Transcript Prezentace aplikace PowerPoint

Role of proteolysis
in physiological and pathological
processes
Zdenka Kučerová
Proteolytic enzymes :
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cleave peptide bonds ( proteins or peptides)
control of the cell cycle
key roles in many pathophysiological processes
targets for therapeutic strategies
speed of the cleavage depends on the strategically
located positions of cleaved bonds
Activity of proteolytic enzymes
• determined by many factors at the post-translational level
• allosteric nature  physiologically important interactions
 explains why the enzymes carry a large
body of structural and functional information
degradom
Regulation by proteolytic enzymes
• limited proteolysis or by proteinase inhibitors
Limited proteolysis:
• post-translational modification
• conversion of an inactive precursor into an active
form
Proteinase inhibitors:
• regulate:  coagulation
 fibrinolysis
 connective tissue turnover
 complement activation
• concentration changes during physiological or
pathological processes
Proteolysis
 complete degradation of proteins to free amino acids
requires series of enzymes with different specificity
not only in the digestive tract, but also in every cell  in
lysosomes, in cytoplasm and other parts of the cell
Division of proteolytic enzymes
peptidasesendopeptidases and exopeptidases (position of
the cleavage site within the substrate molecule)
 endopeptidases (proteinases )  groups according to
similar reaction mechanisms (e.g. serine or cysteine
proteinases)
Mechnism of interaction
• enzymes recognise and bind to a short amino acid
sequences and then specifically hydrolyse bonds
between particular aminoacid residues
Classification of proteinases
A) endopeptidases – hydrolysis within the peptide chain
1) serine proteinases – serine or histidine residues in active
sites, pH optimum 7 – 9
trypsin, chymotrypsin, thrombin, plasmin
2) cysteine proteinases – cysteine residue in active site, pH
optimum 4 – 7
calpain, caspases
3) aspartic proteinases – two aspartic residues in active site,
pH optimum below 5
pepsin, gastricsin, rennin, HIV proteinase
4) metaloproteinases – metal ion important for catalysis, pH
opt. 7 – 9
matrix metaloproteinases, collagenase, gelatinase
Homology of various proteinases
B) exopeptidases - cleave off single amino acid one after
another
1) aminopeptidases – cleave off a single amino acid from
amino terminus
2) dipeptidyldipeptidases – cleave off dipeptide from
amino terminus
3) carboxypeptidases – cleave off a single amino acid
from carboxy terminus
4) dipeptidases – hydrolyse dipeptides in two single
amino acids
• Individual proteinases differ in their substrate specifity
• For complete degradation of a protein, enzymes from
each of the above-named groups are required
Characterization of some proteinases
enzym
m.wt
isoelektric point
(pH)
chymotrypsinogen
25.000
8.1
trypsin
24.000
10.8
plasmin
75.000
-
elastase
26.000
9.5
papain
21.000
8.7
pepsin
33.000
1.0
carboxypeptidase A
34.000
6.0
Inhibitors
differ according to individual groups of proteinases
•Serine proteinases…..chlormethylketons,
•Cysteine proteinases…. dithiothreitol, mercaptans,
•Aspartic proteinasess… epoxy- and diazo- compounds, pepstatin
• Metalloproteinases….. chelates
Serine proteinases
Proteinases of digestive
tract – trypsin, chymotrypsin
important for:
fibrinolysis
blood coagulation
complement activation
produced as inactive zymogens
activated by limited proteolysis by another proteinase
Blood clotting
• enzymatic conversion of a soluble plasmatic
fibrinogen into a fibrous network of insoluble fibrin
polymers
• fibrinogen cleaved by thrombin and
fibrin
aggregate into insoluble fibrin polymer
• most of the coagulation factors are proteinases
Initiation of blood clotting
two different ways
• extravascular pathway - triggered by an injury of tissue
• intravascular pathway - initiated by contact with a damaged
inner surface of blood vessel
• cascade of proteolytic cleavages
• requires calcium and phospholipids
• conversion of inactive prothrombin to active thrombin
• series of zymogens of serine proteinases are sequentially
activated (active serine proteinases arise from inactive
precursors, the active enzymes then activate other inactive
proteins)
Unique aspects of coagulation
• requirement for a protein or a lipoprotein cofactor for
optimal reaction rates
• requirement for a membrane surface
• metal ion requirement
• uniqueness of initiating reactions in comparison to those
which activate the pancreatic zymogens
Blood coagulation cascade
• ended by dissociation of the membrane complex
• regulated by activators and inhibitors in dynamic equilibrium
Antithrombin III
• inhibits activity of thrombin  an anticoagulant factor
• anticoagulant therapy  reduction of formation or reduction of
function of thrombin and other serine proteinases
Protein C
•Serine proteinase…inactivation of factor Va and VIIIa
Fibrinolysis
• fibrin networks are degraded by plasmin
• plasmin is serine proteinase
• plasmin evolves from precursor plasminogen after
activation by plasminogen activator
• plasmin activity inhibited by α-2 antiplasmin
• therapy by fibrinolytic proteinases  thrombus
degradation
Characterization of plasmin, plasminogen and
plasminogen activator
Plasminogen is one-chain glykoprotein
hydrolyse bounds Arg67-Met68, Lys76-Lys77, Lys77-Val78
Plasmin is two chain serine protease (with disulfidic bound)
active site His-603, Asp-646, Ser-741
its main target is fibrin
Human plasminogen activator [PA] is serine protease
(tissue and urokinase)
one polypeptide chain ( single chain-ScuPA)
hydrolyse bounds Arg275-Ileu276 (two chain-TctPA)
plasmin formation is dependent on:
• plasmatic concentration of plasminogen
• availability of activators of plasminogen in the plasma
• surrounding tissue environment
• concentration of naturally present inhibitors
• concentration of fibrin
Pathological fibrinolysis
• abnormal fibrinolysis, bleeding or thrombus formation
Higher fibrinolysis
• increased level of plasminogen activator (PA)
• deficiency of plasminogen activator inhibitor (PAI)
• deficiency of antiplasmin
Thrombus formation
• defective synthesis of plasmin or PA
• PA used to pharmacological digestion of thrombus
Malignant tumors
• disorders of hemostasis - ability of the tumor to alter the
coagulation system
• Tumor malignancies connected with invasive growth and
metastasis  enzymatic breakdown of two proteolytic
systems (fibrinolytic s. and matrix metaloproteinases s.)
Methastatic disease
• disintegrated regulation
• irreversible progression of cell cycle
• resistance to a cell signalling
• understanding of the mechanism  preventing tumor
cell spread
• proteases have proteolytic activity that may affect cancer
progression
Proteinases - Biomarkers
• correspond to changes in the whole organism
changes: - biochemical
- histological
- morphological
- physiological
• very important for early diagnosis
Complement
This system collaborates in recognition and elimination of
pathogens as a part of both the innate and acquired
immune systems.
•The activation of complement pathway -
through
attached serine proteases
•the complement system is one of the most highly
organized innate immune systems
• serine proteinases are responsible for regulation of the
early event of complement
Kallikrein
• plasmatic serine proteinases (15 )
• kalikrein - kinin
is a complex system produced in various
organs
•the kallikreins
must now be considered as important
'hormonal' regulators of tissue function
• important for initiation of cellular fibrinolysis, which is
independent on plasmin, fibrin and tissue plasminogen
activator
•predictive marker for cancer
Cysteine proteinases
lysosomal - cysteine cathepsins
• implicated in tumor spread and metastasis
• prognostic factors for tumor recurrence in human breast
cancer
non-lysosomal – calpain
• cleave
cell-cycle proteins, cytoskeletal and myofibrilar
proteins
• cytoplasmatic enzyme
Apoptosis
• evolutionary conserved form of cell suicide
• requires a proteolytic system - mainly caspases
•caspase family of proteases can be divided into proapoptotic and pro-inflammatory members based on their
substrate specificity and participation in separate
signalling cascades
• caspase regulation  therapeutic purposes
• caspases - minimally requires a tetrapeptide substrate in
which Asp is in absolute requirement in P1 position, the
P4 substrate residues is unique to each homologue.
• defective control of apoptosis  pathogenesis of diseases
• cancer chemotherapy and radiation  cancer cell death by
apoptosis
• Caspases are responsible for crucial aspects of
inflammation and immune-cell death that are disrupted in a
number of genetic autoimmune and autoinflammatory
diseases.
• caspases  proteolytic devitalizing and remodelling of
tumor cells  suppress tumor cell growth invasion and
metastasis
Aspartic proteinases
Acid proteinases from mammalian gastric mucosa
pepsins
Proteinases associated with limited proteolysis
renin blood pressure
cathepsins D,E lysosomal enzyme
proteinase HIV
Renin
• aspartic proteinase
• formed as a precursor (prorenin) in kidneys
• active form (renin) released into the blood
• renin cleaves angiotensinogen to form angiotensin I
• angiotensin-converting enzyme (ACE) then converts
angiotensin I to angiotensin II
Renin
• highly specific
• synthesized in response to decreasing levels of sodium
ions and declining blood pressure
• renin  angiotensin II  affects: - kidney
- brain stem
- hypophysis
- adrenal cortex
- blood vessel walls
- heart
Cathepsins D and E
Cathepsin D
• aspartic proteinase localized in lysosomes
• in most tissues and cells of human organism
• widely distributed among biological species
• influences: - intracellular protein catabolism
- hormone and antigen processing
- pathological processes (neoplasia and
neurodegenerative changes)
Cathepsin E
• in human stomach cells (another stomach cells than
pepsinogen and progastricsin)
Both is over- expressed by cancer cells
Proteinase HIV
• AIDS  retrovirus (human immunodeficiency virus)
• retroviral aspartic proteinases  deeply studied proteins
in today molecular biology chemistry
• three-dimensional structure of active site is similar to that
of eucariotic proteinases
• proteinase (and other enzymes)  design of novel drugs
for the treatment of AIDS
• fast development of resistance of virus towards synthetic
inhibitors and low bioavailability of these inhibitors
• resistance results primarily from multiple mutations of
the proteinase
• drug-resistant HIV proteinase → altered substrate
specifity
• altered substrate specifity understanding → valuable in
the design of new protease inhibitors
•active antiretroviral therapy (HAART) combining potent
drugs that can inhibit reverse transcriptase, integrase and
protease activities
Metaloproteinases
• matrix metaloproteinases (MMPs)
• zinc-dependent enzymes
• regulate tissue remodelling in physiological and
pathological conditions
• MMPs are key enzymes for tumor progression cell
carcinoma
• individual MMPs found in most tumor types
• presence of specific MMPs has been shown to be a
prognostic marker at tumor invasion
• tumor invasion and metastasis → degradation of the
extracellular matrix around tumor cells and cell migration
• cancer metastasis → imbalance between MMPs and its
inhibitor
• MMPs →
regulatory effects on both primary and
secondary tumors
• inhibitors of MMPs were assessed for anticancer
properties
• proteolysis has a central role for cancer metastasis
Exopeptidases
Peptidyl dipeptidases
• angiotensin-converting enzyme in the control of blood
pressure
Human prolylcarboxypeptidase also known as
angiotensinase C inactivates angiotensin II
Inhibitors of dipeptidyl peptidase IV (DPP IV) provide a
strategy for the treatment of type 2 diabetes
Methionine aminopeptidase (MetAP) is a bifunctional
protein that plays a critical role in the regulation of posttranslational processing and protein synthesis
•inhibitors of all peptidases have therapeutic effect
Human gastric proteinases
• digestive proteinases
• adult mammalian gastric mucosa contains the highest
concentration and greatest diversity of gastric
proteinases
• human gastric mucosa contains four aspartic
proteinases: - pepsinogen A (PGA)
- pepsinogen C (PGC)
- chymosin
- cathepsin E
• common biological properties (the ability to hydrolyze
proteins at acid pH) but each is distinguishable by
immunologic technique
• serum contains both PGA and PGC, but only PGA is
detectable in urine
Schematic view of pepsinogen A
Pepsinogen A and pepsinogen C
• aspartic proteinases
• differ in charge → distinguishable by electrophoresis
• heterogeneity of pepsinogens → multiple genes
→ posttranslational
modifications of the primary gene product
Duodenal ulcer
• high level of PGA → subclinical marker of duodenal ulcer
Superficial gastritis
• acute or chronic superficial gastritis → high serum PGA
and PGC levels
• inflammation of the mucosa → increased rates of release
of both zymogens into the circulation → elevated levels of
PGA and PGC
• level of PGC increased much more than level of PGA
Pepsinogen A / pepsinogen C ratio in peptic ulcer, gastric
cancer and control
Gastric ulcer
• changed levels of PGA and PGC
• gastric ulcer usually develops within areas of chronic
gastritis
• pepsinogen A / pepsinogen C ration decreases
Gastric cancer
• very low serum levels of PGA
• low serum PGA level → subclinical marker of increased
risk for gastric cancer → it is possible to identify high-risk
subjects → more effective approaches to diagnosis and
treatment
• serum PGA and PGC levels → subclinical markers of
disorders of gastric mucosal structure and function
Importance of monitorig serum PGA level
• apparently healthy subjects, but:
→ low serum PGA level
→ subclinical marker of increased risk for gastric
cancer
→ better identification of high-risk subjects
→ development of more effective diagnosis
and treatment