An Infectious Disease-Associated Il12b Polymorphism Regulates IL-12/23 p40 Transcription Involving Poly(ADP-Ribose) Polymerase 1.

IL-12 and IL-23 are important host defense factors produced by APCs against certain intracellular and extracellular pathogens. Their dysregulation has also been implicated in several autoimmune diseases. The nucleotide polymorphism in the promoter region of Il12b (rs41292470 consisting of the long or short allele) encoding the shared subunit of IL-12 and IL-23, p40, has been reported to associate with susceptibility to infectious diseases and autoimmune disorders. How these genetic variants impact Il12b expression at the molecular level was unclear. We established an Il12b promoter-luciferase reporter system containing the long or short allele driving the reporter gene expression and found that the long allele (infection-resistant) displayed ∼2-fold higher transcriptional activity than the short allele (infection-susceptible), associated with a selective and differential nuclear binding activity to the two alleles in activated macrophages. DNA pull-down assays coupled with mass spectrometry analyses identified the specific DNA binding activity as poly(ADP-ribose) polymerase 1 (PARP-1). Small hairpin RNA-mediated knockdown of the endogenous PARP-1 expression resulted in reduced p40 mRNA expression and Il12b promoter activity. Bone marrow-derived macrophages from PARP-1-deficient mice had decreased p40 expression at both mRNA and protein levels. Furthermore, selective PARP-1 inhibitors resulted in impaired production of IL-12p40 and IL-23 in bone-marrow derived macrophages and PBMCs. Chromatin immunoprecipitation assay revealed that PARP-1 could bind specifically to Il12b in LPS-stimulated macrophages. Our study opens the way for further elucidating the molecular mechanism whereby allele-specific immune responses to foreign and self-antigens mediated by IL-12/IL-23 are controlled in an individually variable manner.

Mycobacterium tuberculosis PPE family protein Rv1808 manipulates cytokines profile via co-activation of MAPK and NF-κB signaling pathways.

BACKGROUND/AIMS: Mycobacterium tuberculosis is an extremely successful intracellular pathogen armed with multiple tactics to subvert host immunity. PPE (Pro-Pro-Glu) family exclusively distributed in mycobacteria might be responsible for the virulence and pathogenicity of M.tuberculosis. The up-regulation of Rv1808 (PPE32) in many conditions prompted us to define its role in host innate immune response. METHODS: The Rv1808 encoding gene was expressed in nonpathogenic fast growing Mycobacterium smegmatis, mycobacteria- Escherichia coli shuttle plasmid pNITmyc served as control. RT-PCR and ELISA were used to detect the transcription and translation of host cytokines in culture supernatant from macrophage incubated with purified Rv1808 protein. Pharmacological inhibitors were applied to confirm the specificity of the effector interfering of host signaling. RESULTS: Recombinant Ms_Rv1808 survived better than Ms_pNITmyc within macrophage, accompanied by slightly higher host cell death. Rv1808 protein is associated with the cell wall and exposed on the cell surface. Physical binding of Rv1808 to TLR2 resulted in increase in the secretion of anti-inflammatory cytokine interleukin-10 (IL-10) and pro-inflammatory cytokines tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) possibly via co-activation of NF-κB and MAPK (p38MAPK, JNK and ERK) signalling. CONCLUSION: Cell wall associated Rv1808 protein manipulated the host cytokines via MAPK and NF-κB signaling pathways.

Mycobacterium tuberculosis proteases and implications for new antibiotics against tuberculosis.

Tuberculosis remains a global health concern. Effective novel therapeutics are urgently needed to tackle the inexorable increase of multidrug resistant and extensively drug-resistant strains and HIV coinfection. Most proteases are important for Mycobacterium tuberculosis virulence involving in the evasion or subversion of host defenses and/or tissue degradation, therefore they are ideal candidates for new drug targets. To explore this possibility, we summarize the functions of Mycobacterium tuberculosis proteases, especially their roles in pathogenesis and as inhibitors during different clinical stages.

Discovery of novel BCR-ABL PROTACs based on the cereblon E3 ligase design, synthesis, and biological evaluation.

Protein degradation is a promising strategy for drug development. Proteolysis-targeting chimeras (PROTACs) hijacking the E3 ligase cereblon (CRBN) exhibit enormous potential and universal degradation performance due to the small molecular weight of CRBN ligands. In this study, the CRBN-recruiting PROTACs were explored on the degradation of oncogenic fusion protein BCR-ABL, which drives the pathogenesis of chronic myeloid leukemia (CML). A series of novel PROTACs were synthesized by conjugating BCR-ABL inhibitor dasatinib to the CRBN ligand including pomalidomide and lenalidomide, and the extensive structure-activity relationship (SAR) studies were performed focusing on optimization of linker parameters. Therein, we uncovered that pomalidomide-based degrader 17 (SIAIS056), possessing sulfur-substituted carbon chain linker, exhibits the most potent degradative activity in vitro and favorable pharmacokinetics in vivo. Besides, degrader 17 also degrades a variety of clinically relevant resistance-conferring mutations of BCR-ABL. Furthermore, degrader 17 induces significant tumor regression against K562 xenograft tumors. Our study indicates that 17 as an efficacious BCR-ABL degrader warrants intensive investigation for the future treatment of BCR-ABL+ leukemia.

Structure-Activity Relationships of Noncovalent Immunoproteasome ß5i-Selective Dipeptides.

The immunoproteasome (i-20S) has emerged as a therapeutic target for autoimmune and inflammatory disorders and hematological malignancies. Inhibition of the chymotryptic ß5i subunit of i-20S inhibits T cell activation, B cell proliferation, and dendritic cell differentiation in vitro and suppresses immune responses in animal models of autoimmune disorders and allograft rejection. However, cytotoxicity to immune cells has accompanied the use of covalently reactive ß5i inhibitors, whose activity against the constitutive proteasome (c-20S) is cumulative with the time of exposure. Herein, we report a structure-activity relationship study of a class of noncovalent proteasome inhibitors with picomolar potencies and 1000-fold selectivity for i-20S over c-20S. Furthermore, these inhibitors are specific for ß5i over the other five active subunits of i-20S and c-20S, providing useful tools to study the functions of ß5i in immune responses. The potency of these compounds in inhibiting human T cell activation suggests that they may have therapeutic potential.

Discovery of SIAIS178 as an Effective BCR-ABL Degrader by Recruiting Von Hippel-Lindau (VHL) E3 Ubiquitin Ligase.

The oncogenic fusion protein BCR-ABL is the driving force of leukemogenesis in chronic myeloid leukemia (CML). Despite great progress for CML treatment through application of tyrosine kinase inhibitors (TKIs) against BCR-ABL, long-term drug administration and clinical resistance continue to be an issue. Herein, we described the design, synthesis, and evaluation of novel proteolysis-targeting chimeric (PROTAC) small molecules targeting BCR-ABL which connect dasatinib and VHL E3 ubiquitin ligase ligand by extensive optimization of linkers. Our efforts have yielded SIAIS178 (19), which induces proper interaction between BCR-ABL and VHL ligase leading to effective degradation of BCR-ABL protein, achieves significant growth inhibition of BCR-ABL+ leukemic cells in vitro, and induces substantial tumor regression against K562 xenograft tumors in vivo. In addition, SIAIS178 also degrades several clinically relevant resistance-conferring mutations. Our data indicate that SIAIS178 as efficacious BCR-ABL degrader warrants extensive further investigation for the treatment of BCR-ABL+ leukemia.

A novel crosstalk between TLR4- and NOD2-mediated signaling in the regulation of intestinal inflammation.

Although Toll-like receptor 4 (TLR4)- and nucleotide-binding oligomerization domain 2 (NOD2)-mediated signaling mechanisms have been extensively studied individually, the crosstalk between them in the regulation of intestinal mucosal defense and tissue homeostasis has been underappreciated. Here, we uncover some novel activities of NOD2 by gene expression profiling revealing the global nature of the cross-regulation between TLR4- and NOD2-mediated signaling. Specifically, NOD2 is able to sense the intensity of TLR4-mediated signaling, resulting in either synergistic stimulation of Interluekin-12 (IL-12) production when the TLR signaling intensity is low; or in the inhibition of IL-12 synthesis and maintenance of intestinal mucosal homeostasis when the TLR signaling intensifies. This balancing act is mediated through receptor-interacting serine/threonine kinase 2, and the transcriptional regulator CCAAT/enhancer-binding protein α (C/EBPα) via its serine 248 phosphorylation by Protein Kinase C. Mice deficient in C/EBPα in the hematopoietic compartment are highly susceptible to chemically induced experimental colitis in an IL-12-dependent manner. Additionally, in contrast to the dogma, we find that the major Crohn's disease-associated NOD2 mutations could cause a primarily immunodeficient phenotype by selectively impairing TLR4-mediated IL-12 production and host defense. To restore the impaired homeostasis would be a way forward to developing novel therapeutic strategies for inflammatory bowel diseases.

Mycobacterium tuberculosis serine protease Rv3668c can manipulate the host-pathogen interaction via Erk-NF-κB axis-mediated cytokine differential expression.

Tuberculosis caused by Mycobacterium tuberculosis (MTB) remains a serious global public health concern. About one-third of the global population has been latently infected with this pathogen. MTB proteases are important virulence factors and involve in subverting the host immunity. MTB protease Rv3668c was implicated in drug action and dormancy by Gene Expression Omnibus data. To define the role of Rv3668c in pathogen-host interaction, we constructed recombinant strain Mycobacterium smegmatis-Rv3668c (Ms-Rv3668c). The resultant strains were used to challenge the human macrophage cell line U937. The cytokine levels and the survival of recombinants and macrophages were monitored. The results showed that recombinant Ms-Rv3668c specifically upregulated the secretion of proinflammatory cytokines TNF-α, IL-1ß, and IL-6 and downregulated the secretion of anti-inflammatory cytokine IL-10 by U937 cells, consistent with the upregulated transcription of TNF-α and IL-1ß. Rv3668c recombinants demonstrated prolonged survival within the U937 cells and accelerated the death of the host cells. Inhibitor experiments showed that the ERK-NF-κB axis was involved in the Rv3668c-triggered TNF-α and IL-1ß changes. These results provided evidence for the engagement of Rv3668c in the interaction between Mycobacterium and host.

Mycobacterium tuberculosis Rv3402c enhances mycobacterial survival within macrophages and modulates the host pro-inflammatory cytokines production via NF-kappa B/ERK/p38 signaling.

Intracellular survival plays a central role in the pathogenesis of Mycobacterium tuberculosis, a process which depends on an array of virulence factors to colonize and replicate within the host. The M. tuberculosis iron regulated open reading frame (ORF) rv3402c, encoding a conserved hypothetical protein, was shown to be up-regulated upon infection in both human and mice macrophages. To explore the function of this ORF, we heterologously expressed the rv3402c gene in the non-pathogenic fast-growing Mycobacterium smegmatis strain, and demonstrated that Rv3402c, a cell envelope-associated protein, was able to enhance the intracellular survival of recombinant M. smegmatis. Enhanced growth was not found to be the result of an increased resistance to intracellular stresses, as growth of the Rv3402c expressing strain was unaffected by iron depletion, H2O2 exposure, or acidic conditions. Colonization of macrophages by M. smegmatis expressing Rv3402c was associated with substantial cell death and significantly greater amount of TNF-α and IL-1ß compared with controls. Rv3402c-induced TNF-α and IL-1ß production was found to be mediated by NF-κB, ERK and p38 pathway in macrophages. In summary, our study suggests that Rv3402c delivered in a live M. smegmatis vehicle can modify the cytokines profile of macrophage, promote host cell death and enhance the persistence of mycobacterium within host cells.

Mycobacterium sulfur metabolism and implications for novel drug targets.

New antibiotic targets are urgently needed to tackle the multidrug resistant and latent Mycobacterium tuberculosis, the causative agent of the most formidable infectious disease tuberculosis. Sulfur metabolism is essential for the survival and virulence of many pathogens including M. tuberculosis. The absence of most genes involved in microbial sulfur metabolism in human beings suggests abundant novel potential antibiotic targets in pathogen sulfur metabolism. In this article, a comparative genomic landscape of Mycobacterium sulfur metabolism, such as the uptake, activation, and reduction of sulfate and allied enzymes, the biosynthesis pathway of some sulfated metabolites, and the enzymes involved in these pathways were presented. Novel clues for antibiotic targets are put forward.

Mycobacterium tuberculosis PE_PGRS17 promotes the death of host cell and cytokines secretion via Erk kinase accompanying with enhanced survival of recombinant Mycobacterium smegmatis.

Tuberculosis (TB) remains a serious threat to global public health, largely due to the successful manipulation of the host immunity by its etiological agent Mycobacterium tuberculosis. The PE_PGRS protein family of M. tuberculosis might be a contributing factor. To investigate the roles of PE_PGRS17, the gene of PE_PGRS 17 was expressed in nonpathogenic fast growing Mycobacterium smegmatis. We found that the recombinant strain survives better than the control in macrophage cultures, accompanied by more host cell death and a marked higher secretion of tumor necrosis factor-alpha by a recombinant strain compared with control. Blocking the action of Erk kinase by an inhibitor can abolish the above effects. In brief, our data showed that PE_PGRS 17 might facilitate pathogen survival and disserve the host cell via remodeling the macrophages immune niche largely consisting of inflammatory cytokines. This furnishes a novel insight into the immune role of this mycobacterium unique gene family.