ABSTRACT
Yersinia outer protein M (YopM) belongs to the group of Yop effector proteins, which are highly conserved among pathogenic Yersinia species. During infection, the effectors are delivered into the host cell cytoplasm via the type 3 secretion system to subvert the host immune response and support the survival of Yersinia. In contrast to the other Yop effectors, YopM does not possess a known enzymatic activity and its molecular mechanism(s) of action remain(s) poorly understood. However, YopM was shown to promote colonization and dissemination of Yersinia, thus being crucial for the pathogen's virulence in vivo. Moreover, YopM interacts with several host cell proteins and might utilize them to execute its anti-inflammatory activities. The results obtained so far indicate that YopM is a multifunctional protein that counteracts the host immune defense by multiple activities, which are at least partially independent of each other. Finally, its functions seem to be also influenced by differences between the specific YopM isoforms expressed by Yersinia subspecies. In this review, we focus on the global as well as more specific contribution of YopM to virulence of Yersinia during infection and point out the various extra- and intracellular molecular functions of YopM. In addition, the novel cell-penetrating ability of recombinant YopM and its potential applications as a self-delivering immunomodulatory therapeutic will be discussed.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Immune Evasion , Virulence Factors/metabolism , Yersinia Infections/microbiology , Yersinia Infections/pathology , Yersinia/physiology , Humans , Immune Tolerance , Yersinia Infections/immunologyABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) has a grim prognosis with <5% survivors after 5 years. High expression levels of ADAM8, a metalloprotease disintegrin, are correlated with poor clinical outcome. We show that ADAM8 expression is associated with increased migration and invasiveness of PDAC cells caused by activation of ERK1/2 and higher MMP activities. For biological function, ADAM8 requires multimerization and associates with ß1 integrin on the cell surface. A peptidomimetic ADAM8 inhibitor, BK-1361, designed by structural modelling of the disintegrin domain, prevents ADAM8 multimerization. In PDAC cells, BK-1361 affects ADAM8 function leading to reduced invasiveness, and less ERK1/2 and MMP activation. BK-1361 application in mice decreased tumour burden and metastasis of implanted pancreatic tumour cells and provides improved metrics of clinical symptoms and survival in a Kras(G12D)-driven mouse model of PDAC. Thus, our data integrate ADAM8 in pancreatic cancer signalling and validate ADAM8 as a target for PDAC therapy.
Subject(s)
ADAM Proteins/metabolism , Membrane Proteins/metabolism , Molecular Targeted Therapy , Pancreatic Neoplasms/drug therapy , ADAM Proteins/antagonists & inhibitors , Animals , Blotting, Western , Carcinoma, Pancreatic Ductal/enzymology , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Movement/drug effects , Extracellular Space/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Knockdown Techniques , Humans , Hydroxamic Acids/pharmacology , Hydroxamic Acids/therapeutic use , Integrin beta1/metabolism , Kaplan-Meier Estimate , Matrix Metalloproteinase 14/metabolism , Matrix Metalloproteinase 2/metabolism , Membrane Proteins/antagonists & inhibitors , Mice , Neoplasm Invasiveness , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/pathology , Peptides, Cyclic/pharmacology , Peptides, Cyclic/therapeutic use , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Multimerization/drug effects , Protein Processing, Post-Translational , Signal Transduction/drug effectsABSTRACT
The effector protein Yersinia outer protein M (YopM) of Yersinia enterocolitica has previously been identified and characterized as the first bacterial cell-penetrating protein (CPP). We found that recombinant YopM (rYopM) enters different eukaryotic cell types and downregulates the expression of several pro-inflammatory cytokines (e.g., tumor necrosis factor-α [TNF-α]) after autonomous translocation. After infection with Y. enterocolitica or transfection of host cells, YopM interacts with isoforms of the two kinases ribosomal S6 protein kinase (RSK) and protein kinase C-related kinase (PRK). This interaction caused sustained RSK activation due to interference with dephosphorylation. Here we demonstrate by co-immunoprecipitation that rYopM interacts with RSK and PRK following cell-penetration. We show that autonomously translocated rYopM forms a trimeric complex with different RSK and PRK isoforms. Furthermore, we constructed a series of truncated versions of rYopM to map the domain required for the formation of the complex. The C-terminus of rYopM was identified to be essential for the interaction with RSK1, whereas any deletion in rYopM's leucin-rich repeat domains abrogated PRK2 binding. Moreover, we found that the interaction of cell-penetrating rYopM with RSK led to enhanced autophosphorylation of this kinase at serine 380. Finally, we investigated whether downstream signaling of the trimeric rYopM-RSK/PRK complex modulates the expression of pro-inflammatory TNF-α. Here, we could exclude that interaction with RSK1 and PRK2 is essential for the anti-inflammatory effects of rYopM.
