Alpha-2-Macroglobulin in Inflammation, Immunity and Infections

Alpha-2-macroglobulin is an extracellular macromolecule mainly known for its role as a broad-spectrum protease inhibitor. By presenting itself as an optimal substrate for endopeptidases of all catalytic types, alpha-2-macroglobulin lures active proteases into its molecular cage and subsequently ‘flags’ their complex for elimination. In addition to its role as a regulator of extracellular proteolysis, alpha-2-macroglobulin also has other functions such as switching proteolysis towards small substrates, facilitating cell migration and the binding of cytokines, growth factors and damaged extracellular proteins. These functions appear particularly important in the context of immune-cell function. In this review manuscript, we provide an overview of all functions of alpha-2-macroglobulin and place these in the context of inflammation, immunity and infections.

Worrying drives cell migration in mechanically unrestrained environments

Migratory cells employ numerous strategies to navigate the very diverse 3D microenvironments found in vivo. These strategies are subdivided into those that create space by pericellular proteolysis of extracellular matrix (ECM) proteins and those that navigate existing spaces. We find that cells can employ an alternative mechanism by digging tunnels through 3D collagen networks without extracellular proteolysis. This is accomplished by persistent polarization of large dynamic membrane blebs at the closed end of the tunnel that repeatedly agitate the collagen, a process we termed mechanical worrying. We find that this agitation promotes breakage and internalization of collagen at the cell front along with extracellular fluid in a macropinocytosis-driven manner. Membrane blebs are short-lived relative to the timescale of migration, and thus their polarization is critical for persistent ablation of the ECM. We find that sustained interactions between the collagen at the cell front and small but persistent cortical adhesions induce PI-3 Kinase (PI3K) signaling that drives polarized bleb enlargement via the Rac1 – Arp2/3 pathway. This defines a mechanism for the reinforcement of bleb expansion against load, which enables precise ablation of mechanically unrestrained environments, such as those encountered in very compliant tissue.

Substrate-biased activity-based probes identify the urokinase-plasminogen axis as a master regulator of metastatic signaling by orphan membrane receptor CDCP1

CDCP1 is an oncogenic orphan transmembrane receptor and a promising target for detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a novel approach for rapid identification of proteases responsible for key proteolytic events exploiting a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl-diphenyl-phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, both by direct cleavage and via activation of CDCP1-cleaving plasmin. We show that co-expression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.

Long-term plasticity of inhibitory synapses in the hippocampus and spatial learning depends on matrix metalloproteinase 3

Learning and memory are known to depend on synaptic plasticity. Whereas the involvement of plastic changes at excitatory synapses is well established, plasticity mechanisms at inhibitory synapses only start to be discovered. Extracellular proteolysis is known to be a key factor in glutamatergic plasticity but nothing is known about its role at GABAergic synapses. We reveal that pharmacological inhibition of MMP3 activity or genetic knockout of the Mmp3 gene abolishes induction of postsynaptic iLTP. Moreover, the application of exogenous active MMP3 mimics major iLTP manifestations: increased mIPSCs amplitude, enlargement of synaptic gephyrin clusters, and a decrease in the diffusion coefficient of synaptic GABAA receptors that favors their entrapment within the synapse. Finally, we found that MMP3 deficient mice show faster spatial learning in Morris water maze and enhanced contextual fear conditioning. We conclude that MMP3 plays a key role in iLTP mechanisms and in the behaviors that presumably in part depend on GABAergic plasticity.

Cooperation and Cheating through a Secreted Aminopeptidase in the Pseudomonas aeruginosa RpoS Response

ABSTRACT The global stress response controlled by the alternative sigma factor RpoS protects enteric bacteria from a variety of environmental stressors. The role of RpoS in other, nonenteric bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, is less well understood. Here, we employed experimental social evolution to reveal that cooperative behavior via secreted public goods is an important function in the RpoS response of P. aeruginosa. Using whole-genome sequencing, we identified rpoS loss-of-function mutants among isolates evolved in a protein growth medium that requires extracellular proteolysis. We found that rpoS mutants comprise up to 25% of the evolved population and that they behave as social cheaters, with low fitness in isolation but high fitness in mixed culture with the cooperating wild type. We conclude that rpoS mutants cheat because they exploit an RpoS-controlled public good produced by the wild type, the secreted aminopeptidase PaAP, and because they do not carry the metabolic costs of expressing PaAP and many other gene products in the large RpoS regulon. Our results suggest that PaAP is an integral part of a proteolytic sequence in P. aeruginosa that permits the utilization of protein as a nutrient source. Our work broadens the scope of stress response functions in bacteria. IMPORTANCE Bacterial stress responses are generally considered protective measures taken by individual cells. Enabled by an experimental evolution approach, we describe a contrasting property, collective nutrient acquisition, in the RpoS-dependent stress response of the opportunistic human pathogen P. aeruginosa. Specifically, we identify the secreted P. aeruginosa aminopeptidase (PaAP) as an essential RpoS-controlled function in extracellular proteolysis. As a secreted “public good,” PaAP permits cheating by rpoS mutants that save the metabolic costs of expressing RpoS-controlled genes dispensable under the given growth conditions. Proteolytic enzymes are important virulence factors in P. aeruginosa pathogenesis and constitute a potential target for antimicrobial therapy. More broadly, our work contributes to recent findings in higher organisms that stress affects not only individual fitness and competitiveness but also cooperative behavior.

The Role of Stromal Proteolytic Systems in Cancer Progression (Review)

Oncological diseases belong to life-threatening pathologies being the second most frequent cause of morbidity and mortality after cardiovascular diseases. Clarification of carcinogenesis mechanisms makes it possible to expand the stock of tools available for prevention of critical illness accompanying this pathological condition.Nowadays, proteolytic systems of tumor microenvironment (ТМЕ) are regarded as key regulators of a tumor progression including tumor growth, invasion and metastazing. The review discusses ТМЕ structure and role in cancer progression.Recent data decipher the role of proteolytic systems in the interaction stromal cells with tumor cells in different types of cancer in humans. The most known proteolytic systems contributed to cancer progression are matrix metalloproteinase system (MMP), urokinase-type plasminogen activator system (uPA-system), various cathepsins, granzymes, and elastase. Inhibition of extracellular proteolysis in the course of an oncological process is considered an effective approach to cancer therapy.

Formation of functional, non-amyloidogenic fibres by recombinantBacillus subtilisTasA

Bacterial biofilms are communities of microbial cells encased within a self-produced polymeric matrix. In theBacillus subtilisbiofilm matrix the extracellular fibres of TasA are essential. Here a recombinant expression system allows interrogation of TasA, revealing that monomeric and fibre forms of TasA have identical secondary structure, suggesting that fibrous TasA is a linear assembly of globular units. Recombinant TasA fibres form spontaneously, and share the biological activity of TasA fibres extracted fromB. subtilis, whereas a TasA variant restricted to a monomeric form is inactive and subjected to extracellular proteolysis. The biophysical properties of both native and recombinant TasA fibres indicate that they are not functional amyloid-like fibres. A gel formed by TasA fibres can recover after physical shear force, suggesting that the biofilm matrix is not static and that these properties may enableB. subtilisto remodel its local environment in response to external cues. Using recombinant fibres formed by TasA orthologues we uncover species variability in the ability of heterologous fibres to cross-complement theB. subtilis tasAdeletion. These findings are indicative of specificity in the biophysical requirements of the TasA fibres across different species and/or reflect the precise molecular interactions needed for biofilm matrix assembly.ContributionsConceived and designed the experiments: CE, EE, RG, CEM, RJM, MS, NSW; Performed the experiments: KB, LC, CE, EE, PKF, RG, CEM, RJM, MS, TS; Contributed new analytical tools: CE, EE, RG, TS; Analysed the data: CE, EE, CEM, RJM, MS, LCS, NSW; Wrote the paper: EE, RJM, CEM, MS, NSW.