Conversely, activating the AKT1 cascade with FBS down‐regulated ABCG2 expression. Replacement of the S195 residue with the phosphomimetic aspartic acid results in significant reduction in expression, localization of ABCG2 protein to peri‐nuclear compartments, and significant sensitivity to the proteasome inhibitor MG132; marking the S195 residue as a Phospho‐degron. Phosphorylation of the well conserved S195 residue may interfere with ATP binding and/or nucleotide‐binding domain sandwich formation, suggesting a novel regulatory mechanism for function and trafficking in ABC transporters.Support or Funding InformationAmerican Heart Association: 14SDG18060004 & Ardea BioSciences

Erratum to: Successful treatment with adalimumab for severe multifocal choroiditis and panuveitis in presumed (early-onset) ocular sarcoidosis.

Early-onset sarcoidosis (EOS) and Blau syndrome are rare auto-inflammatory diseases characterized by a triad of skin rash, granulomatous uveitis, and symmetrical polyarthritis occurring in early childhood. In this paper, we describe a case report very interesting for the multidisciplinary management (pediatric rheumatologist and ophthalmologist), the challenging diagnosis and the difficult choice of the best treatment. We describe a case report of an 8-year old with recurrent episodes of acute uveitis that developed bilateral granulomatous panuveitis initially treated with topical and systemic steroids. Genetic testing for NOD2/CARD15 revealed a heterozygous mutation on exon 4 in the NBD domain (P268S/SNP5). Therefore, an incomplete EOS was suspected. Because uveitis worsening with multifocal chorioretinitis aggravation, intravenous boluses of methylprednisolone were administered. During the steroids tapering, she flared again, and methotrexate was started along with corticosteroids pulse therapy. However, new ocular granuloma appeared, macular oedema with poor visual outcome occurred, and therefore, adalimumab was added to MTX and steroids. After 6 months since the new therapy started, she had a complete visual recovery, and she was able to stop steroid treatment. At 2 years of follow-up, she is still in remission on treatment, and her visual acuity is normal. No side effects were observed. In our patient, we found a heterozygous mutation on exon 4 in the NBD domain (P268S/SNP5) of NOD2/CARD15 gene and an incomplete EOS was hypothesized. The role of this variant is currently under study. Adalimumab use dramatically changed the course of eye disease, prompting to stop steroid treatment and preserving visual acuity.

A Computational Approach towards the Understanding of Plasmodium falciparum Multidrug Resistance Protein 1

The emergence of drug resistance in Plasmodium falciparum tremendously affected the chemotherapy worldwide while the intense distribution of chloroquine-resistant strains in most of the endemic areas added more complications in the treatment of malaria. The situation has even worsened by the lack of molecular mechanism to understand the resistance conferred by Plasmodia species. Recent studies have suggested the association of antimalarial resistance with P. falciparum multidrug resistance protein 1 (PfMDR1), an ATP-binding cassette (ABC) transporter and a homologue of human P-glycoprotein 1 (P-gp1). The present study deals about the development of PfMDR1 computational model and the model of substrate transport across PfMDR1 with insights derived from conformations relative to inward- and outward-facing topologies that switch on/off the transportation system. Comparison of ATP docked positions and its structural motif binding properties were found to be similar among other ATPases, and thereby contributes to NBD domains dimerization, a unique structural agreement noticed in Mus musculus Pgp and Escherichia coli MDR transporter homolog (MsbA). The interaction of leading antimalarials and phytochemicals within the active pocket of both wild-type and mutant-type PfMDR1 demonstrated the mode of binding and provided insights of less binding affinity thereby contributing to parasite's resistance mechanism.

Simulation of the opening and closing of Hsp70 chaperones by coarse-grained molecular dynamics.

Heat-shock proteins 70 (Hsp70s) are key molecular chaperones which assist in the folding and refolding/disaggregation of proteins. Hsp70s, which consist of a nucleotide-binding domain (NBD, consisting of NBD-I and NBD-II subdomains) and a substrate-binding domain [SBD, further split into the β-sheet (SBD-β) and α-helical (SBD-α) subdomains], occur in two major conformations having (a) a closed SBD, in which the SBD and NBD domains do not interact, (b) an open SBD, in which SBD-α interacts with NBD-I and SBD-β interacts with the top parts of NBD-I and NBD-II. In the SBD-closed conformation, SBD is bound to a substrate protein, with release occurring after transition to the open conformation. While the transition from the closed to the open conformation is triggered efficiently by binding of adenosine triphosphate (ATP) to the NBD, it also occurs, although less frequently, in the absence of ATP. The reverse transition occurs after ATP hydrolysis. Here, we report canonical and multiplexed replica exchange simulations of the conformational dynamics of Hsp70s using a coarse-grained molecular dynamics approach with the UNRES force field. The simulations were run in the following three modes: (i) with the two halves of the NBD unrestrained relative to each other, (ii) with the two halves of the NBD restrained in an "open" geometry as in the SBD-closed form of DnaK (2KHO), and (iii) the two halves of NBD restrained in a "closed" geometry as in known experimental structures of ATP-bound NBD forms of Hsp70. Open conformations, in which the SBD interacted strongly with the NBD, formed spontaneously during all simulations; the number of transitions was largest in simulations carried out with the "closed" NBD domain, and smallest in those carried out with the "open" NBD domain; this observation is in agreement with the experimentally-observed influence of ATP-binding on the transition of Hsp70's from the SBD-closed to the SBD-open form. Two kinds of open conformations were observed: one in which SBD-α interacts with NBD-I and SBD-β interacts with the top parts of NBD-I and NBD-II (as observed in the structures of nucleotide exchange factors), and another one in which this interaction pattern is swapped. A third type of motion, in which SBD-α binds to NBD without dissociating from SBD-β was also observed. It was found that the first stage of interdomain communication (approach of SBD-β, to NBD) is coupled with the rotation of the long axes of NBD-I and NBD-II towards each other. To the best of our knowledge, this is the first successful simulation of the full transition of an Hsp70 from the SBD-closed to the SBD-open conformation.