Theoretical course: Basic biochemical methods and ischemic heart models

Zymography
Prepared by: Krisztina Kupai 2011

Supported by: HURO/0901/069/2.3.1 HU-RO-DOCS

Definition of Zymography
Zymography is an electrophoretic technique, based on SDS PAGE for measuring enzyme activity The technique is particularly useful for analyzing the proteinase composition of complex biological samples Zymography is also widely used to study various aspects of matrix metalloproteinase (MMP) function
Prepared by Krisztina Kupai

Types of zymography
SUBSTRATE ZYMOGRAPHY:
Gelatin Zymography Casein Zymography Collagen Zymography Heparin-Enhanced Substrate Zymography

REVERSE ZYMOGRAPHY In Situ ZYMOGRAPHY

Prepared by Krisztina Kupai

SUBSTRATE ZYMOGRAPHY: when specific substrate is co-polymerized with the acrylamide

In zymography, the proteins are separated by electrophoresis under denaturing [sodium dodecyl sulfate (SDS)], nonreducing conditions The separation occurs in a polyacrylamide gel containing a specific substrate that is co-polymerized with the acrylamide

Source:http://www.invitrogen.com

Prepared by Krisztina Kupai

SUBSTRATE ZYMOGRAPHY: gelatin or casein in the gel

Gelatin Zymography
Gelatin zymography is mainly used for the detection of the gelatinases, MMP-2 and MMP-9

Casein zymography
suitable for the detection of MMP-1, MMP-7, MMP-12, and MMP-13

Prepared by Krisztina Kupai

SUBSTRATE ZYMOGRAPHY: collagen in the gel

Collagen zymography
used for the detection of MMP-1 and MMP-13, but MMP-2 and MMP-9 can also be detected The incorporation of native collagen fibers in polyacrylamide gels appears unsuitable for zymography because of their complicated structure, but SDS disrupts most of the fibrillar organization of the collagen, allowing proteins to run into the gel.
Prepared by Krisztina Kupai

Heparin-Enhanced Substrate Zymography

It is known that the extraction of MMPs from tissue in the presence of heparin results in an enhancement of MMP activity. The addition of heparin to the samples during or prior to electrophoresis also enhances MMP activity. Used for MMP-7 The mechanisms by which heparin seem to enhance MMP-7 activity in zymography are: (i) the induction of a conformational change (ii) the facilitation of refolding (iii) the reduction of autolysis (iv) the increase of anchorage of the MMP in the gel
Prepared by Krisztina Kupai

Reverse Zymography
TIMPs can be detected by reverse zymography, which is a modification of zymography for MMPs Besides gelatin, an MMP is also incorporated into the gel, usually MMP-2. During the activation step after electrophoresis, the MMP-2 only digests the gelatin in areas where TIMPs are absent. Thus, after staining, the gel will be colorless, except for the TIMP bands

Prepared by Krisztina Kupai

In Situ ZYMOGRAPHY
In situ zymography allows the localization of MMPs in tissue sections in situ zymography uses a substrate that is deposited on or under a frozen section of an unfixed tissue sample During incubation, the substrate will be digested by the activated MMPs in a time- and dose-dependent manner The degradation of the substrate is detected by light microscopy or fluorescence microscopy, depending on the type of substrate. Prepared by Krisztina Kupai

In Situ ZYMOGRAPHY

The red and green signal indicates the immunolocalization of MMP-2

Prepared by Krisztina Kupai

Main steps in gelatin substrate zymography:

1. Sample homogenization without a reducing agent or boiling in order for the enzyme to retain its native state (and therefore its proteolytic activity).

2. Preparation of a gel with a final concentration of acrylamide of 8%, containing 2 mg/mL gelatin 3. Electrophoresis at 90 V constant voltage (no boiling, no use of reducing agents in order to preserve enzyme activity)

Prepared by Krisztina Kupai

Main steps in gelatin zymography:

4. Gel washing in Triton X-100 for 40 min at room temperature on an orbital shaker 5. Gel washing in incubation buffer for 20 min 6. Incubation of gels for 20 h at 37 C 7. Staining of the gels with Coomassie Blue for 30 min at room temperature. Areas of digestion appear as clear bands against a darkly stained background where the substrate has been degraded by the enzyme 8. Destaining with a methanol/acetic acid solution for 1 h at room temperature 9. Densitometry of gelatinolytic activity appearing as clear bands on the dark background

MMP-9 proMMP-2 MMP-2

Prepared by Krisztina Kupai

Advantages of substrate Zymography

Expensive materials are not required (e.g., antibodies) Proteases with different molecular weights showing activity towards the same substrate can be detected and quantified on a single gel.
For example, MMPs are released from cells in a proteolytically inactive proform (zymogen) which is approximately 10 kD larger than the activated form Because the proform becomes activated during the process of denaturation and renaturation after gel electrophoresis, the active form and the originally inactive forms degrade gelatin, and both forms can therefore be detected on zymograms.
Prepared by Krisztina Kupai

Advantages of substrate Zymography

MMPs in solution are often associated with endogenous tissue inhibitors of metalloproteases (TIMPs). During electrophoresis the inhibitors dissociate from the MMP and do not interfere with detection of the enzymatic activity. On the other hand, sandwich ELISA can discriminate between MMP/TIMP complexes and free MMPs, resulting in determination of a potential active fraction (Zucker et al. 1992; Ratnikov et al. 2002; Catterall and Cawston 2003).
Prepared by Krisztina Kupai

Advantages of substrate Zymography

On the basis of molecular weight markers, the molecular weight of the proteolytic band can be determined, and by comparison with recombinant proteins and the use of specific protease inhibitors the type of protease can be established

Prepared by Krisztina Kupai

Disadvantages of substrate Zymography

Information on the localization of the proteolytic activity in cells or tissues cannot be obtained on the basis of zymography.

Prepared by Krisztina Kupai

MMPs Introduction
MMPs were discovered in 1962 (ECM degradation is requiered for resorption of the tadpole tail) Play important roles:
Physiological processes: embriogenesis angiogenesis activation of cell surface receptors MMP-2 Pathological processes: tumour metastasis inflammation arthritis Cardiovascular diseases 1. atherosclerosis 2. heart failure 3. aneurism

MMP classification
Based on the ECM substrate which they proteolyse Groups of MMPs include : 1. Collageneses (MMP-1,-8) 2. Stromelysins (MMP-10,-11) 3. Membrane-type MMP (MMP-14,-15,-17) 4. Matrilysins (MMP-26) 5. Gelatinases (MMP-2,-9)
heart

MMP in the heart: MMP-2/gelatinase A

In the normal heart MMPs are present as proMMP and often expressed complex with their endogenous inhibitors ,the tissue inhibitors of MMPs (TIMP) MMP expressed and found:
1. Cardiac myocites 2. Endothelium 3. Vascular smooth muscle cells 4. fibroblasts Localized to the sarcomere within the myocyte

MMP-2 structre
Inactive/zymogen enzyme SH Pre Pro Catalytic (Zn 2+ ) Fibronectin- Zn 2+ type activation Active enzyme Catalytic (Zn 2+ ) Fibronectin- Zn 2+ type Hinge S S 64 kDa Hinge
HEMOPOEXIN

C-terminal S S 72 kDa

MMP-2 structure
Inactive/zymogen enzyme SH Pre Pro Catalytic (Zn 2+ ) Fibronectin- Zn 2+ type Hinge
HEMOPOEXIN

C-terminal S S

The propeptide domain contains a conserved cysteine that chelates the zinc in the active site fibronectin type II domain: enhance substrate binding Hinge: confers specificity hemopexin domain: regulatory subunit substrate specificity involved in activation as well as inhibition of MMPs calcium binding site (Ca required for full activity)

72 kDa

64 kDa

72 kDa

Proteolytic activation of MMP-2 by MT1-MMP/TIMP or by other proteases occurs by removal of the autoinhibitory propeptide domain (left arrow) resulting in an active truncated MMP-2. presence of oxidative stress (ONOO-) and cellular glutathione (GSH) causes the Sgluathiolation of the critical cysteine residue in the propeptide domain, disrupting its binding to the catalytic Zn2+ ion, resulting in an active full-length enzyme

Activation of MMPs and inactivation by ONOO

Peroxynitrite is formed in vivo by the reaction of NO with superoxide. It is a powerful oxidizing agent that can initiate lipid peroxidation, oxidize sulfhydryls, and nitrate the aromatic residues of proteins. The cysteine residue of propeptide domain containing the sulphhydryl groups coordinated to the catalytic Zn2+ Highly sensitive to changes in the redox environment 1-20 M ONOO activates MMP-1,-8,-9 >100 M ONOO inactivate MMP-2 likely via the nitration of tyrosine residues in the sequence

SH Pre Pro Catalytic (Zn 2+ ) Fibronectin- Zn 2+ type Hinge

HEMOPOEXIN/FIB RONECTIN

Modulation of MMP activity by TIMPs

TIMP: tissue inhibitor of metalloproteinase TIMPs are comprised of 4 family members:
TIMP-1: expression is response to signals such as proinflammatory cytokines TIMP-2: expressed constitutively in most of the cell types found in the heart TIMP-3: found in greatest concentration in the ECM TIMP-4: also known as cardiac inhibitor of MMPs; most abundant in the heart

Modulation of MMP activity by TIMPs

all four TIMPs have been observed in the heart and in cardiac myocytes Each ~23 kDa in size They inhibit the activity by forming tight binding complexes in a 1:1 ratio with MMPs TIMP-4 is localized to the sarcomers and to the thin myofilaments, where MMP-2 is also found

Therapeutic potential of MMP inhibition

Since various MMP isoenzymes are involved in the human pathologies, close to 60 MMP inhibitor (MMPi) have been pursued as clinical candidates since the first drug discovery program began The clinical developments of most of the MMPi have discontinued due to safety reasons

Therapeutic potential of MMP inhibition

Doxicycline (Periostat ) (approved for periodontal disease)
Able to protect the isolated heart from I/R Inhibit MMP-2 Occured in a concentration range Thought to have antioxidant properties (scavenge ONOO)

Other synthetic MMP-2 inhibitors

O-phenantroline Ilomastat (GM-6001, gelardin)
Zn 2+ chelators

Structure of ilomastat,the Znbinding group is highlighted in box