Jixi Li

Structural Biology of Innate Immunity

Innate immune responses depend on timely recognition of pathogenic or danger signals by multiple cell surface or cytoplasmic receptors and transmission of signals for proper counteractions through adaptor and effector molecules. At the forefront of innate immunity are four major signaling pathways, including those elicited by Toll-like receptors, RIG-I-like receptors, inflammasomes, or cGAS, each with its own cellular localization, ligand specificity, and signal relay mechanism. They collectively engage a number of overlapping signaling outcomes, such as NF-κB activation, interferon response, cytokine maturation, and cell death. Several proteins often assemble into a supramolecular complex to enable signal transduction and amplification. In this article, we review the recent progress in mechanistic delineation of proteins in these pathways, their structural features, modes of ligand recognition, conformational changes, and homo- and hetero-oligomeric interactions within the supramolecular complexes. Regardless of seemingly distinct interactions and mechanisms, the recurring themes appear to consist of autoinhibited resting-state receptors, ligand-induced conformational changes, and higher-order assemblies of activated receptors, adaptors, and signaling enzymes through conserved protein-protein interactions.

Structural Insights into the Assembly of Large Oligomeric Signalosomes in the Toll-Like Receptor–Interleukin-1 Receptor Superfamily

Structural studies show that Toll-like receptors assemble into large oligomeric intracellular signaling complexes upon ligand binding. Innate immunity and inflammation constitute the initial responses to injury or infection. Toll-like receptors (TLRs) participate in the recognition of invading viruses and microorganisms by recognizing molecules that are either not present or uncommon in the host cell, known as pathogen-associated molecular patterns (PAMPs). The potent proinflammatory cytokine interleukin-1β (IL-1β) is recognized by IL-1 receptor I (IL-1RI) and the co-receptor IL-1RAcP. Upon recognition of their cognate ligand, these receptors initiate a shared cytosolic signaling cascade. Structural studies have begun to reveal that these signaling cascades coalesce into large oligomeric signaling complexes or “signalosomes” for signal propagation. Through phosphorylation and ubiquitination reactions, this signaling results in the activation of the inhibitor of nuclear factor κB (NF-κB) kinase (IKK), which then phosphorylates the NF-κB inhibitor IκB, promoting its degradation. Degradation of IκB enables the transcription factor NF-κB to translocate to the nucleus and promote the expression of genes whose products are involved in innate and adaptive immunity, inflammation, cell survival, and proliferation. The Toll-like receptor (TLR)–interleukin 1 receptor (IL-1R) superfamily plays fundamentally important roles in innate immune and inflammatory responses. Structural studies have begun to show that upon ligand stimulation, TLRs and IL-1Rs assemble large oligomeric intracellular signaling complexes, or “signalosomes,” to induce the activation of kinases and E3 ubiquitin ligases, leading eventually to the activation of the transcription factors that are responsible for the expression of genes whose products mediate immune and inflammatory responses. The different scaffolds identified by these structural studies provide a molecular foundation for understanding the formation of microscopically visible signaling clusters that have long been known to cell biologists. Here, we illustrate the potential mechanisms of step-by-step assembly from the membrane-proximal interactions to the more downstream events. Formation of large oligomeric signalosomes may help to establish a digital threshold response in TLR and IL-1R signaling.

Cloning and Analysis of Human Apg16L

Autophagy is an intracellular bulk degradation system, which delivers cytoplasmic components to the lysosome/vacuole. In yeast and mammalian cells, the Apg12-Apg5 conjugate, together with Apg16, form a multimeric complex, which plays an essential role in autopihageosome formation. By large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a putative protein with 607 amino acid residues, which shows 90% identity and 93% similarity to mouse Apg16L. This protein, designated human Apg16L, contains a coiled-coil domain and a motif with seven WD repeats, which are also shared by mouse Apg16L. Database searching revealed that Apg16L is mapped to chromosome 2q37.1 and there exist at least four splice variants.

Structural study of the RIPoptosome core reveals a helical assembly for kinase recruitment.

Receptor interaction protein kinase 1 (RIP1) is a molecular cell-fate switch. RIP1, together with Fas-associated protein with death domain (FADD) and caspase-8, forms the RIPoptosome that activates apoptosis. RIP1 also associates with RIP3 to form the necrosome that triggers necroptosis. The RIPoptosome assembles through interactions between the death domains (DDs) of RIP1 and FADD and between death effector domains (DEDs) of FADD and caspase-8. In this study, we analyzed the overall structure of the RIP1 DD/FADD DD complex, the core of the RIPoptosome, by negative-stain electron microscopy and modeling. The results show that RIP1 DD and FADD DD form a stable complex in vitro similar to the previously described Fas DD/FADD DD complex, suggesting that the RIPoptosome and the Fas death-inducing signaling complex share a common assembly mechanism. Both complexes adopt a helical conformation that requires type I, II, and III interactions between the death domains.

Identification and characterization of human uracil phosphoribosyltransferase (UPRTase)

AbstractUracil phosphoribosyltransferase, which catalyzes the conversion of uracil and 5-phosphoribosyl-1-R-diphosphate to uridine monophosphate, is important in the pyrimidine salvage pathway and is an attractive target for rational drug design by incorporation of prodrugs that are lethal to many parasitic organisms specifically. So far, uracil phosphoribosyltransferase has been reported in Arabidopsis thaliana only, not in mammals. In this study, a novel uracil phosphoribosyltransferase family cDNA encoding a 309 amino acid protein with a putative uracil phosphoribosyltransferase domain was isolated from the human fetal brain library. It was named human UPRTase (uracil phosphoribosyltransferase). The ORF of human UPRTase gene was cloned into pQE30 and expressed in Escherichia coli M15. The protein was purified by Ni-NTA affinity chromatography, but UPRTase activity could not be detected by spectrophotometry. RT-PCR analysis showed that human UPRTase was strongly expressed in blood leukocytes, liver, spleen, and thymus, with lower levels of expression in the prostate, heart, brain, lung, and skeletal muscle. Subcellular location of UPRTase-EGFP fusion protein revealed that human UPRTase was distributed in the nucleus and cytoplasm of AD293 cells. Evolutional tree analyses of UPRTases or UPRTase-domain-containing proteins showed that UPRTase was conserved in organisms. UPRTases of archaebacteria or eubacterium had UPRTase activity whereas those higher than Caenorhabditis elegans, which lacked two amino acids in the uracil-binding region, had no UPRTase activity. This means that human UPRTase may have enzymatic activity with another, unknown, factor or have other activity in pyrimidine metabolism.

Crystal structure and versatile functional roles of the COP9 signalosome subunit 1

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) plays key roles in many biological processes, such as repression of photomorphogenesis in plants and protein subcellular localization, DNA-damage response, and NF-κB activation in mammals. It is an evolutionarily conserved eight-protein complex with subunits CSN1 to CSN8 named following the descending order of molecular weights. Here, we report the crystal structure of the largest CSN subunit, CSN1 from Arabidopsis thaliana (atCSN1), which belongs to the Proteasome, COP9 signalosome, Initiation factor 3 (PCI) domain containing CSN subunit family, at 2.7 Å resolution. In contrast to previous predictions and distinct from the PCI-containing 26S proteasome regulatory particle subunit Rpn6 structure, the atCSN1 structure reveals an overall globular fold, with four domains consisting of helical repeat-I, linker helix, helical repeat-II, and the C-terminal PCI domain. Our small-angle X-ray scattering envelope of the CSN1–CSN7 complex agrees with the EM structure of the CSN alone (apo-CSN) and suggests that the PCI end of each molecule may mediate the interaction. Fitting of the CSN1 structure into the CSN–Skp1-Cul1-Fbox (SCF) EM structure shows that the PCI domain of CSN1 situates at the hub of the CSN for interaction with several other subunits whereas the linker helix and helical repeat-II of CSN1 contacts SCF using a conserved surface patch. Furthermore, we show that, in human, the C-terminal tail of CSN1, a segment not included in our crystal structure, interacts with IκBα in the NF-κB pathway. Therefore, the CSN complex uses multiple mechanisms to hinder NF-κB activation, a principle likely to hold true for its regulation of many other targets and pathways.

Structural basis of cell apoptosis and necrosis in TNFR signaling.

The tumor necrosis factor receptors (TNFRs) play essential roles in innate and adaptive immunity. Depending on conditions, TNFR induces multiple cell fates including cell survival, cell apoptosis, and cell programmed necrosis. Here, we review recent progress in structural studies of the TNFR signaling pathway. The structural basis for the high order signal complexes, including the DISC, ripoptosome, necrosome, and RIP3/MLKL complex, may provide novel insights for understanding the biophysical principles of cell signaling cascades.

Structural insights of a hormone sensitive lipase homologue Est22.

Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate. The crystal structures of wild type and mutated Est22 with its product p-nitrophenol are solved with resolutions ranging from 1.4 Å to 2.43 Å. The Est22 exhibits a α/β-hydrolase fold consisting with a catalytic domain and a substrate-recognizing cap domain. Residues Ser188, Asp287, and His317 comprise the catalytic triad in the catalytic domain. The p-nitrophenol molecule occupies the substrate binding pocket and forms hydrogen bonds with adjacent residues Gly108, Gly109, and Gly189. Est22 exhibits a dimeric form in solution, whereas mutants D287A and H317A change to polymeric form, which totally abolished its enzymatic activities. Our study provides insights into the catalytic mechanism of HSL family esterase and facilitates the understanding for further industrial and biotechnological applications of esterases.

Molecular cloning and characterization of a novel human putative transmembrane protein homologous to mouse sideroflexin associated with sideroblastic anemia.

Sideroflexin1 (Sfxn1), the prototype of a novel family of evolutionarily conserved proteins present in eukaryotes, has been found mutated in mice with siderocytic anemia. It is speculated that this protein facilitates the transport of a component required for iron utilization into mitochondrial. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA encoding a novel sideroflexin protein (SFXN4), which showed 59% identity and 71% similarity to mouse sideroflexin4. According to the search of the human genome database, SFXN4 gene is mapped to chromosome 10q25-26 and spans more than 24.7 kb of the genomic DNA. It is 1428 base pair in length and the putative protein contains 305 amino acids with a conserved predicted five-transmembrane-domains structure. RT-PCR result shows that the SFXN4 gene is expressed in many tissues.

A human homolog of the yeast gene encoding tRNA 2'-phosphotransferase: cloning, characterization and complementation analysis

The Saccharomyces cerevisiae TPT1 gene plays a role in removing the 2′-phosphate from ligated tRNA during the maturation of pre-tRNA. Here we reported the cloning and characterization of the human TRPT1 gene as a homolog of yeast TPT1. The TRPT1 gene is located at human chromosome 11q13 and encodes a polypeptide of 253 amino acids. BLAST searches with its amino acid sequence revealed the ubiquitous occurrence of TRPT1 homologs and their functional relationships with the presence of the DUF60/KptA domain. Northern analysis demonstrated that the gene is primarily expressed in heart and skeletal muscle, with lower or undetectable levels in other tissues studied. A plasmid-shuffling experiment showed that the human TRPT1 gene could complement the tpt1 mutation in S. cerevisiae