Chemotherapy has been the standard treatment for liver cancer. However, intrinsic or acquired drug resistance remains a major barrier to successful treatment. At present, the underlying molecular mechanisms of chemoresistance in liver cancer have not been elucidated. Dipeptidyl peptidase 9 (DPP9) is a member of the dipeptidyl peptidase IV family that has been found to be highly expressed in a variety of tumors, including liver cancer. It is unclear whether DPP9 affects chemoresistance in liver cancer. In this study, we find that DPP9 weakens the responses of liver cancer cells to chemotherapy drugs by up-regulating NQO1 and inhibiting intracellular ROS levels. In terms of mechanism, DPP9 inhibits ubiquitin-mediated degradation of NRF2 protein by binding to KEAP1, up-regulates NRF2 protein levels, promotes mRNA transcription of NQO1, and inhibits intracellular ROS levels. In addition, the NQO1 inhibitor dicoumarol can enhance the efficacy of chemotherapy drugs in liver cancer cells. Collectively, our findings suggest that inhibiting DPP9/NQO1 signaling can serve as a potential therapeutic strategy for liver cancer.

Most genetic variants identified through genome-wide association studies (GWAS) are suspected to be regulatory in nature, but only a small fraction colocalize with expression quantitative trait loci (eQTLs, variants associated with expression of a gene). Therefore, it is hypothesized but largely untested that integration of disease GWAS with context-specific eQTLs will reveal the underlying genes driving disease associations. We used colocalization and transcriptomic analyses to identify shared genetic variants and likely causal genes associated with critically ill COVID-19 and idiopathic pulmonary fibrosis. We first identified five genome-wide significant variants associated with both diseases. Four of the variants did not demonstrate clear colocalization between GWAS and healthy lung eQTL signals. Instead, two of the four variants colocalized only in cell-type and disease-specific eQTL datasets. These analyses pointed to higher ATP11A expression from the C allele of rs12585036, in monocytes and in lung tissue from primarily smokers, which increased risk of IPF and decreased risk of critically ill COVID-19. We also found lower DPP9 expression (and higher methylation at a specific CpG) from the G allele of rs12610495, acting in fibroblasts and in IPF lungs, and increased risk of IPF and critically ill COVID-19. We further found differential expression of the identified causal genes in diseased lungs when compared to non-diseased lungs, specifically in epithelial and immune cell types. These findings highlight the power of integrating GWAS, context-specific eQTLs, and transcriptomics of diseased tissue to harness human genetic variation to identify causal genes and where they function during multiple diseases.

COVID-19 has a broad clinical spectrum, ranging from asymptomatic-mild form to severe phenotype. The severity of COVID-19 is a complex trait influenced by various genetic and environmental factors. Ethnic differences have been observed in relation to COVID-19 severity during the pandemic. It is currently unknown whether genetic variations may contribute to the increased risk of severity observed in Latin-American individuals The aim of this study is to investigate the potential correlation between gene variants at CCL2, OAS1, and DPP9 genes and the severity of COVID-19 in a population from Quito, Ecuador. This observational case-control study was conducted at the Carrera de Biologia from the Universidad Central del Ecuador and the Hospital Quito Sur of the Instituto Ecuatoriano de Seguridad Social (Quito-SUR-IESS), Quito, Ecuador. Genotyping for gene variants at rs1024611 (A>G), rs10774671 (A>G), and rs10406145 (G>C) of CCL2, OAS1, and DPP9 genes was performed on 100 COVID-19 patients (43 with severe form and 57 asymptomatic-mild) using RFLP-PCR. The genotype distribution of all SNVs throughout the entire sample of 100 individuals showed Hardy Weinberg equilibrium (P=0.53, 0.35, and 0.4 for CCL2, OAS1, and DPP9, respectively). The HWE test did not find any statistically significant difference in genotype distribution between the study and control groups for any of the three SNVs. The multivariable logistic regression analysis showed that individuals with the GG of the CCL2 rs1024611 gene variant had an increased association with the severe COVID-19 phenotype in a recessive model (P = 0.0003, OR = 6.43, 95% CI 2.19-18.89) and for the OAS1 rs10774671 gene variant, the log-additive model showed a significant association with the severe phenotype of COVID-19 (P=0.0084, OR=3.85, 95% CI 1.33-11.12). Analysis of haplotype frequencies revealed that the coexistence of GAG at CCL2, OAS1, and DPP9 variants, respectively, in the same individual increased the presence of the severe COVID-19 phenotype (OR=2.273, 95% CI: 1.271-4.068, P=0.005305). The findings of the current study suggests that the ethnic background affects the allele and genotype frequencies of genes associated with the severity of COVID-19. The experience with COVID-19 has provided an opportunity to identify an ethnicity-based approach to recognize genetically high-risk individuals in different populations for emerging diseases.

Dipeptidyl peptidase 9 (DPP9) is an intracellular amino-dipeptidase with physiological roles in the immune system, DNA repair and mitochondria homeostasis, while its deregulation is linked to cancer progression and immune-associated defects. Through its rare ability to cleave a peptide bond following the imino-acid proline, DPP9 acts as a molecular switch that regulates key proteins, such as the tumor-suppressor BRCA2. In this review we will discuss key concepts underlying the outcomes of protein processing by DPP9, including substrate turn-over by the N-degron pathway. Additionally, we will review non-enzymatic roles and the regulation of DPP9 by discussing the interactome of this protease, which includes SUMO1, Filamin A, NLRP1 and CARD8.

The cytosolic dipeptidyl-aminopeptidases 8 (DPP8) and 9 (DPP9) belong to the DPPIV serine proteases with the unique characteristic of cleaving off a dipeptide post-proline from the N-termini of substrates. To study the role of DPP8 and DPP9 in breast cancer, MCF-7 cells (luminal A-type breast cancer) and MDA.MB-231 cells (basal-like breast cancer) were used. The inhibition of DPP8/9 by 1G244 increased the number of lysosomes in both cell lines. This phenotype was more pronounced in MCF-7 cells, in which we observed a separation of autophagosomes and lysosomes in the cytosol upon DPP8/9 inhibition. Likewise, the shRNA-mediated knockdown of either DPP8 or DPP9 induced autophagy and increased lysosomes. DPP8/9 inhibition as well as the knockdown of the DPPs reduced the cell survival and proliferation of MCF-7 cells. Additional treatment of MCF-7 cells with tamoxifen, a selective estrogen receptor modulator (SERM) used to treat patients with luminal breast tumors, further decreased survival and proliferation, as well as increased cell death. In summary, both DPP8 and DPP9 activities confine macroautophagy in breast cancer cells. Thus, their inhibition or knockdown reduces cell viability and sensitizes luminal breast cancer cells to tamoxifen treatment.

Genetic defects in components of inflammasomes can cause autoinflammation. Biallelic loss-of-function mutations in dipeptidyl peptidase 9 (DPP9), a negative regulator of the NLRP1 and CARD8 inflammasomes, have recently been shown to cause an inborn error of immunity characterized by pancytopenia, skin manifestations, and increased susceptibility to infections.
We sought to study the molecular basis of autoinflammation in a patient with severe infancy-onset hyperinflammation associated with signs of fulminant hemophagocytic lymphohistiocytosis.
Using heterologous cell models as well as patient cells, we performed genetic, immunologic, and molecular investigations to identify the genetic cause and to assess the impact of the identified mutation on inflammasome activation.
The patient exhibited pancytopenia with decreased neutrophils and T, B, and natural killer cells, and markedly elevated levels of lactate dehydrogenase, ferritin, soluble IL-2 receptor, and triglycerides. In addition, serum levels of IL-1β and IL-18 were massively increased, consistent with inflammasome activation. Genetic analysis revealed a previously undescribed de novo mutation in DPP9 (c.755G>C, p.Arg252Pro) affecting a highly conserved amino acid residue. The mutation led to destabilization of the DPP9 protein as shown in transiently transfected HEK293T cells and in patient-derived induced pluripotent stem cells. Using functional inflammasome assays in HEK293T cells, we demonstrated that mutant DPP9 failed to restrain the NLRP1 and CARD8 inflammasomes, resulting in constitutive inflammasome activation. These findings suggest that the Arg252Pro DPP9 mutation acts in a dominant-negative manner.
A de novo mutation in DPP9 leads to severe infancy-onset autoinflammation because of unleashed inflammasome activation.

The inflammasome pathway is a critical early response mechanism of the host that detects pathogens, initiates the production of inflammatory cytokines, and recruits effector cells to the infection site. Nonetheless, the mechanism of inflammasome activation in coronavirus infection and its biological functions in host defense remain unclear. Transmissible gastroenteritis virus (TGEV), a member of the genus Alphacoronavirus, is a significant pathogen that mainly infects piglets and causes intestinal inflammation and inflammatory cell infiltration. Here, we investigated the mechanism of inflammasome activation in intestinal epithelial cells (IECs) infected with TGEV. We observed a substantial increase in interleukin 1β (IL-1β) and IL-18 levels in both IECs and TGEV-infected porcine intestinal tissues. Furthermore, TGEV infection resulted in increased activation of caspase-1 and the NLRP1 (NOD-like receptor [NLR]-containing pyrin domain [PYD]) inflammasome. Our findings revealed that TGEV infection impeded the interaction between porcine NLRP1 (pNLRP1) and porcine dipeptidyl peptidases 9 (pDPP9), yet it did not reduce the expression of pDPP9. Importantly, the ZU5 domain, not the function-to-find domain (FIIND) reported in human NLRP1, was identified as the minimal domain of pNLRP1 for pDPP9 binding. In addition, the robust type I IFN expression induced by TGEV infection also upregulated pNLRP1 expression and pNLRP1 itself acts as an interferon-stimulated gene to counteract TGEV infection. Our data demonstrate that pNLRP1 has antiviral capabilities against coronavirus infection, which highlights its potential as a novel therapeutic target for coronavirus antiviral therapy. IMPORTANCE Coronavirus primarily targets the epithelial cells of the respiratory and gastrointestinal tracts, leading to damage in both humans and animals. NLRP1 is a direct sensor for RNA virus infection which is highly expressed in epithelial barrier tissues. However, until recently, the precise molecular mechanisms underlying its activation in coronavirus infection and subsequent downstream events remained unclear. In this study, we demonstrate that the alphacoronavirus TGEV induces the production of IL-1β and IL-18 and upregulates the expression of pNLRP1. Furthermore, we found that pNLRP1 can serve as an interferon-stimulated gene (ISG) to inhibit the infection of enterovirus TGEV. Our research highlights the crucial role of NLRP1 as a regulator of innate immunity in TGEV infection and shows that it may serve as a potential therapeutic target for the treatment of coronavirus infection.

Proline residues highly impact protein stability when present either in the first or second N-terminal position. While the human genome encodes for more than 500 proteases, only few proteases are capable of hydrolyzing a proline-containing peptide bond. The two intra-cellular amino-dipeptidyl peptidases DPP8 and DPP9 are exceptional as they possess the rare ability to cleave post-proline. By removing N-terminal Xaa-Pro dipeptides, DPP8 and DPP9 expose a neo N-terminus of their substates, which can consequently alter inter- or intra-molecular interactions of the modified protein. Both DPP8 and DPP9 play key roles in the immune response and are linked to cancer progression, emerging as attractive drug targets. DPP9 is more abundant than DPP8 and is rate limiting for cleavage of cytosolic proline-containing peptides. Only few DPP9 substrates have been characterized; these include Syk, a central kinase for B-cell receptor mediated signaling; Adenylate Kinase 2 (AK2) which is important for cellular energy homeostasis; and the tumor suppressor Breast cancer type 2 susceptibility protein (BRCA2) that is critical for repair of DNA double strand breaks. N-terminal processing of these proteins by DPP9 triggers their rapid turn-over by the proteasome, highlighting a role for DPP9 as upstream components of the N-degron pathway. Whether N-terminal processing by DPP9 leads to substrate-degradation in all cases, or whether additional outcomes are possible, remains to be tested. In this chapter we will describe methods for purification of DPP8 and DPP9 as well as protocols for biochemical and enzymatic characterization of these proteases.

The inflammasome sensor NLRP1 (nucleotide-binding oligomerization domain-like receptor containing a pyrin domain 1) detects a variety of pathogen-derived molecular patterns to induce an inflammatory immune response by triggering pyroptosis and cytokine release. A number of mutations and polymorphisms of NLRP1 are known to cause autoinflammatory diseases, the functional characterization of which contributes to a better understanding of NLRP1 regulation. Here, we assessed the effect of the common NLRP1 variant M1184V, associated with asthma, inflammatory bowel disease, and diabetes, on the protein level. Our size-exclusion chromatography experiments show that M1184V stabilizes the \"function-to-find\" domain (FIIND) in a monomeric conformation. This effect is independent of autoproteolysis. In addition, molecular dynamics simulations reveal that the methionine residue increases flexibility within the ZU5 domain, whereas valine decreases flexibility, potentially indirectly stabilizing the catalytic triad responsible for autocleavage. By keeping the FIIND domain monomeric, formation of a multimer of full-length NLRP1 is promoted. We found that the stabilizing effect of the valine further leads to improved dipeptidyl peptidase 9 (DPP9)-binding capacities for the FIIND domain as well as the full-length protein as determined by surface plasmon resonance. Moreover, our immunoprecipitation experiments confirmed increased DPP9 binding for the M1184V protein in cells, consistent with improved formation of an autoinhibited complex with DPP9 in activity assays. Collectively, our study establishes a molecular rationale for the dichotomous involvement of the NLRP1 variant M1184V in autoimmune syndromes.

Dipeptidyl peptidase 8 (DPP8) and 9 (DPP9) are widely expressed in mammals including humans, mainly locate in the cytoplasm. The DPP8 and DPP9 (DPP8/9) belong to serine proteolytic enzymes, they can recognize and cleave N-terminal dipeptides of specific substrates if proline is at the penultimate position. Because the localization of DPP8/9 is different from that of DPP4 and the substrates for DPP8/9 are not yet completely clear, their physiological and pathological roles are still being further explored. In this article, we will review the recent research advances focusing on the expression, regulation, and functions of DPP8/9 in physiology and pathology status. Emerging research results have shown that DPP8/9 is involved in various biological processes such as cell behavior, energy metabolism, and immune regulation, which plays an essential role in maintaining normal development and physiological functions of the body. DPP8/9 is also involved in pathological processes such as tumorigenesis, inflammation, and organ fibrosis. In recent years, related research on immune cell pyroptosis has made DPP8/9 a new potential target for the treatment of hematological diseases. In addition, DPP8/9 inhibitors also have great potential in the treatment of tumors and chronic kidney disease.