Native architecture of a human GBP1 defense complex for cell-autonomous immunity to infection.

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.

Progerin, an Aberrant Spliced Form of Lamin A, Is a Potential Therapeutic Target for HGPS.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder caused by the mutant protein progerin, which is expressed by the abnormal splicing of the LMNA gene. HGPS affects systemic levels, with the exception of cognition or brain development, in children, showing that cellular aging can occur in the short term. Studying progeria could be useful in unraveling the causes of human aging (as well as fatal age-related disorders). Elucidating the clear cause of HGPS or the development of a therapeutic medicine could improve the quality of life and extend the survival of patients. This review aimed to (i) briefly describe how progerin was discovered as the causative agent of HGPS, (ii) elucidate the puzzling observation of the absence of primary neurological disease in HGPS, (iii) present several studies showing the deleterious effects of progerin and the beneficial effects of its inhibition, and (iv) summarize research to develop a therapy for HGPS and introduce clinical trials for its treatment.

Progerinin, an Inhibitor of Progerin, Alleviates Cardiac Abnormalities in a Model Mouse of Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford Progeria Syndrome (HGPS) is an ultra-rare human premature aging disorder that precipitates death because of cardiac disease. Almost all cases of HGPS are caused by aberrant splicing of the LMNA gene that results in the production of a mutant Lamin A protein termed progerin. In our previous study, treatment with Progerinin has been shown to reduce progerin expression and improve aging phenotypes in vitro and in vivo HGPS models. In this record, cardiac parameters (stroke volume (SV), ejection fraction (EF), fractional shortening (FS), etc.) were acquired in LmnaWT/WT and LmnaG609G/WT mice fed with either a vehicle diet or a Progerinin diet by echocardiography (from 38 weeks to 50 weeks at various ages), and then the cardiac function was analyzed. We also acquired the tissue samples and blood serum of LmnaWT/WT and LmnaG609G/WT mice for pathological analysis at the end of echocardiography. From these data, we suggest that the administration of Progerinin in the HGPS model mouse can restore cardiac function and correct arterial abnormalities. These observations provide encouraging evidence for the efficacy of Progerinin for cardiac dysfunction in HGPS.

Splicing Variants, Protein-Protein Interactions, and Drug Targeting in Hutchinson-Gilford Progeria Syndrome and Small Cell Lung Cancer.

Alternative splicing (AS) is a biological operation that enables a messenger RNA to encode protein variants (isoforms) that give one gene several functions or properties. This process provides one of the major sources of use for understanding the proteomic diversity of multicellular organisms. In combination with post-translational modifications, it contributes to generating a variety of protein-protein interactions (PPIs) that are essential to cellular homeostasis or proteostasis. However, cells exposed to many kinds of stresses (aging, genetic changes, carcinogens, etc.) sometimes derive cancer or disease onset from aberrant PPIs caused by DNA mutations. In this review, we summarize how splicing variants may form a neomorphic protein complex and cause diseases such as Hutchinson-Gilford progeria syndrome (HGPS) and small cell lung cancer (SCLC), and we discuss how protein-protein interfaces obtained from the variants may represent efficient therapeutic target sites to treat HGPS and SCLC.

RKIP Induction Promotes Tumor Differentiation via SOX2 Degradation in NF2-Deficient Conditions.

Loss of NF2 (merlin) has been suggested as a genetic cause of neurofibromatosis type 2 and malignant peripheral nerve sheath tumor (MPNST). Previously, we demonstrated that NF2 sustained TGFß receptor 2 (TßR2) expression and reduction or loss of NF2 activated non-canonical TGFß signaling, which reduced Raf kinase inhibitor protein (RKIP) expression via TßR1 kinase activity. Here, we show that a selective RKIP inducer (novel chemical, Nf18001) inhibits tumor growth and promotes schwannoma cell differentiation into mature Schwann cells under NF2-deficient conditions. In addition, Nf18001 is not cytotoxic to cells expressing NF2 and is not disturb canonical TGFß signaling. Moreover, the novel chemical induces expression of SOX10, a marker of differentiated Schwann cells, and promotes nuclear export and degradation of SOX2, a stem cell factor. Treatment with Nf18001 inhibited tumor growth in an allograft model with mouse schwannoma cells. These results strongly suggest that selective RKIP inducers could be useful for the treatment of neurofibromatosis type 2 as well as NF2-deficient MPNST. IMPLICATIONS: This study identifies that a selective RKIP inducer inhibits tumor growth and promotes schwannoma cell differentiation under NF2-deficient conditions by reducing SOX2 and increasing SOX10 expression.

Novel chemical inhibitor against SOD1 misfolding and aggregation protects neuron-loss and ameliorates disease symptoms in ALS mouse model.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu, Zn-superoxide dismutase (SOD1) causing the gain of its toxic property are the major culprit of familial ALS (fALS). The abnormal SOD1 aggregation in the motor neurons has been suggested as the major pathological hallmark of ALS patients. However, the development of pharmacological interventions against SOD1 still needs further investigation. In this study, using ELISA-based chemical screening with wild and mutant SOD1 proteins, we screened a new small molecule, PRG-A01, which could block the misfolding/aggregation of SOD1 or TDP-43. The drug rescued the cell death induced by mutant SOD1 in human neuroblastoma cell line. Administration of PRG-A01 into the ALS model mouse resulted in significant improvement of muscle strength, motor neuron viability and mobility with extended lifespan. These results suggest that SOD1 misfolding/aggregation is a potent therapeutic target for SOD1 related ALS.

A human apolipoprotein L with detergent-like activity kills intracellular pathogens.

Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.

Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, and GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D-dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-caspase-4 pathway in antibacterial defense.

NADPH Oxidase and Guanylate Binding Protein 5 Restrict Survival of Avirulent Type III Strains of Toxoplasma gondii in Naive Macrophages.

Phagocytic cells are the first line of innate defense against intracellular pathogens, and yet Toxoplasma gondii is renowned for its ability to survive in macrophages, although this paradigm is based on virulent type I parasites. Surprisingly, we find that avirulent type III parasites are preferentially cleared in naive macrophages, independent of gamma interferon (IFN-γ) activation. The ability of naive macrophages to clear type III parasites was dependent on enhanced activity of NADPH oxidase (Nox)-generated reactive oxygen species (ROS) and induction of guanylate binding protein 5 (Gbp5). Macrophages infected with type III parasites (CTG strain) showed a time-dependent increase in intracellular ROS generation that was higher than that induced by type I parasites (GT1 strain). The absence of Nox1 or Nox2, gp91 subunit isoforms of the Nox complex, reversed ROS-mediated clearance of CTG parasites. Consistent with this finding, both Nox1-/- and Nox2-/- mice showed higher susceptibility to CTG infection than wild-type mice. Additionally, Gbp5 expression was induced upon infection and the enhanced clearance of CTG strain parasites was reversed in Gbp5-/- macrophages. Expression of a type I ROP18 allele in CTG prevented clearance in naive macrophages, suggesting that it plays a role counteracting Gbp5. Although ROS and Gbp5 have been linked to activation of the NLRP3 inflammasome, clearance of CTG parasites did not rely on induction of pyroptosis. Collectively, these findings reveal that not all strains of T. gondii are adept at avoiding clearance in macrophages and define new roles for ROS and Gbps in controlling this important intracellular pathogen.IMPORTANCEToxoplasma infections in humans and other mammals are largely controlled by IFN-γ produced by the activated adaptive immune system. However, we still do not completely understand the role of cell-intrinsic functions in controlling Toxoplasma or other apicomplexan infections. The present work identifies intrinsic activities in naive macrophages in counteracting T. gondii infection. Using an avirulent strain of T. gondii, we highlight the importance of Nox complexes in conferring protection against parasite infection both in vitro and in vivo We also identify Gbp5 as a novel macrophage factor involved in limiting intracellular infection by avirulent strains of T. gondii The rarity of human infections caused by type III strains suggests that these mechanisms may also be important in controlling human toxoplasmosis. These findings further extend our understanding of host responses and defense mechanisms that act to control parasitic infections at the cellular level.

Erratum: E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP.

This corrects the article DOI: 10.1038/ncomms15865.

E3 Ubiquitin ligase ZNRF4 negatively regulates NOD2 signalling and induces tolerance to MDP.

Optimal regulation of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is essential for controlling bacterial infections and inflammatory disorders. Chronic NOD2 stimulation induces non-responsiveness to restimulation, termed NOD2-induced tolerance. Although the levels of the NOD2 adaptor, RIP2, are reported to regulate both acute and chronic NOD2 signalling, how RIP2 levels are modulated is unclear. Here we show that ZNRF4 induces K48-linked ubiquitination of RIP2 and promotes RIP2 degradation. A fraction of RIP2 localizes to the endoplasmic reticulum (ER), where it interacts with ZNRF4 under either 55 unstimulated and muramyl dipeptide-stimulated conditions. Znrf4 knockdown monocytes have sustained nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, and Znrf4 knockdown mice have reduced NOD2-induced tolerance and more effective control of Listeria monocytogenes infection. Our results thus demonstrate E3-ubiquitin ligase ZNRF4-mediated RIP2 degradation as a negative regulatory mechanism of NOD2-induced NF-κB, cytokine and anti-bacterial responses in vitro and in vivo, and identify a ZNRF4-RIP2 axis of fine-tuning NOD2 signalling to promote protective host immunity.

Interferon-induced guanylate-binding proteins in inflammasome activation and host defense.

Traditional views of the inflammasome highlight the assembly of pre-existing core components shortly after infection or tissue damage. Emerging work, however, suggests that the inflammasome machinery is also subject to 'tunable' or inducible signals that might accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these signals operate downstream of interferon receptors to elicit inflammasome regulators, including a new family of interferon-induced GTPases called 'guanylate-binding proteins' (GBPs). Here we investigate the critical roles of interferon-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity to a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential effect of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases.

Functional insight from the tetratricopeptide repeat-like motifs of the type III secretion chaperone SicA in Salmonella enterica serovar Typhimurium.

SicA functions both as a class II chaperone for SipB and SipC of the type III secretion system (T3SS)-1 and as a transcriptional cofactor for the AraC-type transcription factor InvF in Salmonella enterica subsp. enterica serovar Typhimurium. Bioinformatic analysis has predicted that SicA possesses three tetratricopeptide repeat (TPR)-like motifs, which are important for protein-protein interactions and serve as multiprotein complex mediators. To investigate whether the TPR-like motifs in SicA are critical for its transcriptional cofactor function, the canonical residues in these motifs were mutated to glutamate (SicAA44E , SicAA78E , and SicAG112E ). None of these mutants except SicAA44E were able to activate the expression of the sipB and sigD genes. SicAA44E still has a capacity to interact with InvF in vitro, and despite its instability in cell, it could activate the sigDE operon. This suggests that TPR motifs are important for the transcriptional cofactor function of the SicA chaperone.

Enhancement of host immune responses by oral vaccination to Salmonella enterica serovar Typhimurium harboring both FliC and FljB flagella.

Flagellin, the structural component of the flagellar filament in various motile bacteria, can contribute to the activation of NF-κB and proinflammatory cytokine expression during the innate immune response in host cells. Thus, flagellin proteins represent a particularly attractive target for the development of vaccine candidates. In this study, we investigated the immune response by increasing the flagella number in the iacP mutant strain and the adjuvant activity of the flagellin component FljB of Salmonella enterica serovar Typhimurium. We found that the iacP mutant strain expresses two flagellin proteins (FliC and FljB), which result in increased NF-κB-dependent gene expression in bone marrow derived macrophages. Using an oral immunization mouse model, we observed that the administration of a live attenuated S. typhimurium BRD509 strain expressing the FliC and FljB flagellins induced significantly enhanced flagellin-specific IgG responses in the systemic compartment. The mice immunized with the recombinant attenuated S. typhimurium strain that has two types of flagella were protected from lethal challenge with the Salmonella SL1344 strain. These results indicate that overexpression of flagella in the iacP mutant strain enhance the induction of an antigen-specific immune responses in macrophage cell, and both the FliC and FljB flagellar filament proteins-producing S. typhimurium can induce protective immune responses against salmonellosis.

Macrophage migration inhibitory factor (MIF) is a critical mediator of the innate immune response to Mycobacterium tuberculosis.

Macrophage migration inhibitory factor (MIF), an innate cytokine encoded in a functionally polymorphic genetic locus, contributes to detrimental inflammation but may be crucial for controlling infection. We explored the role of variant MIF alleles in tuberculosis. In a Ugandan cohort, genetic low expressers of MIF were 2.4-times more frequently identified among patients with Mycobacterium tuberculosis (TB) bacteremia than those without. We also found mycobacteria-stimulated transcription of MIF and serum MIF levels to be correlated with MIF genotype in human macrophages and in a separate cohort of US TB patients, respectively. To determine mechanisms for MIF's protective role, we studied both aerosolized and i.v. models of mycobacterial infection and observed MIF-deficient mice to succumb more quickly with higher organism burden, increased lung pathology, and decreased innate cytokine production (TNF-α, IL-12, IL-10). MIF-deficient animals showed increased pulmonary neutrophil accumulation but preserved adaptive immune response. MIF-deficient macrophages demonstrated decreased cytokine and reactive oxygen production and impaired mycobacterial killing. Transcriptional investigation of MIF-deficient macrophages revealed reduced expression of the pattern recognition receptor dectin-1; restoration of dectin-1 expression recovered innate cytokine production and mycobacterial killing. Our data place MIF in a crucial upstream position in the innate immune response to mycobacteria and suggest that commonly occurring low expression MIF alleles confer an increased risk of TB disease in some populations.

RaoN, a small RNA encoded within Salmonella pathogenicity island-11, confers resistance to macrophage-induced stress.

Bacterial small non-coding RNAs act as important regulators that control numerous cellular processes. Here we identified RaoN, a novel small RNA encoded in the cspH-envE intergenic region on Salmonella pathogenicity island-11 (SPI-11). RaoN contributes to survival under conditions of acid and oxidative stress combined with nutrient limitation, which partially mimic the intramacrophage environment. Indeed, inactivation of raoN reduces the intramacrophage replication of Salmonella enterica serovar Typhimurium. Genome-wide transcriptome analysis revealed that the lactate dehydrogenase gene ldhA is upregulated in the raoN knockout mutant. Notably, both inactivation and overexpression of ldhA in the WT strain render Salmonella more sensitive to oxidative stress, particularly when combined with nutrient limitation. However, ldhA is not the sole determinant of RaoN function in facilitating intramacrophage survival of Salmonella. Together, our data suggest that balanced regulation of ldhA expression by RaoN is necessary for survival under in vitro stress conditions and contributes to the intramacrophage growth of Salmonella.

Molecular characterization of the InvE regulator in the secretion of type III secretion translocases in Salmonella enterica serovar Typhimurium.

The type III secretion systems (T3SSs) are exploited by many Gram-negative pathogenic bacteria to deliver a set of effector proteins into the host cytosol during cell entry. The T3SS of Salmonella enterica serovar Typhimurium is composed of more than 20 proteins that constitute the membrane-associated base, the needle and the tip complex at the distal end of the T3SS needle. Membrane docking and piercing between the T3SS and host cells is followed by the secretion of effector proteins. Therefore, a secretion hierarchy among the substrates of the T3SS is required. The secretion of the pore-forming translocase proteins SipB, SipC and SipD is controlled by the T3SS regulator protein, InvE. During an attempt to identify the regions of InvE that are involved in T3SS regulation, it was observed that the secretion of SipB, SipC and SipD was inhibited when the C-terminal 52 amino acids were removed from InvE. In addition, InvE derivatives lacking the N-terminal 30 and 100 residues were unable to secrete translocases into the culture medium. Interestingly, in the absence of the N-terminal 180 residues of InvE, SipD is unstable, resulting in the hypersecretion of SipB. We also found that both the type III secretion signals of SipB and SptP were functionally interchangeable with the first 30 amino acids of InvE, which could allow the secretion of a reporter protein. These results indicate that InvE may have two functional domains responsible for regulating the secretion of translocases: an N-terminal secretion signal and a C-terminal regulatory domain.

IFN-inducible GTPases in host cell defense.

From plants to humans, the ability to control infection at the level of an individual cell-a process termed cell-autonomous immunity-equates firmly with survival of the species. Recent work has begun to unravel this programmed cell-intrinsic response and the central roles played by IFN-inducible GTPases in defending the mammalian cell's interior against a diverse group of invading pathogens. These immune GTPases regulate vesicular traffic and protein complex assembly to stimulate oxidative, autophagic, membranolytic, and inflammasome-related antimicrobial activities within the cytosol, as well as on pathogen-containing vacuoles. Moreover, human genome-wide association studies and disease-related transcriptional profiling have linked mutations in the Immunity-Related GTPase M (IRGM) locus and altered expression of guanylate binding proteins (GBPs) with tuberculosis susceptibility and Crohn's colitis.

Expression and delivery of tetanus toxin fragment C fused to the N-terminal domain of SipB enhances specific immune responses in mice.

Live attenuated bacteria can be used as a carrier for the delivery of foreign antigens to a host's immune system. The N-terminal domain of SipB, a translocon protein of the type III secretion system of Salmonella enterica serovar Typhimurium, is required for secretion and outer membrane localization. In the present study, vaccine plasmids for antigen delivery in which the non-toxic tetanus toxin fragment C (TTFC), which contains a T cell epitope, is fused to the N-terminal 160 amino acids of SipB were developed. It was found that the recombinant proteins are secreted into the culture media and localized to the bacterial surface. TTFC-specific antibody responses are significantly increased in mice orally immunized with attenuated S. Typhimurium BRD509 strains carrying TTFC delivery plasmids. When the TTFC delivery cassettes were introduced into a low copy vector, the plasmid was stably maintained in the BRD509 strain and induced an immune response to the TTFC antigen in mice. These results suggest that expression and delivery of heterologous antigens fused to the N-terminus of SipB enhance the induction of antigen-specific immune responses, and that the N-terminal domain of SipB can be used as a versatile delivery system for foreign antigens.

IFN-γ elicits macrophage autophagy via the p38 MAPK signaling pathway.

Autophagy is a major innate immune defense pathway in both plants and animals. In mammals, this cascade can be elicited by cytokines (IFN-γ) or pattern recognition receptors (TLRs and nucleotide-binding oligomerization domain-like receptors). Many signaling components in TLR- and nucleotide-binding oligomerization domain-like receptor-induced autophagy are now known; however, those involved in activating autophagy via IFN-γ remain to be elucidated. In this study, we engineered macrophages encoding a tandem fluorescently tagged LC3b (tfLC3) autophagosome reporter along with stably integrated short hairpin RNAs to demonstrate IFN-γ-induced autophagy required JAK 1/2, PI3K, and p38 MAPK but not STAT1. Moreover, the autophagy-related guanosine triphosphatase Irgm1 proved dispensable in both stable tfLC3-expressing RAW 264.7 and tfLC3-transduced Irgm1(-/-) primary macrophages, revealing a novel p38 MAPK-dependent, STAT1-independent autophagy pathway that bypasses Irgm1. These unexpected findings have implications for understanding how IFN-γ-induced autophagy is mobilized within macrophages for inflammation and host defense.