21ª AULA

Prévia do material em texto

•  Internalização de macromoléculas e partículas: fagocitode, pinocitose e 
endocitose mediada por receptores. 
•  Espécies reativas de oxigénio vs radicais livres de oxigénio. 
•  Fontes de ROS celulares. 
•  Sistemas antioxidantes celulares: enzimáticos e não-enzimáticos 
Bibliografia 
•  Lodish H., Baltimore D., Berk A., Zipursky S.L., Matsudaira P., and Darnell J., Molecular 
Cell Biology (4ª Ed.), Freeman W.H. and Company (USA), Cap 15 (2000). 
•  Voet D. and Voet J.G. (2011) Biochemistry, 4 th edition, Wiley. Inc. 
Formation of phagocytic vesicle 
Respiratory burst prodides a dramatic example of the 
destructivenes of ROS 
REACTIVE OXYGEN SPECIES (ROS) 
NADPH + O2 
Superoxide (O2-.) 
Hydrogen peroxide (H2O2) 
Hydroxyl radical (.OH) 
Hypochlorite (HOCl) 
O2 + H2O 
NADPH oxidase 
Superoxide dismutase 
C
at
al
as
e 
Myeloperoxidase 
Fe
2+
 
Mitochondria

Lysosomes

Peroxisomes

Endoplasmic
Re7culum

Cytoplasm

Microsomal
Oxida7on,


Flavoproteins,


P450
enzymes


Myeloperoxidase

(phagocytes)


Electron
transport

Oxidases,

Flavoproteins

Plasma
Membrane
Lipoxygenases,

Prostaglandin
synthase

NADPH
oxidase

Xanthine
Oxidase,

NOS
isoforms

Fe

Cu

Transi7on

metals

Sources
of
ROS

OXIDATIVE DAMAGES 
As células aeróbias dependem em absoluto do O2 
No entanto, 
Quantidades elevadas de O2 tornam o O2 um agente 
altamente tóxico para a célula 
Esta toxicidade não deriva da acção directa do O2 sobre as 
células,mas sim da acção de espécies químicas derivadas do 
O2, denominadas RADICAIS LIVRES DE OXIGÉNIO 
A free radical is any species capable of independent existence 
that contains one or more unpaired electrons. 
An unpaired electron is one that occupies an atomic or molecular 
orbital by itself. 
The diatomic oxygen molecule qualifies as a radical because two unpaired electrons each located in a different π* orbital 
Oxygen is an oxidizing agent and oxidizes another atom or molecule by taking electrons from it. Electrons must be 
of the antiparallel spin to fit in the vacant spaces in the π* orbitals 
The other two more reactive forms of oxygen are singlet oxygens and can be generated by an input of energy 
If an electron is added to the ground state of oxygen molecule, it must enter one of the π* orbitals. The product 
is the superoxide radical (O2•– ) 
The addition of one more electron yields the peroxide anion (O22–), which is the deprotonated form of H2O2 
RADICAL LIVRE: 
 qualquer espécie capaz de existência independente que 
 contém 1 ou + electrões desemparelhados 
Normalmente muito reactivos! 
ROS – Reactive Oxygen Species 
DEFINIÇÃO 
•  Na verdade, RADICAL LIVRE não é o termo ideal para 
designar esses agentes reativos, pois alguns deles não 
apresentam eletrões desemparelhados na sua última 
camada. Como em sua maioria são derivados do 
metabolismo do O2, costuma-se utilizar o termo “espécies 
reativas de oxigénio” (ROS) para se referir a eles.
Usualmente... 
Oxigénio…. 
Restrição de spin 
•  oxigénio molecular (O2) possui 2 eletrões 
desemparelhados, localizados em 2 orbitais 
p*2p. Esses eletrões têm spins iguais, tornando 
o O2 bastante estável, pois restringe sua 
redução ou a moléculas que possuam 2 eletrões 
de spins paralelos ou a uma redução univalente, 
com um eletrão de cada vez. A este fenómeno 
chamamos restrição de spin. 
•  Esta restrição limita a reatividade do oxigénio, 
permitindo que numa atmosfera de 21% de O2, 
como a da Terra, ocorram moléculas 
constituídas por cadeias carbonadas altamente 
reduzidas (Halliwell, Gutteridge, 1989). 
Origin of ROS
Free radicals formed by neutrophil 
•  Superoxide (O2¯• ) 
–  No direct effects on targets 
–  Penetrates important sites 
–  Subsequently converted to other ROS 
•  Hydrogen Peroxide (H2O2) 
–  Dismutation of superoxide radical 
 2H + + 2O2¯• H2O2 + O2 
–  Reacts with thiols 
–  Bacteriocidal only at higher concentrations 
–  Secondary oxidants from H2O2 responsible for killing 
SOD 
Products of Oxidase: Role in Killing 
•  Hydroxyl Radicals (OH•) – Fenton Reaction 
 Fe2+ +H2O2 Fe3+ + OH¯ + •OH 
–  •OH as a component of neutrophil bacteriocidal arsenal 
•  Singlet Oxygen (O21) – 
–  Electronically excited state of oxygen 
–  Thought to be produced from reaction of H2O2 with HOCl 
–  Can react with a number of biological molecules 
Myeloperoxidase (MPO) 
•  Most H2O2 consumed by MPO 
•  Heme Peroxidase, uses H2O2 to oxidize variety of compounds 
•  Unique property – oxidizes Cl - to HOCl 
 H2O2 + HCl HOCl- + H2O 
MPO 
•  Hypochlorous acid (HOCl) 
–  Most bacteriocidal oxidant known to be produced by 
neutrophils 
–  Bacterial targets – Fe-S proteins, membrane transport 
proteins, ATP generating system 
Myeloperoxidase (MPO) 
 
 
 
 
 
 
 
Under pathological condition the 
physiological balance is lost (oxidative stress)
Hydroxyl radical (·OH) 
•  The most reactive oxygen species known: site specific reaction 
•  Can be produced by high-energy ionizing radiation 
 H2O ·OH + ·H + H2 + H2O2 + H3O+ 
 (ionizing radiation) 
•  Fe2+-dependent decomposition of H2O2 (Fenton reaction) 
 Fe2+ + H2O2 Fe3+ + ·OH + OH- 
 
•  Metal-catalyzed Haber-Weiss reaction 
 Fe3+ + O2.- Fe2+ + O2 
 Fe2+ + H2O2 Fe3+ + .OH + OH- 
Net O2.- + H2O2 O2 + .OH + OH- 
Antioxidant Enzymes and Mechanisms 
2O2.‐
+
2H‐

 








H2O2
+
O2



















(superoxide
dismutase)

2H2O2

 
 






2H2O
+
O2





(catalase)

2GSH
+
H2O2

 
 









GSSG
+
H2O























(glutathione
peroxidase)

GSSG
+
NADPH
 
 







2GSH
+
NADP‐




















(Glutathione
reductase)

GSH:
reduced
glutathione,
GSSG:
oxidized
glutathione

Voltamos aos processos de endocitose celular… 
Pinocytosis 
  “Cellular Drinking” – engulfing liquids and small molecules 
dissolved in liquids. 
  Ex. Intestinal cells, Kidney cells, Plant root cells 
•  In pinocytosis, “cellular drinking”, a cell creates a vesicle around a droplet 
of extracellular fluid. 
–  This is a non-specific process. 
This is the most common form of endocytosis. 
Pinocytosis takes up most proteins and other large molecules. 
Bulk-phase Endocytosis (Pinocytosis) 
•  No receptors involved 
•  Tiny droplets of extracellular fluid are 
taken into the cell. 
•  Resulting smaller molecules leave 
lysosome to be used else where in 
the cell. 
•  Occurs in most cells specially 
absorptive cells in the intestine and 
kidneys. 
•  Receptor-mediated endocytosis is very specific in what 
substances are being transported. 
•  This process is triggered when extracellular substances bind 
to special receptors, ligands, on the membrane surface, 
especially near coated pits. 
•  This triggers the formation of a vesicle 
Receptor-Mediated Endocytosis 
Receptor Mediated Endocytosis 
Also HIV virus enters T-cell by endocytosis after attaching to a receptor called CD4 
Cells use Cholesterol 
to rebuild its membranes 
and for synthesis of 
steroids. 
LDL=Low density lipoprotein 
A clathrin-coated pit on the cytosolic face of the plasma membrane 
Clathrin associates via adaptins with receptors in the donor membrane. 
The receptors bind specific cargo. The clathrin assembles into a cage 
that encapsulates a region of membrane. Then dynamin causes the 
membrane to pinch off forming a vesicle. 
Energy requirements: 
• GTP hydrolysis by dynamin accompanies pinching off. 
Filme- endocitose