The Crystal Structure of Human Pyrin B30.2 Domain: Implications for Mutations Associated with Familial Mediterranean Fever

The inherited autoinflammatory syndrome familial Mediterranean fever (FMF) is characterized by recurrent episodes of fever, which are independent of any bacterial or viral infections. This disease is associated with point mutations in the mefv gene product pyrin. Although the precise molecular functions of pyrin are unknown, it seems to be involved in the maturation and secretion of interleukin-1β. Approximately two thirds of all FMF-associated mutations cluster in the C-terminal B30.2 domain of pyrin. To investigate the molecular consequences of FMF-associated mutations, we determined the crystal structure of the pyrin B30.2 domain at 1.35-Å resolution. The comparison with other B30.2/ligand complex structures revealed a shallow cavity, which seems to be involved in binding the pyrin ligand. The bottom of this cavity is covered mainly with hydrophobic amino acids, suggesting that pyrin recognizes its ligand by hydrophobic contacts and surface complementarities. FMF-associated mutations cluster around two sites on the B30.2 surface. Approximately two thirds, including those mutations with the most severe disease outcomes, are observed in the vicinity of the predicted peptide binding site, suggesting that they will have a direct impact on ligand binding. A second mutational hot spot was observed on the opposite side of the B30.2 domain in the neighbourhood of its artificial N-terminus. Although most FMF-associated mutations are solvent exposed, several will modify the main-chain conformation of loops. The experimental crystal structure of the pyrin B30.2 domain serves as a basis for an accurate modelling of these mutations.     

Fighting disease by selective autophagy of aggregate-prone proteins

Ubiquitinated protein aggregates are hallmarks of a range of human diseases, including neurodegenerative, liver and muscle disorders. These protein aggregates are typically positive for the autophagy receptor p62. Whereas the ubiquitin-proteasome system (UPS) degrades shortlived and misfolded ubiquitinated proteins that are small enough to enter the narrow pore of the barrel-shaped proteasome, the lysosomal pathway of autophagy can degrade larger structures including entire organelles or protein aggregates. This degradation requires autophagy receptors that link the cargo with the molecular machinery of autophagy and is enhanced by certain posttranslational modifications of the cargo. In this review we focus on how autophagy clears aggregate-prone proteins and the relevance of this process to protein aggregate associated diseases.     

TNF‐α regulates the early development of avascular necrosis of the femoral head by mediating osteoblast autophagy and apoptosis via the p38 MAPK/NF‐κB signaling pathway

Previous studies have shown that the tumor necrosis factor‐α (TNF‐α) levels in serum and bone tissues formed in avascular necrosis of femoral head (ANFH) patients were higher than those of normal individuals, indicating TNF‐α might play a role in the pathogenesis of ANFH. However, the underlying mechanisms remain unclear. Hematoxylin and eosin staining was performed to show the pathological changes of ANFH bone tissues. TNF‐α expression in normal and ANFH tissues was examined by quantitative real‐time polymerase chain reaction and western blot analyses. Osteoblast autophagy and apoptosis, as well as signaling pathways activation, were measured by their corresponding marker proteins. Osteoblast proliferation, autophagy, and apoptosis were evaluated using cell counting kit‐8, transmission electron microscopy, and flow cytometry. The structures of bone tissues of ANFH were obviously damaged. TNF‐α expression was significantly upregulated in ANFH bone tissues compared to normal tissues. Autophagy and apoptosis were remarkably promoted, and p38 mitogen‐activated protein kinase (MAPK)/nuclear factor‐κB (NF‐κB) signaling pathways were markedly activated in ANFH. Suppression of the p38 MAPK/NF‐κB pathway significantly attenuated the TNF‐α‐induced autophagy, however, enhanced the TNF‐α‐induced apoptosis in osteoblasts. Increased TNF‐α in ANFH regulated osteoblast autophagy and apoptosis by p38 MAPK/NF‐κB signaling pathways, blocking the pathway by inhibitors exacerbated TNF‐α‐induced apoptosis through impairing autophagy flux.     

Extracellular histones activate autophagy and apoptosis via mTOR signaling in human endothelial cells

Circulating histones have been proposed as targets for therapy in sepsis and hyperinflammatory symptoms. However, the proposed strategies have failed in clinical trials. Although different mechanisms for histone-related cytotoxicity are being explored, those mediated by circulating histones are not fully understood. Extracellular histones induce endothelial cell death, thereby contributing to the pathogenesis of complex diseases such as sepsis and septic shock. Therefore, the comprehension of cellular responses triggered by histones is capital to design effective therapeutic strategies. Here we report how extracellular histones induce autophagy and apoptosis in a dose-dependent manner in cultured human endothelial cells. In addition, we describe how histones regulate these pathways via Sestrin2/AMPK/ULK1-mTOR and AKT/mTOR. Furthermore, we evaluate the effect of Toll-like receptors in mediating autophagy and apoptosis demonstrating how TLR inhibitors do not prevent apoptosis and/or autophagy induced by histones. Our results confirm that histones and autophagic pathways can be considered as novel targets to design therapeutic strategies in endothelial damage.     

microRNA-206 is required for osteoarthritis development through its effect on apoptosis and autophagy of articular chondrocytes via modulating the phosphoinositide 3-kinase/protein kinase B-mTOR pathway by targeting insulin-like growth factor-1

microRNA (miR) has been shown to be involved in the treatment of diseases such as osteoarthritis (OA). This study aims to investigate the role of miR-206 in regulating insulin-like growth factor-1 (IGF-1) in chondrocyte autophagy and apoptosis in an OA rat model via the phosphoinositide 3-kinase (P13K)/protein kinase B (AKT)-mechanistic target of rapamycin (mTOR) signaling pathway. Wistar rats were used to establish the OA rat model, followed by the observation of histopathological changes, Mankin score, and the detection of IGF-1-positive expression and tissue apoptosis. The underlying regulatory mechanisms of miR-206 were analyzed in concert with treatment by an miR-206 mimic, an miR-206 inhibitor, or small interfering RNA against IGF-1 in chondrocytes isolated from OA rats. Then, the expression of miR-206, IGF-1, and related factors in the signaling pathway, cell cycle, and apoptosis, as well as inflammatory factors, were determined. Subsequently, chondrocyte proliferation, cell cycle distribution, apoptosis, autophagy, and autolysosome were measured. OA articular cartilage tissue exhibited a higher Mankin score, promoted cell apoptotic rate, increased expression of IGF-1, Beclin1, light chain 3 (LC3), Unc-51-like autophagy activating kinase 1 (ULK1), autophagy-related 5 (Atg5), caspase-3, and Bax, yet exhibited decreased expression of miR-206, P13K, AKT, mTOR, and Bcl-2. Besides, miR-206 downregulated the expression of IGF-1 and activated the P13K/AKT signaling pathway. Moreover, miR-206 overexpression and IGF-1 silencing inhibited the interleukins levels (IL-6, IL-17, and IL-18), cell apoptotic rate, the formation of autolysosome, and cell autophagy while promoting the expression of IL-1β and cell proliferation. The findings from our study provide a basis for the efficient treatment of OA by investigating the inhibitory effects of miR-206 on autophagy and apoptosis of articular cartilage in OA via activating the IGF-1-mediated PI3K/AKT-mTOR signaling pathway.     

High density lipoprotein inhibits the activation of sterol regulatory element-binding protein-1 in cultured cells

A link between cellular uptake of high density lipoprotein (HDL) and regulation of sterol regulatory element-binding protein-1 (SREBP-1) was investigated in vitro. HDL decreased nuclear SREBP-1 levels as well as SREBP-1 target gene expression in HepG2 and HEK293 cells. However, HDL did not repress an exogenously expressed, constitutively active form of SREBP-1. HDL increased cellular cholesterol levels, and cellular cholesterol depletion by methyl-β-cyclodextrin abolished the effects of HDL. These results suggest that HDL inhibits the activation of SREBP-1 through a cholesterol-dependent mechanism, which may play an important role in regulating lipid synthetic pathways mediated by SREBP-1.     

Discovering differential activation machinery of the Toll-like receptor 4 signaling pathways in MyD88 knockouts

To understand differential time activation of nuclear factor kappaB (NF-kappaB) and the temporal features of the downstream pro-inflammatory cytokines' [tumour-necrosis-factor-alpha (TNF-alpha) and IP-10] mRNA levels in myeloid differentiation primary-response protein 88 (MyD88) knockouts (KOs), I developed a computational model of the TLR4 pathway. The result suggests that the late phase expression of NF-kappaB activity observed in MyD88 KOs is possibly due to a number of novel intermediates acting along the MyD88-independent pathway. I also simulate that the TNF-alpha levels will increase at a longer time in MyD88 KOs, not previously mentioned.     

Crystal structure of the IL-22/IL-22R1 complex and its implications for the IL-22 signaling mechanism

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2. STRUCTURED SUMMARY:     

Distinct roles of the R3H and RRM domains in poly(A)-specific ribonuclease structural integrity and catalysis

Deadenylases specifically catalyze the degradation of eukaryotic mRNA poly(A) tail in the 3′- to 5′-end direction with the release of 5′-AMP as the product. Among the deadenylase family, poly(A)-specific ribonuclease (PARN) is unique in its domain composition, which contains three potential RNA-binding domains: the catalytic nuclease domain, the R3H domain and the RRM domain. In this research, we investigated the roles of these RNA-binding domains by comparing the structural features and enzymatic properties of mutants lacking either one or two of the three RNA-binding domains. The results showed that the R3H domain had the ability to bind various oligonucleotides at the micromolar level with no oligo(A) specificity. The removal of the R3H domain dissociated PARN into monomers, which still possessed the RNA-binding ability and catalytic functions. Unlike the critical role of the RRM domain in PARN processivity, the removal of the R3H domain did not affect the catalytic pattern of PARN. Our results suggested that both R3H and RRM domains were essential for the high affinity of long poly(A) substrate, but the R3H domain did not contribute to the substrate recognition of PARN. Compared to the RRM domain, the R3H domain played a more important role in the structural integrity of the dimeric PARN. The multiple RNA-binding domain architecture endows PARN the property of highly efficient catalysis in a highly processive mode.     

Sera from amyotrophic lateral sclerosis patients induce the non-canonical activation of NMDA receptors "in vitro"

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by the selective loss of both upper and lower motoneurons (MNs). The familial form of the illness is associated with mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD-1) enzyme, but it accounts for fewer than 10% of cases; the rest, more than 90%, correspond to the sporadic form of ALS. Although many proposals have been suggested over the years, the mechanisms underlying the characteristic selective killing of MN in ALS remain unknown. In this study we tested the effect of sera from sporadic ALS patients on NMDA receptors (NMDAR). We hypothesize that an endogenous seric factor is implicated in neuronal death in ALS, mediated by the modulation of NMDAR.Sera from ALS patients and from healthy subjects were pretreated to inactivate complement pathways and dialyzed to remove glutamate and glycine. IgGs from ALS patients and healthy subjects were obtained by affinity chromatography and dialyzed against phosphate-buffered saline. Human NMDAR were expressed in Xenopus laevis oocytes, and ionic currents were recorded using the two-electrode voltage clamp technique.Sera from sporadic ALS patients induced transient oscillatory currents in oocytes expressing NMDAR with a significantly higher total electrical charge than that induced by sera from healthy subjects. Sera from patients with other neuromuscular diseases did not exert this effect. The currents were inhibited by MK-801, a noncompetitive blocker of NMDAR. The PLC inhibitor, U-73122, and the IP₃ receptor antagonist, 2-APB, also inhibited the sera-induced currents. The oscillatory signal recorded was due to internal calcium mobilization. Isolated IgGs from ALS patients significantly affected the activity of oocytes injected with NMDAR, causing a 2-fold increase over the response recorded for IgGs from healthy subjects.Our data support the notion that ALS sera contain soluble factors that mobilize intracellular calcium, not opening directly the ionic conductance, but through the non-canonical activation of NMDAR.     

Characterization of interleukin-1 receptor-associated kinase 1 binding protein 1 gene in small abalone Haliotis diversicolor

Interleukin receptor-associated kinase (IRAK)-1 binding protein 1 (IRAK1BP1) is a critical factor in preventing dangerous overproduction of proinflammatory cytokines by the innate immune system and in influencing the specificity of TLR responses. In this study, a first molluscan IRAK1BP1 gene, saIRAK1BP1, was cloned from the small abalone (Haliotis diversicolor). Its full-length cDNA sequence is 1047bp, with a 747bp open reading frame encoding a protein of 249 aa. The molecular mass of the deduced protein is approximately 28.1kDa with an estimated pI of 8.87, and shows highest identity (52%) to acorn worm Saccoglossus kowalevskii. Amino acid sequence analysis revealed that saIRAK1BP1 shares a conserved SIMPL domain. Quantitative real-time PCR was employed to investigate the tissue distribution of saIRAK1BP1 mRNA, and its expression in abalone under bacteria challenge and larvae at different developmental stages. The saIRAK1BP1 mRNA could be detected in all examined tissues, with the highest expression level in hemocytes, and was up-regulated in gills, kidneys and hemocytes after bacteria injection. Additionally, saIRAK1BP1 was constitutively expressed at all examined developmental stages. These results indicate that saIRAK1BP1 play an important role in the adult abalone immune system and might be essential in embryo and larval development in abalone.     

Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment

Initially understood for its physiological maintenance of self-tolerance, the immune checkpoint molecule has recently been recognized as a promising anti-cancer target. There has been considerable interest in the biology and the action mechanism of the immune checkpoint therapy, and their incorporation with other therapeutic regimens. Recently the small-molecule inhibitor (SMI) has been identified as an attractive combination partner for immune checkpoint inhibitors (ICIs) and is becoming a novel direction for the field of combination drug design. In this review, we provide a systematic discussion of the biology and function of major immune checkpoint molecules, and their interactions with corresponding targeting agents. With both preclinical studies and clinical trials, we especially highlight the ICI + SMI combination, with its recent advances as well as its application challenges.