UNIVERSITÀ DI PISA
Dipartimento di Farmacia
Corso di Laurea Specialistica in Chimica e Tecnologia Farmaceutiche
Tesi di Laurea:
Synthesis and X-ray analysis of Metzincin inhibitors
Relatori: Prof. Armando Rossello,
Dott.ssa Elisa Nuti,
Dott. Enrico A. Stura
Candidata: Livia Tepshi (N° MATRICOLA 441654)
SDD: CHIM/08
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Identificazione per presa visione *
Summary
Abstract...p.5 Aims of the thesis ...p.7 Preface ...p.8
CHAPTER 1
ADAMs
1.1 Introduction-Cancer Pathways ...p.9 1.2 ADAMs ...p.12 1.3 Regulation of ADAMs ...p.16 1.4 ADAMs Structure ...p.20 1.5 Mechanism of activation of ADAMs ...p.22
CHAPTER 2
Matrix Metalloproteinases(MMPs)
2.1 Introduction ...p.24 2.2 Functions ...p.24 2.3 (Q)SAR Interactions ...p.26 2.4 Structure ...p.26 2.5 Mechanism of the proteolysis in the MMPs ...p.27CHAPTER 3
Expression and purification of the mutant MMP8A
3.1 Introduction ...p.29 3.2 Different systems used for protein's expression...p.29 3.2.1 Expression using E.Coli...p.31 3.2.2 Importance of expressing the mutant...p.31 3.3 Experimental part ...p.31 3.3.1 Introduction ...p.32 3.3.2 Materials and methods ...p.32 3.4 Experimental data ...p.35 3.5 Results ...p.35
CHAPTER 4
Biological crystallogenesis
4.1 Introduction ...p.37 4.2 Importance of 3D structure determination ...p.38 4.3 Macromolecular Protein Crystallization ...p.38 4.4 Sitting drop, Hanging drop, Sandwich drop ...p.41 4.5 Streak seeding technique ...p.42 4.6 Cryosolutions ...p.43 4.7 X-Ray Crystallography ...p.44 4.8 Structure Definition and Refinement ...p.45
CHAPTER 5
Experimental part introduction
5.1 Design and Synthesis of selective ADAM-10 inhibitors ...p.47 5.1.1 Scheme1 ...p.53 5.2 Crystallography ...p.54 5.2.1 Introduction ...p.54 5.2.2 Inhibitors tested ...p.55 5.2.3 Complexes of 2 with MMPs ...p.56 5.2.4 Distances from the Zn atom ...p.58
5.2.5 Comparison between the compound 2 (LT4) binding the catalytic site
and MMP-8,-9 and -12 ...p.60 5.2.6 Polymorphism ...p.60
CHAPTER 6
Experimental Part
6.1 Chemistry experiments ...p.63 6.1.1 Materials and methods ...p.63 6.1.2 Synthesis ...p.63 6.2 Crystallization Experiments ...p.66 6.2.1 Crystallization Procedure ...p.66 6.2.2 Materials and Methods ...p.66 6.2.3 Crystallization experiments ...p.67 Bibliography ...p.70
Abstract
The selectivity of zinc-metalloproteases inhibitors is crucial as broad spectrum inhibitors can cause severe adverse effects. In both laboratories where I have carried out my thesis work, the development of new selective inhibitors is a major endeavor. The work has involved the synthesis of inhibitors for ADAM10 and the crystal structure determination of these ligands complexed to the catalytic domains of different MMPs with the aim to design inhibitors with enhanced specificity. The ADAMs family of trans-membrane proteins belongs to the zinc protease superfamily. Members of this family have a modular design, characterized by metalloprotease and integrin receptor-binding activities. The cytoplasmic domain in many family members specifies receptor-binding sites for various signal transducing proteins. The ADAMs family has been implicated in the control of membrane fusion, cytokine and growth factor shedding, cell migration, as well as processes such as muscle development, fertilization, and cell fate determination. Pathologies such as inflammation and cancer may involve ADAMs family members. Zinc proteases are subdivided according to the primary structure of their catalytic sites and include gluzincin, metzincin, inuzincin and certain carboxypeptidases [1]. The ADAMs belong to the metzincin subgroup that is subdivided into serralysins, astacins, matrixins, and adamalysins [2]. The matrixins comprise the matrix metalloproteases, or MMPs. These enzymes are the main agents responsible for extracellular matrix degradation and remodeling, and play relevant roles in development, wound healing and in the pathology of diseases such as arthritis and cancer [3]. Adamalysins differ from the matrixins by the presence of a unique integrin receptor-binding disintegrin domain but have similar metalloprotease domains. ADAMs derive their name from the presence of these two domains (A Disintegrin And
Metalloprotease). Their involvement in such a broad spectrum of diseases is due to the large variety
of substrates that ADAMs are able to cleave. They can activate growth factors or inactivate receptors by shedding their extracellular domain from the cell membrane. Similarly, they can break off cells by cleaving cell adhesion molecules. Some of these proteolytic events are part of cleavage cascades known as Regulated Intramembrane Proteolysis that leads to intracellular signaling. Thus ADAMs can fulfill a key role in diverse processes and pathologies, making them prime targets for developing therapies. This is the main reason for pharmaceutical research investment in zinc protease inhibitors (MMP and ADAM inhibitors).
My thesis started with the aim to design and synthesize selective ADAM10 inhibitors:
• The synthesis of LT2 (Fig.1)
All the new inhibitors, were tested in vitro in human recombinant ADAM-10 and ADAM-17 (TACE1)and on MMPs (MMP-8, MMP-9, MMP-12, MMP-13) by fluorimetric assay. The results are shown in Table 1.
IC50(μM) IC50(μM)
Compd MMP8h MMP9h MMP12h MMP13h MMP14h ADAM 10 TACE*
LT2 >10 7 >10 7.8 6.4 5.5 10
LT4 2 2.7 0.29 0.7 8.1 0.004 0.5
LR43 - 2.9 31 0.97 - -
-Table 1: In vitro activity (IC50) for MMPs and ADAMs
The inhibitors' activity was in the micro-molar range, except for LT4 that is nanomolar and selective for ADAM-10 and able to discriminate against TACE. The reason why this hydroxamate based inhibitor has such good selectivity needs to be understood. Referring to Tape et al [4] the “C-shaped” form of the ADAM proteins implies that large catalytic-cleft inhibitors such as TIMPs and ADAM pro domains cannot bind ADAM ectodomains efficiently. (Further described in chapter 1).
1 1Tumor necrosis factor-alpha converting enzyme.
During the course of my thesis, crystallographic studies were carried out only using the catalytic domains of MMP-8, MMP-9, MMP-12 and MMP-13 since MMP-14, ADAM-10 and TACE were not yet available for crystallographic studies. This gives an initial idea regarding how the ligands are stabilized in the metzincin catalytic sites and of the interactions made by the substituents within the pockets. This allows a comparison with theoretical predictions and other crystallographic studies.
Aims of the thesis:
This study focuses on selectivity of LT4 for ADAM-10 and aims to understand the interactions made within the catalytic site to design new inhibitors with better specificity toward MMPs or ADAMs, both part of the metzincin family. The design of the new molecules is possible because of the similarities in the catalytic domains of MMPs and TACE. During my thesis, active compounds were tested on different types of MMPs, ADAM-10 and TACE. X-ray structure determination was carried out for the complexes that could be crystallized. This was determined by the availability of the catalytic domains and the success of the crystallization efforts. The structures obtained of the various MMP inhibitor complexes give sufficient insight to guide the design of new inhibitors. The synthesized inhibitors can then be tested so as to verify that the insights have lead to improved selectivity for the targeted enzyme (ADAM-10) and better discrimination against TACE and the other MMPs. Although the amino acidic sequence of ADAM-10 is well-known, no three-dimensional structure has yet been determined. This is the reason why the models may become very useful in the future for the progression of the research.
Preface:
This thesis contains work done from April 2013 to February 2014. Starting with the organic synthesis of the inhibitors described in chapter 6 that was finished in October. The development of selective ADAM-10 and TACE inhibitors is summarized in chapter 5 using models of inhibitors developed before. Some of the inhibitor models that were considered, made use of a strong zinc chelating moiety, the hydroxamate group, in some other articles the arylsulfonamides were retained important for the selective inhibition of TACE in ovarian cancer[5][6].
The work proceeded in the next three months at the Commissariat à L'Energie Atomique (CEA) with efforts to crystallize MMP-inhibitor complexes. This involved learning how to obtain crystals that diffract at high resolution, how to prepare the crystals for X-ray data collection at two different synchrotron facilities (Soleil, St. Aubin and European Synchrotron Research Facility (ESRF), Grenoble, France) described in Chapter 3.
During my experience at CEA I also assisted in the production of the catalytic domain of the inactive mutant form of the human MMP8 using the Escherichia coli expression system (Chapter 4).
CHAPTER 1
ADAMs (A Disintegrin and Metalloproteinase)
1.1 Introduction-Cancer Pathways
Cancer begins when cells of our body start to grow out of control. Some of the earliest evidence of cancer is found among fossilized bone tumors, in human mummies from ancient Egypt, dating back to about 3000 BC. There are several disease pathways followed during the progression of cancer. Research tends to choose the one that brings results faster and with less side-effects. This is not an easy task. The following are the cancer pathways:
➢ AKT signaling pathways – serine/threonine kinase AKT and PI3K2 are the two proteins involved. The activation of AKT by receptor tyrosine kinases, integrins, B and T cell receptors, cytokine receptors, G-protein-coupled receptors and other stimuli leads to the production of PtdIns(3,4,5)P33 by PI3K. These lipids serve as plasma membrane docking sites for proteins, including AKT which is over-expressed in pathological conditions such as cancer [7].
➢ Inhibition of MMPs and ADAMs
➢ VEGF4 family ligands and receptor interactions – They are involved in angiogenesis stimulating tyrosine kinase receptors (VEGFr). They stimulate endothelial cell mitosis and their migration. Their production is frequently induced in case of hypoxia (in tumor cells that don't receive enough oxygen) [8].
➢ EGF pathways (Fig.3)
➢ Cyclines and cell cycle regulation- Cyclines are proteins that control the cell cycle by forming the complex Cyclin-Cdk5 making the cell to pass to the next cycle phase by phosphorylation.
➢ Targets for ATM6 phosphorylation and p53 pathway– In case of DNA damage, ATM plays an important role on the cell cycle delay. The ATM stabilizes and activates the tumor suppressor p53 [9].
2 5AKT= Protein kinase B, PI3K = phosphoinositide 3-kinase
36 Phosphatidylinositol 3,4,5 triphosphates
4 7Vascular Endothelial Growth Factor
5 8Cdk = Cyclin-Dependent Protein Kinase
➢ HIF 7 regulation of transcription – HIF1 is a heterodimeric protein composed of HIF1-α and HIF1-β able to respond in conditions of hypoxia, maintaining the oxygen levels normal. Angiogenesis, the process of sprouting new blood vessels from existing blood vessels is one of the requirements for tumor development. Angiogenesis occurs following an inflammation event, ischemia, wound healing and in more critical conditions like tumor growth or rheumatoid arthritis. Metalloproteases are involved in proteolytic events that contribute to the process of angiogenesis. ADAMs participate in a wide variety of cell surface remodeling processes, including ectodomain shedding (Fig.2)8, regulation of growth factor availability and in mediating cell-matrix interactions. ADAM-17 and ADAM-15 have been identified in endothelial cells [1].
The ectodomain shedding is followed by the activation of three specific receptors such as Notch, ErbB and Tie-1 (angiopoietin receptor).
The ErbB receptor family, was discovered to have a crucial role in the study of tumors progression. This because an over-expression of the ErbB receptors were noticed in numerous human tumors [10]. Four receptors are members of the ErbB family:
● EGFR9(HER1 or ErbB1)
7 10HIF = Hypoxia-Inducible Factor
8 Image 2 available at: Nature Reviews Molecular Cell Biology 6, 32-43 (January 2005) doi:10.1038/nrm1548 9 3Epidermal Growth Factor Receptor
Figure 2: A schematic representation of the process of ectodomain shedding by an ADAM (a disintegrin and
metalloprotease) protein or other protease, which results in the release of its soluble ectodomain. the ectodomains belong to membrane-anchored growth factors, cytokines and receptors. b | A receptor–ligand pair is used to illustrate possible roles of ectodomain shedding. In the absence of shedding, a membrane-anchored ligand might only engage its receptor in a juxtacrine or autocrine (although there might be impediments to autocrine receptor stimulation, such as improper orientation of the ligand and receptor). However, to reach a receptor at a distance and to participate in paracrine signaling, a membrane-anchored ligand must be shed. Receptors might also be shed, which could result in their activation or inactivation. Signaling through Notch is a prime example of a role for proteolysis in activating a receptor.
● HER2(ErbB2) ● HER3(ErbB3) ● HER4(ErbB4)
When their appropriate ligands bind to their specific receptors homodimer or heterodimer complexes are formed. The ligands that have been reported to bind to the ErbB receptor family are: the epidermal growth factor (EGF), transforming growth factor-alpha (TGF-α), heparin binding EGF-like ligand (HB-EGF), amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), epigen (EPG) and epiregulin (ERG)/heregulin (HRG) family member (Cancer Res 2008, september 1, 2008). Heregulin is the only ligand that binds HER3. Heterodimerization of HER3 with HER2 brings to the formation of the most oncogenically active ErbB receptor complex upon heregulin stimulation (Fig.3)10
Members of the metzincins superfamily of Zn-metalloproteinases share many structural and functional commonalities. The differences among its members are responsible for the fine tuning of
10 Image 3 available at: Expression and function of ErbB receptors and ligands in the pituitary, Odelia Cooper et al, 14 September 2011, Society for endocrinology.
Figure 3: The family of ErbB is represented, composed by four members: HER1, HER2, HER3 and HER4. ErbB2 has
no ligand-binding capacity and ErbB3 has no active kinase domain. Ligand binding to ErbB receptors induces the formation of receptor homo- and heterodimers and activation of the intrinsic kinase domain, resulting in phosphorylation on specific tyrosine residues.
key physiological functions in mammals. Deregulation of their activity is directly connected to numerous inflammatory and degenerative diseases such as arthritis or cancer. The metzincin super-family of metalloproteinases are extracellular matrix (ECM)-degrading enzymes that encompass the most studied matrix metalloproteinases (MMPs), the membrane anchored proteins: ADAMs, and the secreted-type: ADAMTs (ADAMs with thrombospondin repeats). Several ADAMs genes encode for more than one protein, only 40% have proteolytic domains, the remaining 60% have other functions. The catalytic domains share the common HEXGHXXGXXHD sequence in their active sites including the typical MMP ''Met-turn''.
There are four important families involved in the superfamily of Zn-metalloproteinases.
● The reprolysins or adamalysins (snake venom metalloproteinases and a disintegrin and metalloproteinase (ADAMs)).
● The serralysins (bacterial proteinases like Pseudomonas aeruginosa aeruginolysin ).
● The astacins ( The first member of this family was identified in the crayfish Astacus astacus. A second member of the family, bone morphogenetic protein 1 (BMP-1) was found in vertebrates as bone-inducing factor) [5].
● The matrix metalloproteinases (MMPs) also known as matrixins (vertebrate collagenasis). There is also a fifth family consisted in two members. The features they both demonstrate, suggest their belonging to the metzincin superfamily. The two members of this family are: pregnancy-associated plasma protein-A1 (PAPP-A1) and pregnancy-pregnancy-associated plasma protein-A2 (PAPP-A2). They are most similar to the serralysin family [11].
1.2 ADAMs
Although members of the metalloprotease family have been considered promising targets in cancer therapy from over 27 years no molecule has passed clinical trials yet [12]. They are noted for their resemblance to snake venom proteins. Researchers working on MMP inhibitors and those who work on ADAMs, concur that the reason of the lack of results may be related to the side-effects revealed during long term clinical trials, due to poor selectivity. One of the most frequent side effect of MMPs inhibitors being musculo-skeletal syndrome (MSS). This has become the main focus point in the design of both ADAM and MMP inhibitors. The ability of the inhibitors to bind the zinc moiety, present in all the metalloproteases, is also the feature that can make them poorly selective. This could be addressed by refocusing the attention to the development of inhibitors that exploit secondary binding sites (exosites). Although there are only a few reports of potential exosites in ADAM proteases, there are articles in the literature where inhibitors that bind exosites show good selectivity against TACE (Tape et al, The use of antibody). Currently, X-ray structures have been determined for the catalytic domains of ADAM-17 (TACE), 33 and 8. For ADAM-10, a crystal structure has been determined for portions of the disintegrin and cysteine-rich domains.
Regarding the catalytic domain, the active site is different from ADAMs whose structures have been determined, and these differences may be important for selectivity.
The structural similarity among metalloproteases is shown in Fig.4 for the typical MMP, ADAM or ADAMTS11. ADAMs are structurally more complex than MMPs, but all three groups contain an inhibitory pro-domain that after excision at a furin cleavage site results in an active enzyme. The interaction between a cysteine in the pro-domain and the zinc in the catalytic site (-SH-Zn) that causes inactivation is relieved by the removal of the pro-domain by furin-like convertase or by auto-catalysis. The metalloprotease-like domain situated after the cleavage site is not sufficient to render ADAMs proteolitically active. Only 50% of them (ADAM-8, ADAM-9, ADAM-10, ADAM-12, ADAM-15, ADAM-17, ADAM-19, ADAM-28, ADAM-33) become active. (Michael Duffy et al, Clinical Proteomics 2011). Persistent inactivity is conferred by the disintegrin domain (present only in ADAMs and ADAMTs but not in MMPs).
11 Image 4 was taken from this article: Paulissen et al. Respiratory Research 2009 10:127 doi:10.1186/1465-9921-10-127
ADAM Function/Potential Function Proteolitically inactive
ADAM-2 Sperm-egg fusion
ADAM-7 Sperm maturation
ADAM-11 Integrin ligand, neural adhesion, tumor suppression
ADAM-18 Ovocyte recognition
ADAM-22 Adhesion
ADAM-23 Tumor suppression
ADAM-29 Unknown
Proteolitically active
ADAM-8 Shedding of adhesion molecules, leukocyte receptors, neutrophil infiltration, osteoclast
stimulation
ADAM-9 α-secretase activity, cellular adhesion
ADAM-10 α-secretase activity, shedding of TNF α, EGF, betacellulin, HER2, Notch, and collagen
IV, cellular adhesion
ADAM-12 Cellular adhesion, shedding of HB-EGF
ADAM-15 Cellular adhesion
ADAM-17 Release of several growth factor ligands, e.g, TNF-alpha and specific EGFR/HER
ligands, cellular adhesion
ADAM-19 Unknown
ADAM-28 Shedding of IGFBP3
ADAM-33 Involved in pathogenesis of gastric cancer via IL-18 secretion
Table 2: Potential Functions of human ADAMs*
The disintegrin domain that binds to integrin provoking cell adhesion is important for migration and proliferation. The other domains present in metalloproteases are: the cysteine-rich and EGF-like domains and the trans-membrane and the cytoplasmic regions. There are data to show that the cysteine-rich domain of ADAM17 undergoes phosphorylation at some sites, in particular Thr735 and Ser819. The phosphorylation at Thr735 induces the shedding of TGF-α from TACE [13].
** LPL; lipoprotein lipase, CLL; chronic lymphocytic leukemia, TNFα; tumour necrosis factor-alpha, EGF; epidermal
growth factor, HB-EGF; heparin -binding-EGF, IGFBP3; insulin-like growth factor-binding protein 3, IL-18; interleukin-18.
There are known 40 different types of ADAM in various species. Only 15 are found in humans but 35 of them are expressed in Mus musculus 12 [14]. Together with the ADAMTs and snake venom metalloproteinases they constitute the adamalysins subfamily. While studies on MMPs began in 1962 following the discovery of the collagenase in the tadpole tail, interest in the family of ADAMs, discovered only recently, is growing rapidly [15].
The resemblance of the ADAM's catalytic domain to that from the MMPs was used to obtain models for the development of compounds that inhibit ADAM10 selectively (an example is compound LT4). The MMP catalytic domain is characterized by the presence of three histidines involved in binding two zinc ions. Of these ions, one is structural while the other plays a catalytic role. In the pro-enzyme both are tetra-coordinated, that makes them stable. The fourth group coordinating the zinc, the thiol from cysteine 73 is lost when the pro-domain is shed exposing the active site to substrates [16]. In the case of the ADAMs like for the MMPs the prodomain maintains the protein inactive but its activation consists on the recognition of a furin site (an RXXR
sequence). Mostinhibitors are designed to bind the active site zinc and occupy one or more specificity pockets. The argument will be better expanded in Chapter 2 specific for the metalloproteinases.
12 11Mus musculus = mouse
Figure 4: Structure of ADAMs, ADAMTs proteinases and Matrixmetalloproteinases. The structure of MMP is made of a
prodomain, a furin cleavage site, a catalytic metalloproteinase domain with a fibronectin repeats (MMP-2 and MMP-9), a linker peptide and a haemopexin-like domain), a linker peptide, a transmembrane domain and cytoplasmatic tail. ADAMs structure is similar to that of the MMPs except that it has an additive disintegrin domain more. The ADAMTS family members have numerous thrombospondin-like motifs (TS).
1.3 Regulation of ADAMs
Being involved in both proteolysis and cell adhesion mechanisms, the manner in which ADAMs contribute to cancer progression varies as classified below:
1. Activation of positively-stimulating pathways
This mechanism consists in the release of stimulating growth factors. To reach the maximum of their activity these pro-growth factors (precursor proteins), initially synthesized in their inactive form need to be first converted to their active state. The ADAMs play a role in the activation of growth factors. Two types of ADAMs, ADAM-10 and TACE are involved in the conversion from inactive toactive of ligands binding to the ErbB family of receptors. This mechanism is represented in the Fig.5. ADAM-10 and ADAM-17 shed different ligands. ADAM-17 appears to be the physiological sheddase for TGF-α, amphiregulin, HB-EGF, and epiregulin. ADAM-10, on the other hand, appears to be the major sheddase for the release of EGF and betacellulin. In certain situations however, other ADAMs including ADAM -8, -9, -12, -17 and -19 can activate one or more of these ligands [17].
Their active form does not bind only one receptor. It can bind one ore more of the EGFR/HER family of receptors. They are classified as HER-1 (c-erbB1), HER-2 (c-erbB2), HER-3 (c-erbB3)
Figure 5: Activation of EGRF/HER receptor signaling by ADAMs. In a first phase ADAM (10 or 17) shed the
ectodomain of the pro-ligand expulsing the active ligand (EGF, TGF-α ,heregulin, betacellulin) that once it is activated binds an activated receptor (homodimer or heterodimer). After this process, signaling through many pathways like MAPK, PI3K and JAK/STAT is activated. The MAPK pathway causes cell proliferation bringing in an increment of tumoral cells translated with the progression of the disease.
and HER-4 (c-erbB4). Their general structure is very similar and consists in: an extracellular domain, a trans-membrane domain and an intracellular domain [18]. All of these receptors, excluding HER2, can be directly activated just after ligand binding. The further process of homo- or heterodimerisation plays an import role on, activating several different pathways including the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K) pathway and janus kinase/signal transducer and activator of transcriptional (JAK/STAT) pathway. The signaling obtained from the activation of these three pathways becomes a marker on the malignancy, bringing to further enhanced cell proliferation, increased cell motility and increased cell survival.
Evidences are nowadays available from the release of ADAM-mediated growth factor ligand release and EGFR signaling in cancer cell proliferation or migration. One example is the study by Singh et al [19]where it was shown that by UV irradiation of skin cancer cells ADAM-9 and -17 were activated, followed by amphiregulin shedding, EGFR trans-activation and increased cell proliferation. Another study demonstrated the role of ADAM-17, where by releasing the ligand, in promoting the proliferation and invasion in vitro by breast cancer cells [20]. These examples give sense to the search for selective ADAM inhibitors, promising future success in targeting these disintegrin metalloproteases.
Another interesting process of interest is the translation of HER's C-terminal fragment after binding the ligand. This translation, from the cell membrane to the nucleus, is followed by a cell proliferation regulation. The ligand for which such an effect was proved is heparin binding-epidermal growth factor [HB-EGF], in keratinocytes and gastric cancer cells and is thus another plausible mechanism by which ADAM-catalyzed shedding of growth factors can affect cell proliferation.
2. Inactivation of growth-inhibitory pathways
Inactivation of growth inhibitory signaling systems would be expected to produce the same end result as activation of positively-activating growth factors. An inhibition of proliferation in both normal and early malignant cells, was reported for β due to its binding of βR1 and TGF-βR2, while in progressive malignancy, TGF-β can promote proliferation. ADAM-17 has also been reported to mediate shedding of the type 1 TGF-β receptor. This decreases the TGF-β signaling with a concomitant reduction in growth inhibition. Thus ADAM-17 mediated reduction in growth inhibition complements the growth stimulation, resulting from increased release of the EGFR/HER ligands.
3. Shedding of adhesion proteins
In addition to being mediators of shedding of adhesion proteins, ADAMs activation can also cause cell proliferation. β-catenin was found to translocate from the cell membrane to the nucleus after ADAM-10 shedding of the extracellular domain of cadherin E [21]. More than one study substantiate that shedding of adhesion proteins from the ADAMs increases proliferation and migration in cancer cells. Najy et al have reported the ADAM-15 mediated the shedding of a form of cadherin E that could bind to and activate HER-2 in breast cancer cells[22]. The complex of the shed form of cadherin E with HER-2 and HER-3 resulted in enhanced ERK signaling, increased cell proliferation and migration. In addition to cell proliferation this form of cadherin E may cause a reduction in cell-cell interactions and massive dissociation of potential invasive and metastatic cells, dangerous in terms of malignancy because of its potential to promote metastasis. Other adhesion proteins such as L-selectin, ICAM-1 or VCAM can be shed. From their sheddase is expected a modulation in binding of tumor cells to the vascular wall and thus convolution in intravasation [i.e., exiting of tumor cells from the vasculature into distant organs].
4. Potential involvement in mediating angiogenesis
ADAMs may promote cancer growth and metastasis by mediating angiogenesis. Angiogenesis is described as the process of the capillary outgrowth from pre-existing blood vessels, also referred to as pathological neovascularization. A similar growth out of the what are called limits, becomes a help in cell proliferation and tumor invasion because it brings ''nutrition'' with the blood to the tumor cells. Early evidence highlighting the role of ADAMs in angiogenesis was found in a pulmonary hypervascularization in mice by exposing them to catalytically inactive ADAM-17. In another work the gene encoding for ADAM-17 was deleted in a mouse model, resulting in pathological neovascularization and reduced growth of injected tumor cells [23]. What makes the study more interesting is that developmental and vascular homeostasis was not affected. Other ADAMs implicated in pathological neovascularization include ADAM-9 and ADAM-15.
Figure 6: Shedding of cadherins from the ADAMs provokes the translation of the β-catenin from the subcellular space to
Four separate pathways regulate ADAM activity: Inhibition by inhibitors, control of gene expression, zymogen activation and intracytoplasmic and pericellular regulation.
1. Inhibition by Inhibitors
This inhibition by inhibitors is the best known. The ADAMs, like MMPs have endogenous inhibitors that control their activity, particularly in cases of over-expression. MMPs and ADAMs are inhibited by four different TIMPs of 21 to 48 kDa, with a homology of 40-50% among them. TIMP-3 is the endogenous inhibitor the most used, ADAM-10, ADAM-12, ADAM-17, ADAM-28, ADAM-33, ADAMTS-4 and ADAMTS-5 are inhibited by it. Not all ADAMs are inhibited by a TIMP, this list includes ADAM-8, -9 and -19.
2. Gene expression
The levels of different ADAMs are regulated in vivo by transcription factors. Foxm1 is responsible for TACE (ADAM-17) regulation [24]. ADAM-8 expression is controlled differently; it is induced by the interferon-regulating factor 1. For ADAM-9 and ADAM-12 two different regulation mechanisms have been observed. The first through reactive oxygen species like hydrogen peroxide and super-oxide ions generated during cellular stress; these induce the expression of ADAM-9. Alternatively, the treatment of hepatic stellate cells with TGF-β resulted in up-regulation of ADAM-12 expression. The process involves PI3K and MAPK kinase activities.
3. Zymogen activation
The furin recognition site is responsible for the activation of the protein through proteolytic removal of the pro-domain. Interestingly, MMPs can start a cascade of ADAM activation.
4. Intra-cytoplasmic and pericellular regulation
The fourth mechanism of ADAMs regulation concerns the composition of their intra-cytoplasmic tail regions specialized in binding to signaling proteins and captors. These proteins are involved in the regulation of ADAM activity or simply affect their sub-cellular localization.
1.4 ADAMs Structure
From the studies of Snake Venom Metalloproteinases it was discovered the structure of about 750 amino acid length of the ADAMs, composed by a domain, consisting of an N-terminal signal sequence followed by a pro-domain, a metalloprotease domain, a disintegrin sequence with a cysteine-rich region, an EGF domain, a trans-membrane domain and a cytoplasmic tail (Fig.7), typical of the metzincin clan.
Until it is removed by autocatalysis or by a furin-type pro-protein convertase, the pro-domain of ADAMs, possessing a protease domain, remains catalytically inactive. As for some MMPs, there is a cysteine switch mechanism on the base of activation of the ADAMs [25][26]. It preferentially coordinates the required active zinc site atom, maintaining the protease domain in a latent state. The metalloproteinase catalytic domain in the ADAMs is similar to MMPs. From the crystallographic results obtained from the crystallization of several metzincin family members, ADAM-17 included, a catalytic zinc and water atoms, necessary for the hydrolytic processing of protein substrates were found. As for the MMPs, the zinc atom results coordinated by three histidine residues binding consensus sequence (HExGHxxGxxHD) and a methionine one [27].The methionine lies in a Met turn motif that is positioned so as to face the consensus HExxHxxGxxH site, creating a 1,4-β-turn that constitutes the active cleft. It is assumed that the ones having the HexxH sequence are proteolytically active. The globular structure provides a catalytic domain composed by the active site running between the two subdomains: the upper N-terminal, 5-stranded-β-pleated sheet
Figure 7: General domain structure of the ADAM family. (PRO)amino-terminal propeptide, (MP) metalloproteinase domain,
three distinct regions carboxy-terminal to the catalytic domain: disintegrin domain (DIS), disulphide bonded to a cysteine-rich region (CR) often containing an epidermal growth factor-like repeat (EGF) constitute the remainder of the ADAM ectodomain. DIS and CR domains form a disulphide bonded entity exposing a hypervariable region (HVR) in the CR domain that is likely to be a major interaction site with other molecules, including substrates. (CD) cytoplasmic domain; (TM) transmembrane domain.
structure subdomain and the lower domain, being an αβ helix structure containing the HExxH of the catalytic Zn-binding site. The strands of the upper domain are highly twisted and lie parallel to each other. The catalytic Zn atom stands between the subdomains, at the bottom of the groove, in which the subsites: S3S2S1S'1S'2S'3 determine the specificity for particular amino acid sequences binding P3P2P1P'1P'2P'3 in the substrate, where cleavage occurs between the P1 and P'1 residues. The depth and hydrophobicity of the S'1 specificity pocket was revealed to be in particular critical for building selective inhibitors of the metalloproteinase domain. The disintegrin domain is 90 aa long in which 14 of them form the so-called "disintegrin loop", considered responsible for the interactions between ADAMs and integrins. The disintegrin domain can be further divided into two sub-domains, which have been called, by the way they are disposed: the shoulder (Ds) and the arm domain (Da). The N-terminus region of the Cys-rich (C) domain following Ds and Da is called a ''wrist'' domain (Cw), and together the Ds, Da and Cw domains shape the whole molecule into the form of a C. In this structure, the Cys-rich domain is close to the disintegrin domain and faces towards the catalytic side. Following the same logic in the denomination the Ch segment of the Cys-rich domain was given the name ''hand'' containing the hypervariable region (HVR) important for the recognition of the target, at its distal portion. It has been identified a consensus motif present in the disintegrin loops able to interact with the integrins: CRxxxxxCDxxExC. This interactions have shown to be important in many cases for cell-cell interactions and cell adhesion. Next to the Ch-domain comes the EGF-like domain (~60 aa) working as a linker between the three upper domains (metalloprotease, disntegrin and C-shaped domain) also called MDC-domains and the membrane-spanning region. Cytoplasmic domain is considered important for regulating the catalytic activity of the function of the ADAMs but also for trafficking the protein to the correct cellular location. The transmembrane ADAMs have cytoplasmic domains that vary a lot in length and in sequence, counting from the 11 residues of ADAM-11 to 231 residues for ADAM-19. The principle motif responsible for binding the SH3 domain present in some protein families like: PI3-Kinase, Ras GTP-ase activating protein, CDC24 and CDC25, named PxxP was found in the cytoplasmatic domain of several ADAMs [28]. There is a great number of ADAMs containing an SH3 binding site: ADAM-7, -8, -9, 10, -12, -15, -17, -19, -22, -29 and -33. ADAM-12 and -15 are the ones containing the most wide repertoires of Pro-rich motifs. Cytoplasmic domain is revealed to have an important role in coupling ADAM-17 and other ADAMs to specific signalling. One example is the event of ''triple membrane-passing signal'' where GPCR (G- protein coupled receptors) activate ADAM that releases EGFR ligand.
1.5 Catalytic Mechanism of activation of ADAMs
The catalytic site of ADAMs contain a zinc atom bound by three histidines and a fourth ligand to stabilize a tetrahedral structure. In the zymogen the fourth ligand is a cysteine residue. After proteolytic activation whereby inactivating cysteine is removed together with the propeptide, water becomes the fourth ligand. The same mechanism as in MMPs. The positioning of the water molecule is controlled by a Glu residue that interacts with it. On substrate binding, activation starts
with the deprotonation of the water molecule while the glutamate residue passes from the ionic form to the non protonated form (COOH). The generation of the hydroxide species leads a nucleophilic attack on a carbonyl in the peptidic chain forming a tetrahedral intermediate with a negative charge on an oxygen atom.
The positioning of the natural substrate in the active site exposing the carbonyl group of its peptidic chain towards the Zn atom aids the nucleophilic attack by the transition of the Zn ion from tetra- to penta-coordinated (unstable condition). The interaction with the Zn atom polarizes the carbonyl group causing its carbon to change hybridization state from sp2 to sp3. Formation of the tetrahedral intermediate, allows the glutamate to release the proton acquired during first step of the reaction from the water molecule; leaving a cleaved peptidic chain. This mechanism is similar to that used by carboxypepetidases and thermolysins.
Figure 8: Catalytic cycle for the proteolytical mechanism of stromelysin 1 ( Vladimir Pelmenschikov- Catalytic mechanism of
CHAPTER 2
MMPs (Matrix Metalloproteinases)
2.1 Introduction
The MMPs are a large class of Zn metalloproteinases that include different subclasses of enzymes: collagenases, stromelysins and gelatinases. Their principal function is the degradation of the ECM. Pathological states are related with an unbalance between the activation and inhibition of the MMPs. This leads to the phenomenon of the ECM degradation that is the main cause of different pathologies as: congestive heart failure, osteoarthritis rheumatoid and tumor malignancies (that is the target of this study). By matrix remodeling, tumor cells would have their free path, that means it would be easy for them to access blood and lymphatic vessels..This would bring to an increase of the invasiveness of cell lines that over-express MMPs. The matrix metalloproteinases can help the tumor growth by inducing the formation of new blood vessels by angiogenesis.
2.2 Functions
They are co-involved with ADAMs in extracellular membrane degradation by hydrolyzing the group of three proteins like: fibronectin, collagen IV and laminin. The three of them have an important role in the reorganization of the connective tissue. Because of their important role, a short description for each of them follows:
1.Fibronectin
It is a glycoprotein of high molecular weight (440kDa), that binds to ''integrins'' (receptors binding to ECM (extracellular matrix). It also binds collagen, fibrin and heparan sulphate proteoglycans (syndecans). It is a dimer where the two monomers are connected by sulphide bonds. There are two main types of fibronectin: The first is the soluble plasma fibronectin, produced by the hepatocites in the liver, while the second is the insoluble component of the ECM. Fibroblasts produce the soluble form of fibronectin. The protein starts interacting with the integrin receptor creating protein-receptor complexes. An increase of the concentrations of these complexes is followed by an increase of the interaction of bound molecules with each another promoting the formation of short fibronectin fibrils between adjacent cells, leading to large insoluble fibrils. This fibronectin-fibronectin interaction allows cell associated fibrils to ramify and form the fibronectin-fibronectin matrix insoluble complex. There are three ways in which fibronectin can contribute to tumor progression.
• A decreased fibronectin expression.
• A decreased expression of fibronectin binding receptors.
These are three conditions that lead towards tumor progression will be considered during the study.
2. Collagen
Collagen is the most important component of the connective tissues in animals (25-35% of the body proteins is collagen). Collagen can be produced by fibroblasts, pro-inflammatory cells that start functioning as a result of an inflammatory event. It is a triple-helical protein composed by two identical chains (α1) and another chain that has differs a little chemically from the other ones (α2) [29]. The most frequent sequences found in the collagen's helices are Gly-Pro-X and Gly-X-HyP where X stands for any amino acid different from glycine, proline or hydroxyproline (HyP). There are five different types of collagen classified according to the functional distinctions they were found to have and their mechanism of action. Type IV collagen forms the basal lamina, the epithelium-secreted layer of the basement membrane. The degradation of collagen IV by the ADAMs contributes to the degradation of the ECM.
3. Laminin
Laminin is the third protein degraded from the ADAMs. It is part of the connective tissue, component of one of the layers of the basement membrane, the basal lamina. Laminin is a trimeric protein composed by 3 α helixes (α,β,γ chains) forming a structure that reminds the shape of a cross. This particular shape makes it easier to connect the cells with one another bindig them and consecutively funding the connective tissue. They form independent networks. They are connected with collagen type IV networks and fibronectin and bind to cell membranes through integrin receptors.
Figure 9: In this picture the structure of laminin is shown . It is a trimeric protein composed by three chains : α,β,γ that
2.3 (Q)SAR Interactions
The main components present in the MMP's structure responsible of their (Q)SAR interactions are the below listed:
1. ZBG (Zinc Binding Group) 2. Peptidic Chain
3. Pocket occupying the side chain
Most efforts revolve around molecules with functional groups able to bind the Zn atom. The functional groups used for zinc chelation, hydroxamates, the carboxylic acids, the thiols and phosphinic acids differentiates among the inhibitors. Two main strategies to produce inhibitors with ZBG are followed. The first aims to reproduce molecules similar to the TIMPs (Tissue Inhibitor of Metalloprotease, the endogenous inhibitor). Inhibitors like Batimastat or Merimastat follow this strategy; they have have a ZBG and a peptidic chain. Their complex structures require significant effort. The alternative strategy, also used by Prof. Rossello and his team work at the University of Pisa, focuses on tailoring smaller structures to the catalytic pocket so as to make specific interactions with the specific subsites P1', P2' and P3').
The nature of the zinc binding group is important. A hydroxamate moiety (NHOH) gives a powerful interaction with the zinc atom, but its strength precludes selectivity, and by indiscriminate inhibition of unintended zinc-containing targets becomes responsible for the toxicity of these molecules. On the other hand a carboxylic moiety (COOH) that establishes a weaker grip on the zinc can achieve specificity from other interactions. With better oral bioavailability and a more stable functional group this approach may lead to effective inhibitors. The weaker hold on the zinc must be compensated by a longer P1' substituent better tailored to fit the S1' pocket [30].
2.4 Structure
MMPs are composed by three principle domains and a hinge region important for the selectivity. The first is an N-terminal pro-domain cleaved during the activation. The second, that is also the place where the atom of Zn stands, is the catalytic domain and the last is a C-terminal hemopexin like domain, important in the macromolecular substrate recognition and for the interaction with the TIMPs (the endogenous inhibitors).
Catalytic Domain
It is folded into a single globular unit with a diameter of 35 Å. It is composed by one five-stranded β-sheet, composed of one anti-parallel and four parallel strands, and three α-helices. In the inactive form, before cleavage of the propeptide a cysteine stabilizes the catalytic zinc. The sequence of the propeptide is well defined: Pro-Arg-Gly-Cys-X-Pro-Asp where X can be any amino acid. After the
replaces the thiol. In the catalytic domain one to three structural calcium molecules help to maintain the structural integrity of the domain. The presence of a sequence containing a Met residue close to the catalytic Zn ion exerts a protective function for the zinc[31].
The active site of the MMPs is characterized by the presence of a groove in the protein surface in the center of which stands the catalytic Zn and a site named S1' important for its specificity, being different for different types of MMPs.
2.5 Mechanism of the proteolysis in the MMPs
The mechanism that results in the degradation of the substrate starts with a nucleophilic attack by a molecule of water. It gets in contact with a carbonyl generating a negative charge on the oxygen. This partial charge is stabilized by an interaction with the catalytic Zn atom. Meanwhile the molecule of water, at the same time, stabilizes hydrogen bonds with two amino acids: Glu198 and Ala161 in the catalytic domain (Fig.9). In the third step of this mechanism the nitrogen of the amide gets protonated, carrying a positive charge on itself. This charge is stabilized by interactions with Ala161.
Composition of the active site of the MMPs
The small differences between different forms of MMPs makes it difficult to find selective inhibitors. Nevertheless these small differences make it possible to differentiate one MMP from another. A major difference is the shape of the S1 pocket. The MMPs posses some subsites: three primed (S1', S2', S3') positioned to the right of the catalytic zinc and three unprimed ones (S1, S2,
S3) on the left. Depending on their disposition with respect to the Zn atom inhibitors are classified as: the right-hand side or left-hand side inhibitors. In Fig. 11 the numbering of the subsites in the enzyme substrate is represented from P1 to P3'. The most important subsites for interactions are those at the right of the zinc [32].
Most of the modifications done in the structure of the inhibitors concern the S1' subsite responsible for the selectivity. It is a very deep pocket in most of the MMPs except for MMP-1 and MMP-7 where an Arg and a Tyr reduce the size of the S1' pocket. This conclusion that it is important is for the P1' group to be hydrophobic was reached when the introduction of hydrophilic groups at this level reduced potency and selectivity. To differentiate between family members the trend is to focus on finding inhibitors that distinguish between deep pocket enzymes and short pocket ones. The MMPs with a spacious S1' subsite are: 2, -3, -8, -9 and 13. Instead, 1 and MMP-7 have small S1' pockets. The S1' in MMP-12 for example is unique, because it can also accommodate polar groups because of the presence of Thr215. It is also known that an increase of the rigidity in the inhibitors structure, increases the selectivity towards MMP-12. The MMP-13 the S1' pocket is defined by a very flexible loop especially in the stretch 245-253 that confers to the MMP-13 the possibility of accommodating large P1' substituents. Specific MMP-MMP-13 inhibitors are characterized by large backbones able to fit inside the deep S1' pocket.
An induced fit mechanism is noticed in some cases. Residues like Arg424 and Thr426 of MMP-9 and MMP-2 respectively cause variations in the size and shape of the S1' pocket. MMP inhibitors with long P1' chain inhibit MMP-2 better than MMP-9 mainly because Arg424 occludes the S1' pocket. in the MMP-9. This peculiarity is insufficient to effectively distinguish between these two MMP. They are very homologous for in amino acidic composition except within the 425-431 stretch. Not all MMP inhibitors carry a ZBG. There are also potent and selective inhibitors without a ZBG. For MMP-13, inhibitors only require a long hydrophobic group that fits in the wide S1' pocket. The use of non ZBG is based inhibitors exploit the intrinsic flexibility of S1' loop, a characteristic of MMP-13.
Figure 11: The binding subsites inside the active site of the MMPs together with the residues of the substrate.
CHAPTER 3
Expression and purification of the mutant MMP8A
3.1 Introduction
The production of purified proteins becomes an important process for several experimental procedures including crystallization ones. Through these studies, small molecules drug discovery can be improved, by crystallizing complexes protein-inhibitor and interpreting their electron density maps. There are some characteristics of proteins, such as sub-cellular localization, and localization the presence of signal peptides, considered as the most critical factors for expression, solubility and purification.
Proteases are part of those protein families that play critical roles in different cellular functions and life annuity in all organisms. Their biological roles vary from signal transduction to post-translational modification, proliferation, apoptosis and pathogenicity using both specific processing or non-specific degradation. Proteases can be divided into two classes, secreted proteases and intracellular ones.
The metalloproteasis, and the MMP-8 in specific, classified as intracellular proteases clear damaged proteins contributing in regulatory pathways through the degradation of specific substrates. Proteases also cover crucial roles in the immune system for both defense mechanisms of host cells and pathogenicity in a variety of pathogens from viruses to higher parasites.
3.2 Different systems used for protein's expression
The enzyme industry started to grow faster between the 1980s and the 1990s with the microbal enzymes. Microbal enzymes are enzymes produced by bacteria such as Escherichia coli,
Streptomyces, Pseudomonas chlorapis. This resulted to be a goal in the production of protein, due to
the fact that the cultivation of microbes is much more easier and faster than the same work in plants and animals. Statistics show that proteasis are the enzymes produced the most. Their contribution to the market is 57% [33]. Except of the class of bacteria (E.coli, different bacteria belonging to Gram-positive and Gram -negative classes), other systems are used for the protein's expression.
Yeasts
For recombinant proteins that are not produced well in E. coli, because of problems with folding or the need to have them in a glycosilated form, yeasts, become the alternative system. They are single-celled eukaryotic fungal organisms. The use of Yeasts as expression system has some advantages respect the use of E. coli. The principal advantages are: they're able to handle S-S proteins, assist their folding, glycosylate them, give rapid growth in the chemically defined media, stable production stains and the high productivity. This doesn't mean there aren't any exceptions.
Glycosylation by S. cerevisiae, for example doesn't often occur for mammalian proteins because of different O-linked chains. The sialylated O-linked chains present in high eucariotic proteins are not present in the O-linked oligosaccharides which contain only mannose.
Filamentous fungi
Filamentous fungi, also known as molds are used for recombinant DNA technology because they're able to produce high levels of bioactive proteins applying post-translational processing such as glycosylation. Foreign genes can be introduced using plasmids, into chromosomes of the filamentous fungi where they integrate stably into the chromosome while tandem providing superior long-term genetic stability by repeating. A. niger is one representant of this class.
Insect cells
Insect cells are better to carry out more complex post-translational modifications than what can be achieved with fungi. They also are able to fold the mammalian proteins and this makes them quite suitable for making soluble protein of mammalian origin. Baculovirus is the vector system for recombinant protein expression in insects most commonly used.
Mammalian cells
Mammalian cell cultures become especially useful because producing well-folded and in their glycosylated form, so, there is no need to renature them. This system does also have its own drawbacks, like the low productivity and the high costs.
Transgenic animals
Transgenic animals such as goats, cows, mice, pigs, sheep and rabbits are being used as production systems used to obtain recombinant proteins in milk, egg white, blood, urine, seminal plasma and silk worm cocoons. Milk and urine are considered the best in terms quality and quantity of production. This system has also its own disadvantages like the difficulty on the maintenance of the cells in a semi-defined medium.
Transgenic plants
The use of plants becomes interesting because of the big number of advantages that it offers in confront also with other systems like the transgenic animals or the mammalian cells. Being grown in an agricultural scale they only require, water, sunlight and minerals. No plant viruses have shown to be pathogens to humans, that gives to this system a low risk of contamination. Some more advantages of plant systems are the production of glycosylated proteins and targeting, compartmentalization and natural storage stability in certain organs.
3.2.1 Expression using E.Coli
In the production of large variant of proteins using Escherichia coli inducible expression systems can be used. In these systems it is the T7 RNA polymerase that transcribes coding sequences cloned under control of a T7lac promoter. Auto-induction permits efficient screening of many clones in parallel to select for expression and solubility. Cultures are inoculated and grown to saturation, making possible the reach of higher yields than those obtained by conventional IPTG induction [34]. Escherichia coli has proved to be an excellent medium for the expression of non-glycosilated proteins. Its broad use comes from the fact that their genetics are better understood permitting this an easier culture and genome modifications but also because of the fast mass production, the high yield, affordable prices. However, production of different proteases in E. coli host cells brings to a critical stress often correlated with the formation of inclusion bodies, non-expression, or cytotoxicity. In the protocol used for the production of MMP8hA (the mutant form with a Val114 at the place of Glu114 in the catalytic pocket of MMP-8) has been taken count of all this disadvantages using denaturant agents that unfold the proteins like urea or the application of extreme heat or pH, and reducing agents to break the disulfide bonds like β-mecaptoethanol. Afterward refolding is obtained eliminating the denaturant and reducing agent, the protein renaturation by air oxidation occurs. Between a large choice of protein expression systems such as: Cell based systems, Yeast systems, Insect cell systems or eukaryotic systems, Bacteria system of Escherichia Coli, one of the most widely used systems was used. Below in Table 4 the advantages and disadvantages of using this system were listed.
Advantages Disadvantages
-Rapid expression with high yields. -Proteins with disulfide bonds are difficult to get produced. -Ease of culture and genome modifications. -Proteins are produced together with endotoxins.
-Affordable price. -Production of non-glycosilated proteins.
-Fast mass production. -Cell toxicity caused from the formation of acetate.
-The proteins produced as inclusion bodies require further refolding because they are inactive.
Table 4: Characteristics of E.Coli expression systems
3.2.2 Importance of expressing the mutant
The presence of glutamic acid in the MMPs seems to play a crucial role in their activation. It is believed to contribute into the charge of a molecule of water that provokes a nucleophilic attack, giving to the MMPs a catalytic activity. The substitution of the glutamate residue with a glutamine residue in the case of the mutant Q or with an alanine one, in the case of the mutant A makes the enzyme inactive and more stable for the crystallization experiments [35].It is interesting to obtain crystals of the same inhibitor binding enzymes in their mutant form or as wild-type and compare the results between them.
3.3 Experimental Part
3.3.1 IntroductionProjects that make use of DNA sequencing have provided coding sequences for an estimable number of proteins from organisms across the evolutionary spectrum. Recombinant DNA technology makes it possible to clone these coding sequences into expression vectors that can direct the production of the corresponding proteins in suitable host cells. An inducible T7 expression system is highly effective and widely used to produce RNAs and proteins from cloned coding sequences in the bacterium Escherichia coli. The coding sequence for T7 RNA polymerase is present in the chromosome under control of the inducible lacUV5 promoter in hosts such as BL21(DE3). The coding sequence for the desired protein (referred to as the target protein) is placed in a plasmid under control of a T7 promoter, that is, a promoter recognized specifically by T7 RNA polymerase. In the absence of induction of the lacUV5 promoter, little T7 RNA polymerase or target protein should be present and the cells should grow well. However, upon addition of an inducer, typically isopropyl-b-D-thiogalactoside (IPTG) the target protein will be expressed.
3.3.2 Materials and methods
MMP8h without pro-domain was expressed using the plasmid pET11, having the gene responsible for the production of hMMP8 inactive in E. coli host cells: BL21(DE3)(Novagen). All proteins were expressed as 1-L cultures from ZY media, supplemented with 100 μg/mL ampicillin. One-liter cultures were inoculated with cells from 20 mL of a starter culture (250 mL flask containing 50 mL
Figure 12: Representation of the exponential growth of
bacteria(image reproduced from Michal Komorniczak)
Figure 13: Escherichia Coli cell. The representation of a
DMSO stocks and grown overnight. Following a chemical induction protocol made at CEA, 1-L cultures of E. coli were grown at 37°C with shaking (~250 rpm), and were induced with IPTG at an optical density at 600 nm (OD600) of 0.8. After induction, the E. coli were cultured for an additional
5 h, and were then harvested by centrifugation.
DAY 1- Preparing the pre-culture Transformation
The plasmid pET11, having the gene responsible for the production of hMMP8 inactive is added to the E. coli cells of the production strain (BL21 Star DE3). Transformation was carried out by thermic shock: competent bacteria were then incubated with the vector 30 min on ice, followed by transfer to 42 ºC for 30 sec followed by a final step of 2 min on ice. The transformants were suspended in 350 μl of SOC culture media and incubated for an hour at 37 ºC.
Pre-culture
The liquid transformation was then added to 10 mL of ZY medium* containing antibiotic (ampicilline) and incubated at 37ºC O.N.
DAY 2- Expression induction with IPTG
The pre-culture becomes turbid (slightly cloudy, but not too cloudy) after overnight incubation. The culture media is fortified under sterile conditions under a laminar flow hood: 925 mL ZY inside a Fernbach with 50 mL NPS solution containing glycerol 50%, 1 mL MgSO4 (concentration) and ampicillin at 100 µg/mL. The culture is seeded at 1/50th with the pre-culture. with the pre-culture and the optical density measured at 600 nm at time 0 (OD0 = 0.05). The cell culture is placed in a shaker under controlled temperature and left to grow for 3 hours. An aliquot is removed to measure the OD, using ZY [no cells but with antibiotics] as a blank.
Once the desired OD is reached, the culture is induced with 0.5mM IPTG and left to shake vigorously for 5h. The OD is measured again after the addition of IPTG.
After incubation, the 2L volume is divided in 4x500mL flasks and centrifuged at 4000 rpm (2969g; Rotor=25.50 JA) for 30 min at 4 ºC. The pellets are suspended in 20mL buffer 1 (50mM TRIS HCl, 10mM calcium chloride, 30mM sodium chloride, pH 7.5), and then centrifuged again inside falcons at 10000 rpm for 5 min at 4 ºC. The pellets are then frozen under liquid nitrogen (-176ºC) and stored at -20ºC.
*NZY Broth composition per liter: 5 g NaCl, 2 g MgSO4 • 7H20, 5 g Yeast extract, 10 g NZ amine (Casein
DAY 3- Cell Lysis and protein extraction
The most common preparation method of protein extraction from inclusion bodies (IB) involves mechanical or chemical cell disruption followed by differential centrifugation to separate the dense IB from the lighter cell membrane components and soluble contaminants. Repeated washes with denaturants or detergents can considerable reduce contaminant levels before IB solubilization. These washing steps are important not only to facilitate protein purification, but also to increase refolding yield as contaminants present in the preparation of IB can affect refolding.
- Cell Disruption
After suspending the pellets in 20 mL of Buffer A (100 mM Tris pH 8,5 / 5 mM benzamidino chloride / 5 mM ß-mercaptoethanol), 500 µl lysozyme at a concentration of 10 mg/ml and 50µl of PMSF (2,5 µg/ml) is added. The solution is left under incubation in ice for 30 min and then add a second aliquot of 50 µl of PMSF (2,5 µg/ml). The cell disruptor is switched on and after another 30 min in ice, the cells are broken at a pressure of 1.5 bar in order to recover the protein. The solution is suspended in buffer A with 2 µl benzonase and 1 ml MgCl2 (1M). Finally, 100 µl of this solution is used for analysis by SDS-PAGE electrophoresis (So).
- IB Washings
Four centrifugation runs at 8100 rpm(6013g ; Rotor=25.50) for 30 min at 4°C are carried out. During the first three, the pellet is suspended in 30 ml of buffer B (100 mM Tris pH 8,5 / 2 M Urea / 5 mM ß-mercapto)and 1mL of the supernatant is checked each time (S1- S3') The pellets appear white (the protein) and grey (the cell membrane). After each centrifugation the grey part becomes smaller until it disappears.
- IB Solubilisation
The pellet obtained after the last centrifugation run is suspended in 80 mL of a solubilization buffer (6M Urea (360 g/l) / 100 mM ß-mercapto (7 ml stock/l) / 20 mM Tris pH 8,5 (2,42 g/l). After leaving the solution for 30 min at 37°C, it is centrifuged at 18000 rpm(13363g; Rotor=25.50 JA) for 20 min. The supernatant should contain the solubilized protein (S4).
- Purification
For purification by dialysis, sacks are prepared. A first dialysis is done in buffer D (50 mM Tris (pH 7.5) – 10 mM CaCl2 – 200 mM NaCl – 0,1 mM ZnCl2 – 3M urea) for 3 h at 4°C. An aliquot of 1 ml (S5) is taken. The second dialysis is done in buffer D1 (20mM Tris (pH 7.5) – 5 mM CaCl2 – 200 mM NaCl – 0,1 mM ZnCl2) and left O.N at 4°C.
DAY 4- Concentration of the protein
After the third dialysis is done in Buffer E (20 mM Tris (pH 7.5) – 3 mM CaCl2 – 200 mM NaCl – 0,1 mM ZnCl2) for 3h at 4°C, the last aliquot is taken and analysed (S6). After the centrifugation at
18 000 rpm (13363g; Rotor=25.50 JA) for 40 min the supernatant is recovered and stored at -80°C. The recovered volume was 300 mL, of these, 100 mL was concentrated under a pressure of 2 bar. 450 µl of protein at 457.9 µM was obtained. The protein, due to the aromatic aminoacids absorb at 280 nm and the nucleic acids at 260 nm. From the UV Spectrum: A280 >1.5, so the protein doesn't need to be diluited. From the values of A280/A260=1.15 that shows the presence of only 2% of nucleic acid (The value is irrelevant to affect the purity of the protein) [36]. The concentration is calculated from the absorbance at 280 nm on a UV spectrometer).
3.4 Experimental Data
The values of the OD measured after each step are reported below: -t0 ODfernbach1 = 0.05 -t1 = 1h40 ODfernbach1 = 0.10 -t2 = 3h15 ODfernbach1 = 0.73 ODfernbach2 = 0.76 -t3 = 8h40 ODfernbach1 = 1.89 ODfernbach2 = 1.94
Applying Lambert&Beer's law it was possible to calculate the final concentration of the protein. Having the MWprot=17597 g/mol, ε=28420 g/L, A=0,6507, it is possible to calculate the concentration of the protein; c=457,9 μM.
The samples collected during the protein expression in E.coli were analysed using an SDS PAGE so as to see if the process of purification went to conclusion and to check the purity of the final protein.
3.5 Results
At the end the pure protein was expressed and purified and was ready for the crystallization experiments. The results of the SDS Page after the process of production of the catalytic domain of MMP-8 are represented in the images above (Fig. 14). From the images we can say that the protein produced was pure and from the further crystallization experiments it showed to be able to crystallize. Crystals obtained in complex with hMMP8A still haven't been analyzed.
Figure 14: The samples collected after each step of the protein's production were tested with SDS-Page. In the last step
corresponding to S6 sample, we have the protein pure and concentrated. Ultraviolet absorption spectra of the MMP8A. Protein absorbs strongly at 280 nm due to a number of its aromatic amino acids (tyrosine and tryptophan) meanwhile the nucleic acids at 260 nm. Absorbance at 310 nm is ~0 as it should be, considering that aromatic amino acids do not absorb above 300 nm.
CHAPTER 4
Biological crystallogenesis
4.1 Introduction
Crystallization is the process by which molecules from a saturated liquid state interact to forming a regular repetitive lattice. The manner in which they pack determines the crystal system (Tab 3). The macroscopic external shape of a crystal is a reflection of the symmetry of the packing of the sub-microscopic molecules that form it. The unit cell is the complete assembly of molecules that can repeat by translation to confers the crystal its visual shape. The asymmetric unit is the smallest repeating unit that builds the unit cell according to crystal symmetry operations that determine the space group of the crystal. All crystals belong to one of seven systems: cubic, hexagonal, orthorhombic, rhombohedral, tetragonal, monoclinic or triclinic. In the triclinic system, the asymmetric unit and the unit cell are identical.
