Proteases and Signaling Sherwin Wilk, Ph.D. Mount Sinai School of Medicine Department of Pharmacology & Biological Chemistry Cell Signaling Systems Course

Proteases and SignalingProteases and Signaling

Sherwin Wilk, Ph.D.Sherwin Wilk, Ph.D.Mount Sinai School of MedicineMount Sinai School of Medicine

Department of Pharmacology & Biological ChemistryDepartment of Pharmacology & Biological Chemistry

Cell Signaling Systems CourseCell Signaling Systems Course

Traenckner et al, EMBO J. 1994 Nov 15;13(22):5433-5441.

Phosphorylation and proteolysis are Phosphorylation and proteolysis are required in the activation of NF-required in the activation of NF-BB

Proteolysis is a hydrolytic reactionProteolysis is a hydrolytic reaction

R1

C

O

N

R2

+ H20 R1 C

O

O

- + R2 NH3+

Classes of proteolytic enzymes:Classes of proteolytic enzymes:

1.1. SerineSerine

2.2. CysteineCysteine

3.3. MetalloMetallo

4.4. AspartylAspartyl

5.5. ThreonineThreonine

Protease-Activated ReceptorsProtease-Activated Receptors

Fig. 1. Mechanism of G protein-coupled receptor (GPCR) activation by a reversible binding of a soluble ligand, such as a neuropeptide, and irreversible cleavage by a protease.

Déry et al, Am J Physiol. 1998 Jun;274(6 Pt 1):C1429-1452.

Fig. 2. A: protein structure of protein-activated receptor (PAR)-1, PAR-2, and PAR-3. Amino acid sequences in NH2-terminus and second extracellular loop that are important for receptor activation are shown. Boxed residues indicate tethered ligand domains (PAR-1, PAR-2, and PAR-3) and anion binding sites (PAR-1 and PAR-3). Arrows indicate cleavage sites. Bold residues in second extracellular loop are conserved. # Intron/exon border. * Glycosylation site. B: genomic organization and chromosomal localization of PAR-1 and PAR-2. Both genes consist of 2 exons and 1 large intron. Exon 1 encodes NH2-terminal domains proximal to cleavage sites, and exon 2 encodes the rest of the receptors. Both receptors are localized within 100 kb on chromosome 5q13.

Déry et al, Am J Physiol. 1998 Jun;274(6 Pt 1):C1429-1452.

TABLE 1Structure/activity relationships for TRAPs

Peptide Cell/Tissue Type Effect (EC50/IC50) References

SFLLRNPNDKYEPF (TRAP-14)

Xenopus oocytes, platelets, rat aortic rings (endothelium denuded or intact), guinea pig gastric longitudinal smooth muscle, CCL39 hamster fibroblasts, rat glomerular mesangial cells, rat astrocytes

4-30 µM Chao et al., 1992; Coller et al., 1992; Kawabata et al., 1999c; Vouret-Craviari et al., 1992; Vu et al., 1991a; Yang et al., 1992

SFLLR-NH2 Platelets, transfected mammalian cells, endothelium denuded and intact RA, CCL39 fibroblasts

0.5-6 µM Ceruso et al., 1999; Hollenberg et al., 1996; Kawabata et al., 1999c; Laniyonu and Hollenberg, 1995; Natarajan et al., 1995; Scarborough et al., 1992

NPNDKYEPF short peptides (<5)

PlateletsCCL39 fibroblasts, various species platelets, rat glomerular mesangial cells, endothelium denuded RA and gastric LM

>200 µMLoss of function

Vassallo et al., 1992Albrightson et al., 1994; Bernatowicz et al., 1996; Chao et al., 1992; Connolly et al., 1994; Vouret-Craviari et al., 1992

Macfarlane et al, Pharmacol Rev. 2001 Jun;53(2):245-282.

Substituted peptides

Acetyl-SFLLR-NH2 Platelets, transfected mammalian cells, rat gomerular mesengial, SH-EP cells

>1000 µM Albrightson et al., 1994; Coller et al., 1992; Sakaguchi et al., 1994; Scarborough et al., 1992

H-SFLLR-NH2 Platelets, transfected mammalian cells, SH-EP cells

Low activity Scarborough et al., 1992; Shimohigashi et al., 1994; Van Obberghen-Schilling et al., 1993

XFFLR-NH2 Various cell types Charged amino acids not tolerated, size/shape important; Thr substitution yields a PAR-1-specific peptide

Bischoff et al., 1994; Ceruso et al., 1999; Chao et al., 1992; Hollenberg et al., 1997; Natarajan et al., 1995; Sakaguchi et al., 1994; Scarborough et al., 1992; Van Obberghen-Schilling et al., 1993; Vassallo et al., 1992; Yang et al., 1992

SXLLR-NH2 Various cell types Only aromatic residues tolerated Albrightson et al., 1994; Ceruso et al., 1999; Chao et al., 1992; Natarajan et al., 1995; Nose et al., 1993; Scarborough et al., 1992; Van Obberghen-Schilling and Pouyssegur, 1993; Vassallo et al., 1992

SFXLR-NH2 Various cell types No loss of activity, more active with 3-(2-naphthyl)-L-alanine

Bischoff et al., 1994; Blackhart et al., 1996; Ceruso et al., 1999; Chao et al., 1992; Laniyonu and Hollenberg, 1995; Natarajan et al., 1995; Scarborough et al., 1992; Shimohigashi et al., 1994; Van Obberghen-Schilling and Pouyssegur, 1993; Vassallo et al., 1992

SFLXR-NH2 Various cell types Acidic and basic amino acids not tolerated

Blackhart et al., 1996; Ceruso et al., 1999; Chao et al., 1992; Natarajan et al., 1995; Scarborough et al., 1992; Vassallo et al., 1992

SFLLX-NH2 Various cell types Wide range of residues tolerated, but reduced activity

Blackhart et al., 1996; Ceruso et al., 1999; Chao et al., 1992; Hollenberg et al., 1997; Natarajan et al., 1995; Nose et al., 1998b; Scarborough et al., 1992; Vassallo et al., 1992

Monocyclic SFLLRN analogues Platelets Significant loss of activity McComsey et al., 1999

Macfarlane et al, Pharmacol Rev. 2001 Jun;53(2):245-282. (Table 1 continued)

Improved agonists

S(p-F)FFLRNP Platelets, SH-EP cells 1.7 µM Nose et al., 1998a; Nose et al., 1993; Shimohigashi et al., 1994

S(p-F)FpGuFLR-NH2 Platelets, smooth muscle, mouse fibroblasts

0.4 µM Bernatowicz et al., 1996

A(p-F)FRCha-HarY-NH2 Platelets 0.01-0.12 µM Ahn et al., 1997; Debeir et al., 1997; Feng et al., 1995; Kawabata et al., 1999c

A(p-F)FRChaCitY-NH2 Platelets 0.2 µM Kawabata et al., 1999c

S(p-F)FHarLRK-NH2 Xenopus oocytes 0.05-0.1 µM Blackhart et al., 1996

S(p-F)F2NaALR-NH2 Platelets, CHRF-288 membranes 80 nM Seiler et al., 1996


Antagonists IC50

1-phenylacetyl-4-(6-guanidohexanoyl)-piperazine

Platelets 50% inhibition of SFLLRNP Alexopoulos et al., 1998

1-(6-guanidohexanoyl)- 4-(phenylacetylamido-methyl)-piperazidine

Platelets 40% inhibition of SFLLRN Alexopoulos et al., 1998

BMS-197525 {N-trans(p-F)FpGuFLR-NH2}

Platelets, smooth muscle, mouse fibroblasts

0.2 µM of SFLLRNP Bernatowicz et al., 1996

BMS-200261 {N-trans(p-F)FpGuFLRR-NH2}

Platelets 20 nM (SFLLRN) 1.6 µM (thrombin) Bernatowicz et al., 1996; Kawabata et al., 1999c

3-Mercapto-propionyl-FChaChaRKNDK-NH2

Platelets 0.7-6.4 µM (thrombin) Kawabata et al., 1999c; Seiler et al., 1995

LVR(D-)CGKHSR Rat astrocytes 180 µM ([3H]thymidine incorporation, by TRAP14 or thrombin)

Debeir et al., 1997

Oxazole-30 Platelets, CHRF membranes

25 µM (thrombin), 6.6 µM (SFLLRN) Hoekstra et al., 1998

S(Npys)- Mp-(p-F)F-NHCH(C6H5)2 Platelets 52 µM (SFLLRNP) Fujita et al., 1999

S(Npys)- Mp-(p-F)F-NHCH2CH(C6H5)2 Platelets 54 µM (SFLLRNP) Fujita et al., 1999

RWJ-56110 series Platelets 0.34 µM (thrombin) 0.16 µM (SFLLRN)

Andrade-Gordon et al., 1999

SCH 79797 Platelets, smooth muscle 70 nM ([3H]haTRAP binding) Ahn et al., 2000

SCH 203099 Platelets, smooth muscle 45 nM ([3H]haTRAP binding) Ahn et al., 2000

FR171113 Platelets 0.29 µM (thrombin) Kato et al., 1999


Labels IC50/Kd

SFp-azido-FLRNPKGGK-biotin HEL cells No effect Bischoff et al., 1994

[3H]haTRAP {[3H]A(p-F)FRChaHarY-NH2}

Platelet membranes, platelets 0.15 µM, Kd = 15 nM Ahn et al., 1997

A(p-F)FRChaHar(125I)Y-NH2 Platelets 0.03 µM Feng et al., 1995

BMS-200661 {N-trans(p-F)FpGuFLROrn}

Platelets Kd = 10-30 nM Bernatowicz et al., 1996

SFLLRNPNDKYEPF-biotin BHK cells Kd = 3 µmol/l Takada et al., 1995

BMS-197525, [3H] and biotinylated derivatives

CHRF-288 cells Kd = 80 nM or less Elliott et al., 1999


Amino acid residues X indicates amino acid scan; Har, homoarginine; (pF)F, parafluorophenylalanine; Cha, cyclohexylalanine; N-trans, trans-cinnomoyl; pGuF, p-guanidino-phenylalanine; Cit, citrulline; Orn, ornithine; 2NaA, 2-naphthylalanine; Npys, S-3-nitro-2-pyridinesulphenyl; β-Mp, β -mercaptopropionyl; [3H]haTRAP, [3H]A(p-F)FRChaHarY-NH2. All EC50 values refer to platelet aggregation unless indicated otherwise. Likewise, all IC50 values refer to inhibition of

TRAP-induced platelet aggregation unless indicated otherwise.

FIG. 2. Structural and functional domains of PARs. The figure shows alignment of domains of human PAR1, PAR2, PAR3, and PAR4. A and B: mechanism of cleavage and interaction of the tethered ligand with extracellular binding domains. C: functionally important domains in the amino terminus, second extracellular loop, and carboxy terminus. Conserved residues in loop II are in bold. [Adapted from Derian et al. (89) and Macfarlane et al. (182).]

Ossovskaya and Bunnett, Physiol Rev. 2004 Apr;84(2):579-621.

FIG. 5. Summary of PAR1 signal transduction. PAR1 couples to Gi, G12/13, and Gq11. Gi inhibits adenylyl cyclase (AC) to reduce cAMP. G12/13 couples to guanine nucleotide exchange factors (GEF), resulting in activation of Rho, Rho-kinase (ROK), and serum response elements (SRE). Gq11 activates phospholipase C(PLC) to generate inositol trisphosphate, which mobilizes Ca2+, and diacylglycerol (DAG), which activates protein kinase C (PKC). PAR1 can activate the mitogen-activated protein kinase cascade by transactivation of the EGF receptor, through activation of PKC, phosphatidylinositol 3-kinase (PI3K), Pyk2, and other mechanisms. G subunits couple PAR1 to other pathways, such as activation of G proteins receptor kinases (GRKs), potassium channels (Ki), and nonreceptor tyrosine kinases (TK). [Modified from Coughlin (68).]

Ossovskaya and Bunnett, Physiol Rev. 2004 Apr;84(2):579-621.

“Cells have capitalized on two highly specific processes --- phosphorylation and ubiquitination --- to control complex signal-transduction pathways,” says Maniatis. “They’ve basically exploited every means of regulating signaling at their disposal.”

The Ubiquitin-Proteasome The Ubiquitin-Proteasome SystemSystem

UbiquitinUbiquitin

Ciechanover et al, J Biol Chem. 1980 Aug 25;255(16):7525-7528.

Ciechanover et al, J Biol Chem. 1980 Aug 25;255(16):7525-7528.

E1SH + Ub + ATP E1S-Ub + PPi + AMP

E1S-Ub + E2SH ES2-Ub + E1SH

E2-Ub + E3-protein Ub-protein conjugate + E2 + E3

Pickart, Cell. 2004 Jan 23;116(2):181-190.

The Ubiquitin-Proteasome The Ubiquitin-Proteasome SystemSystem

The ProteasomeThe Proteasome

Wilk and Orlowski, J Neurochem. 1983 Mar;40(3):842-849.

Groll et al, Nature. 1997 Apr 3;386(6624):463-471.

Wilk and Orlowski, Arch Biochem Biophys. 2000 Nov 1;383(1):1-16.

Ferrell et al, Trends Biochem Sci. 2000 Feb;25(2):83-88.

Tanaka et al, Biochem (Tokyo). 1998 Feb;123(2):195-204.

Impairment of signaling Impairment of signaling by proteolysisby proteolysis

Anthrax lethal factorAnthrax lethal factor

Composition of anthrax toxinComposition of anthrax toxin

protective antigenprotective antigen

edema factoredema factor

lethal factorlethal factor

Collier and Young, Annu Rev Cell Dev Biol. 2003;19:45-70.

Figure 1 Stereo ribbon representation of LF, coloured by domain. The MAPKK-2 substrate is shown as a red ball-and-stick model, and the Zn2+ ion is labelled. The RMSD (C) between the cubic and monoclinic crystal forms is 1.18 ?. The principal differences lie in the position of the helical bundle of domain IV, which undergoes a rigid body shift of ~2 ? relative to the β-sheet, and a smaller shift of the β-sheet of domain I. The third helical element of domain III is invisible in the monoclinic crystals, but can be seen in cubic crystals, albeit with high B-factors. It is possible that this mobile amphipathic helix has a role in the membrane-inserting properties of LF observed in vitro28. Figure prepared with MOLSCRIPT, RENDER and RASTER3D29–31.

Pannifer et al, Nature. 2001 Nov 8;414(6860):229-233.

Tonello et al, Nature. 2002 Jul 25;418(6896):386.

Montecucco et al, Trends Biochem Sci. 2004 Jun;29(6):282-285.

Inhibition of signaling by the Inhibition of signaling by the YersiniaYersinia effector YopJ effector YopJ

Orth, Curr Opin Microbiol. 2002 Feb;5(1):38-43.

Fig. 1. Profile of YopJ inhibition. YopJ is delivered into the target host cytosol via a type III secretion system. YopJ blocks activation of the superfamily of MAPK kinases, including MKKs (which activate the MAPK pathways), and IKKβ (which activates the NFκB pathway). The inhibition results in the inability of the cell to produce cytokines and anti-apoptotic machinery. The possibility exists that YopJ may directly activate the cell death machinery, as denoted by the red arrow with the question mark.


Fig. 2. Amino acid sequence alignment of AVP, the family of YopJ homologues and Ulp1. The figure shows alignment of the catalytic core of cysteine proteases with the catalytic triad (denoted in red) and identities or similarities (outlined and shaded light blue). The alignment includes (protein accession numbers in parentheses): AVP (2781331); YopJ (P31498); AvrA (AAB83970); AvrBst (AAD39255); AvrRxv (AAA27595); AvrXv4 (AAG39033); AvrPpiG1 (CAC16700); ORF5 (AAF71492); ORFB (AAF62400); Y4LO (P55555); and Ulp1 (NP_015305).


Fig. 3. Modification of proteins by ubiquitin or SUMO. Ubiquitin and SUMO are linked to target proteins by an isopeptide bond. The carboxyl terminus of these modifying proteins (blue) is conjugated to the -amine of a lysine residue (LYS) in the target protein. Ubiquitin, unlike SUMO, can conjugate more monomers to itself to form a polyubiquitin chain. De-ubiquitinating enzymes (DUB) and ubiquitin-like protein proteases (for example, Ulp1) can cleave the isopeptide bond in the ubiquitin-conjugates (green arrows) or SUMO-conjugates (pink arrow), respectively.


Fig. 4. Similarities between two reversible post-translational modifications. Phosphorylation is shown on the left and ubiquitination is shown on the right. Addition of phophorylation (P) by kinases and ubiquitin (Ub) by the E1–E2–E3 conjugation machinery to target proteins is shown in blue. The requirement for energy is noted by the purple ATP. The removal of phosphorylation by a phosphatase or of ubiquitin by a de-ubiquitinating enzyme is shown in red. The target protein (green) can cycle from an unmodified to a modified state. The model can be used for ubiquitin-like proteins by replacing the modification with, for example, SUMO or NEDD8, and replacing the protease with a ubiquitin-like protein protease.

Regulated Intramembranous Regulated Intramembranous ProteolysisProteolysis

Notch PathwayNotch Pathway

Mumm and Kopan, Dev Biol. 2000 Dec 15;228(2):151-165.

Golde and Eckman, Sci STKE. 2003 Mar 04;2003(172):RE4.

Fig. 2. Presenilin and presenilin-like MpMPs regulate cleavage of diverse substrates. IP by -secretase (presenilin) requires prior JP by either ß-secretase (shown) or -secretase (not shown). The combination of ß- and -secretase cleavages produce the Alzheimer's disease-associated peptide Aß, whereas the combination of - and -secretase cleavages produce a peptide known as P3 (not shown), whose role in the disease process in currently unknown. Both sequential cleavage events produce CTF [also known as APP intracellular domain (AID or AICD)], which can translocate to the nucleus to form a transcriptionally active complex with Fe65 and Tip60. The nuclear targets of the CTF are not known. Although a single -secretase cleavage is shown, -secretase cleaves the transmembrane domain of APP at multiple sites both near the middle of the membrane and near the cytoplasmic face of the membrane.

Fig. 3. The presenilin -secretase appears to be an aspartyl MpMP. The eight-transmembrane domain model of PS is shown. Although the topology of the first six transmembrane domains is accepted, the exact topology of the COOH-terminal transmembrane remains controversial. The red and orange stars indicate the location of the conserved active site residues within the transmembrane domains 6 and 7. Mutation of either of these aspartates (D) results in a dominant-negative PS that inhibits -secretase activity. A red triangle also indicates the location of the evolutionarily conserved Pro-Ala-Leu (PAL) motif essential for PS stability. The box to the right shows the consensus sequences surrounding these conserved residues. The putative location of these active site aspartates places them in location to cleave near the COOH-terminus of Aß. Y, Trp; G, Gly.



Fig. 4. -Secretase also cleaves the type 1 surface receptor Notch and its family members. Following binding of a ligand present on the surface of the adjacent cell (delta, serrate, or Lag-2) to the extracellular domain of Notch, a conformational change occurs permitting JP by the metalloprotease disintegrin (ADAM17). The Notch COOH-terminal fragment (NEXT) is then cleaved by -secretase to release the Notch intracellular domain (NICD) into the cytoplasm. Upon release, the NICD translocates to the nucleus, where it acts as a transcriptional regulator through its interactions with the transcription factor CSL.


Sterol regulatory element binding Sterol regulatory element binding protein (SREBP)protein (SREBP)

Fig. 1. (A) RIP of SREBP involves an additional protein, SCAP, that regulates SREBP transport. SREBP exists in a hairpin-like conformation with a small luminal loop and two large cytoplasmic domains. The NH2-terminal domain is a basic helix-loop-helix (bHLH) transcription factor and the COOH-terminal domain is a regulatory factor (REG) that binds SCAP. When cells are loaded with sterols, the complex of SCAP and SREBP is sequestered in the ER. Upon sterol deprivation, the SCAP-SREBP complex is transported to a post-ER compartment (thought to be the cis or medial Golgi) where S1P-mediated juxtamemembranous proteolysis (JP) of the small luminal loop of SREBP occurs. The bHLH domain of SREBP is then translocated to the nucleus, where it binds to sterol regulatory elements and controls transcription of a number of genes involved in sterol metabolism. (B) S2P appears to be a metalloprotease-type intramembranous cleaving protease (MpMP). A schematic of the overall topology of S2P is indicated. The position of residues involved in catalysis is indicted by the stars. The conserved residues indicated by these stars are shown in the box. The conserved active site histidines (red star) appear to be located at a site in the membrane that permits cleavage of SREBP within its transmembrane domain close to the cytoplasmic face of the membrane. These residues, along with the remote aspartate, are hypothesized to form an active site similar to that formed by classic metalloprotease. The true three-dimensional structure of S2P is not known, and location of residues is strictly based on hydropathy plots and topology studies.



Rhomboid and EGF signalingRhomboid and EGF signaling

Figure 2. Identification of Putative Catalytic Residues of Rhomboid-1(A) Conserved residues were individually mutated to alanine and the ability of the mutant proteins to mediate Spitz cleavage was determined. The upper panel shows Western blots of cleaved GFP-Spitz in the medium of cells transfected with GFP-Spitz, Star, and mutant or wild-type Rhomboid-1. Mutation of W151, R152, N169, G215, S217, and H281 abolished detectable Rhomboid-1 activity—compare with no Rhomboid-1 control (−). The lower panel shows Rhomboid-1 levels in cells, assessed using N-terminally HA-tagged Rhomboid-1 mutants.(B) All mutants that were unable to mediate Spitz cleavage were, like the wild-type protein, localized to the Golgi apparatus. HA-tagged S217A in a COS cell is shown in green; anti-p115 (Transduction Labs), a Golgi marker, in red.(C) Comparison of the conserved GASGG motif surrounding the putative Rhomboid-1 active serine (S217) with that of serine proteases (family S1A according to MEROPS classification). The subscripts represent the percentage conservation at each residue (total nonredundant sequences for each family are reported as "n" values).(D) Rhomboid-1 with conservative mutations S217C and S217T did not catalyze Spitz cleavage above background levels; panels as in (A).(E) Diagram of Rhomboid-1 residues essential for Spitz cleavage; conserved but nonessential residues are shown in green, essential residues in red. The TMD predictions were agreed by a variety of algorithms, including TMHMM and TMPred (available at http://ca.expasy.org/tools/#transmem)

Urban et al, Cell. 2001 Oct 19;107(2):173-182.

Urban et al, Cell. 2001 Oct 19;107(2):173-182.

Figure 7. Model of Rhomboid-1 ActionWe propose that the conserved asparagine (N), histidine (H), and serine (S) form a serine protease catalytic triad that hydrolyses the Spitz polypeptide within its TMD (although the role of the asparagine is uncertain—see text). Our data imply that this occurs approximately one-third of the distance into the membrane bilayer from the lumenal surface. The lumenal tryptophan (W) and arginine (R) are also essential (although the tryptophan appears less critical in human RHBDL2), but their function in the catalytic mechanism remains to be determined

Fig. 7. RIP by a novel serine MpMP, Rhomboid. (A) Drosophila Rhomboid-1 promotes cleavage of at least three epidermal growth factor (EGF)-like type 1membrane proteins, Spitz, Keren, and Gurken. Rhomboid cleavage does not appear to require prior JP. It also is unique in that it releases the ectodomain from the membrane rather than releasing a cytoplasmic domain. In their uncleaved forms, these EGF-like ligands are sequestered in the ER. In activated cells, the transmembrane protein Star facilitates trafficking to the Golgi where the active Rhomboid protease resides. In the Golgi the ligands are cleaved near the luminal border of their transmembrane domains, releasing them from the membrane. Once released, the ligand is secreted where it binds the EGFR. (B) The predicted topology of Drosophila Rhomboid-1. Rhomboids are serine MpMPs. The stars show the location of the putative catalytic triad and the box describes these consensus sequences (N, Asn; G, Gly; A, Ala; S, Ser; H, His). As is the case for other MpMPs, the active site residues appear to be positioned in an appropriate location to carry out their cleavage, which in this case occurs near the luminal face of the membrane. In addition, the red triangle shows the location of another sequence (W, Trp; R, Arg) essential for function.


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Proteases and Signaling Sherwin Wilk, Ph.D. Mount Sinai School of Medicine Department of Pharmacology & Biological Chemistry Cell Signaling Systems Course