basic seminar thematic area: the cell surface and cell motility b) … · 2016-10-19 · center of...

Center of Physiology and Pharmacology Geiger

Basic SeminarThematic area: The cell surface and cell

motilityB) Extracellular Proteases

Margarethe Geiger


Proteases as hormone-like signal messengers

Extracellular Proteases


From: Physiol Rev 2004

ExtracellularProteases


Protease-activated receptors (PARs)

PAR1: thrombin (50pM), trypsin, Xa

PAR2: trypsin (1nM), tryptase (1nM), TF/VIIa, Xa, MT-SP1, bacterial proteases

PAR3: thrombin (0.2nM)

PAR4: thrombin (5nM), trypsin (1nM)


PAR-signaling

PAR-1, PAR-2, and PAR-4 can also be activated by peptide corresponding to tethered ligand (amino terminus)


PAR-activation

From:Physiol. Rev. 2004


Multiple proteases activate PARs

Coagulation factors

Pancreatic and extrapancreatic trypsins

Mast cell proteases

Leucocyte proteases

Non-mammalian proteases


Blood coagulation

From: Wikipedia


Coagulation factors

Thrombin: PAR-1, PAR-3, PAR-4

on the surface of platelets

→ platelet aggregation, haemostasis

TF-VIIa: PAR-1, PAR-2

many cells

→ on endothelial cells in inflammation

APC on EPCR: PAR-1

→antiinflammatory


Coagulation factors



Pancreatic and extrapancreatic trypsins

• Activation of PAR-2 and PAR-4

- Small intestine

- Inflamed pancreas

- Endothelial and epithelial cells, nervous system, tumors


Mast cell proteases

• Tryptase: PAR-2

Less effective than trypsin

Possible role of posttranslational modification (better activation of unglycosylated receptor )

Local inflammation and pain


Leucocyte proteases

• Cathepsin G: PAR-4

Platelets and other cells at sites of inflammation

Also inactivation of PAR-1 and PAR-2


Cell surface proteases

• MT-SP1(matriptase): Type-2 transmembrane serine protease, active site extracellular, activates PAR-2, coexpression on PC-3 cells

• Others: ????


Non-mammalian proteases

• Mite-allergens (e.g. PAR-2 on lung epithelial cells)

• Bacterial: gingipains-R

• Fungal proteases


PAR-1: Signal transduction



PARs: crosstalk with other receptors

From: Gieseler et al. 2013, Cell Commun Signal


Not all proteases cleave at the same site

From: Gieseler et al. 2013, Cell Commun Signal


Localization of PARs on cell membrane determines intracellular signaling

• Different effects of thrombin (proinflammatory) and APC (cytoprotective) mediated via the same receptor (PAR1)

• APC effect - but not thrombin effect-abolished in CAV1-deficient cells


Soh et al., Br. J. Pharmacol. 2010

Localization of PARs on cell membrane determines intracellular signaling


PARs:termination of the signal

Cell-surface proteolysis

Receptor desensitization

Intracellular proteases


PAR-inactivating proteases

PAR-1: Cathepsin G, plasmin, elastase, proteinase-3, trypsin

PAR-2: Elastase, cathepsin G

PAR-3: Cathepsin G


Desensitation, downregulation, and trafficking of PARs



Biological roles of proteases and PARs



Thrombin receptor signaling on human and mouse platelets


Resting (left) and activated (right) platelets


Consequences of PAR activation in peripheral nerves


Protease signaling in the gastrointestinal tract

Increased chloride secretion from the intestinal mucosa (PAR-1 and PAR-2), partially via submucosal neurons

Effects on motility

Effects on exokrine secretion

Excitatory effects on intestinal neurons

Inflammation


Protease signaling in the airways

Airway resistance (+/- ?)

Inflammation (allergic, tryptase-mediated)

Fibrosis (thrombin, PAR-1)


Protease signaling in the skin

• Pigmentation (phagocytosis of melanosomes by keratinocytes; UV-exposure upregulates PAR-2)

• Wound healing (PAR-1)

• Inflammation


PAR signaling to vessels

Mainly vasodilatation, endothelium-dependent, only partially NO-dependent

Increased permeability (gaps) of endothelial cells (in culture)

Upregulation of PAR-2 by LPS and TNFalpha

Angiogenesis


PAR1-deficient (knockout) mice(Thrombin receptor knockout mice)

~50% die at embryonic day 9-10.

Surviving 50% become grossly normal adult mice.

PAR 1-/- platelets respond to thrombin, PAR 1-/- fibroblasts don‘t.

(Connolly et al., Nature 1996)


PAR2-deficient (knockout) mice

• Viable, fertile, normal size and vitality under pathogen-free conditions

• Delayed onset of inflammation (leukocyte rolling after surgical trauma)

(Lindner et al., J. Immunol. 2000)

• PAR2 mediates eosinophil infiltration and hyperreactivity in in allergic airway inflammation

(Schmidlin et al., J. Immunol. 2002)


PAR3- and PAR4-deficient (knockout) mice

Healthy, normal development

Abnormal platelet responses to thrombin

(Kahn et al., Nature 1998)

(Sambrano et al., Nature 2001)


Hannah Lee , Justin R. Hamilton

Physiology, pharmacology, and therapeutic potential of protease-activated receptors in vascular disease

Pharmacology & Therapeutics Volume 134, Issue 2 2012 246 - 259

http://dx.doi.org/10.1016/j.pharmthera.2012.01.007

PAR expression in the human vascular system


Hannah Lee , Justin R. Hamilton

Physiology, pharmacology, and therapeutic potential of protease-activated receptors in vascular disease

Pharmacology & Therapeutics Volume 134, Issue 2 2012 246 - 259

http://dx.doi.org/10.1016/j.pharmthera.2012.01.007

PAR antagonists


Peptide-mimetic PAR-1 antagonist

• Binds PAR-1, interferes with Ca++-mobilization and cellular functions

• No effect on N-terminal cleavage

• No effect on other PARs

(Andrade-Gordon et al., PNAS 1999)


Transmembrane serine proteases (Type 2)


Transmembrane serine proteases(Type 2)


Digestion


Acivation of ANP


Iron metabolism


Transmembrane serine proteases can activate PARs

From: Szabo & Bugge, 2012)


Activation of the epithelial Na+-channel by membrane associated proteases


In drosophila, clip-SPs form proteolytic cascades participating in the dorsal ventral-patterning during embryonic development and in the humoral immune responses of the adult flies.

Florian Veillard, Laurent Troxler, Jean-Marc Reichhart

Drosophila melanogaster clip-domain serine proteases: Structure, function and regulation

Biochimie, 2015, Available online 8 October 2015http://dx.doi.org/10.1016/j.biochi.2015.10.007


Protein C Inhibitor (PCI) binds to phospholipids and is internalized by cells

PCI can bind to oxidized/negatively charged phospholipids such as phosphatidylserine (PS), oxidized PS, oxidized phosphatidylethanolamine (PE), and phosphoinositides.

PCI can be internalized by cells in a PE-dependent manner and rapidly translocates to the nucleus.

Baumgärtner P. et al.

J Cell Biol 2007


• PCI is cleaved by testisin at the reactive site and a site close to its N-terminus.

• The N-terminus of PCI, corresponding to Helix A+, is released by testisin.

• Testisin cleaved PCI as well as a PCI mutant lacking Helix-A+ cannot be internalized by cells.

• The helix-A+ peptide is a cell penetrating peptide (CPP).

• Testisin or other proteases, which can cleave the helix-A+ peptide of PCI, may arrest the internalization of PCI into cells and thereby prevent its intracellular actions.

Nuclear targets

PCI

Cytosolic targets

Testisin

internalization

cleavage

A membrane-anchored protease regulates cell-membrane permeation of PCI

(Yang H. et al., J. Biol. Chem. 2015)


Proteolytic enzymes:

• Release of cytokines, growth factors, and other mediators from their membrane-bound precursors

• Down-regulation of receptors and other proteins from the cell surface

→ ADAMs (a disintegrin and metalloprotease)


Domain structure of ADAMs


ADAMs

• Mediate cytokine and cytokine receptor shedding (e.g. TNF)

• Mediate growth factor and growth factor receptor processing (HB-EGF)

• Mediate cleavage of other molecules (e.g. IGF-BP, prion precursor, amyloid precursor protein)


ADAMs

• Involvement in cell fate determination by action on:

– Delta and Notch

– Axon growth and guidance

– Cranial neural crest cells

– Adhesion molecules


Matrix Metalloproteases (MMPs)

Associated with normal and

pathological conditions that involve

matrix degradation and tissue

remodelling:

– Reproductive tissues (endometrium, breast, prostate)

– Connective tissue development

– Wound healing

– Tissue destruction in diseases (e.g. periodontitis, rheumatoid arthritis, tumor cell invasion)


MMP substrates that alter cell growth

A. MATRIX SUBSTRATES:

• Perlecan (MMP-1, MMP-3): Release of bound FGF

• Decorin (MMPs-2, 3, or 7): Release of bound TGF-ß


B. NON-MATRIX SUBSTRATES

– IGF-BP: active IGF-ligand

– Transmembrane heparin-binding-EGF (MMP-3): autocrine and paracrine actions

C. Direct activation of growth factors

TGFß (MMP-2, MMP-9)

IL1-ß (MMP-2, 3, and 9)

MMP substrates that alter cell growth


MMP substrates that regulate apoptosis

• Degradation of the basement membrane and loss of cell survival signals (involuting mammary gland)

• Direct proteolysis of death-inducing signaling components (Fas ligand cleavage by MMP-7; prostate involution)


MMP substrates in cell migration

Exposure of adhesion sites by matrix proteolysis

Removal of adhesion sites by matrix proteolysis

Generation of chemotactic signals

Inactivation of chemotactic signals


Functional competence of a protein flagged by protein termini: simplified representation of processing of the amyloid precursorprotein (APP) in the nonamyloidogenic (left, green) and amyloidogenic (right, red) pathways by α-secretases, β-secretases ...

Philipp F Lange, Christopher M Overall

Protein TAILS: when termini tell tales of proteolysis and function

Current Opinion in Chemical Biology, Volume 17, Issue 1, 2013, 73–82

http://dx.doi.org/10.1016/j.cbpa.2012.11.025


Figure 3. Classes of protein modification by limited proteolysis: (a) sequential maturation. Single amino acids or peptides are excised from the main protein backbone by exo-peptidases or endo-peptidases, respectively. One of the fragments undergoes degradatio...

Philipp F Lange, Christopher M Overall

Protein TAILS: when termini tell tales of proteolysis and function

Current Opinion in Chemical Biology, Volume 17, Issue 1, 2013, 73–82

http://dx.doi.org/10.1016/j.cbpa.2012.11.025

basic seminar thematic area: the cell surface and cell motility b) … · 2016-10-19 · center of...

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