Mecanismos intracelulares

de degradação proteica

Dinâmica Molecular da Célula

 O “Turnover” (ciclo de vida) das proteínas

Cerca de 30 % das proteínas recém sintetizadas são rapidamente degradadas devido a

erros (na formação de complexos, conformação, etc…)
 Protein Homeostasis

Ageing

Sometimes, it´s not true…

1914 1974 2015

Protein clearance upon damage (ROS, mutation,…)

DISEASE

Over time
What is protein aggregation?
Relevance of protein modifications in Human disease
Quais os mecanismos conhecidos de

degradação proteica?
 As múltiplas vias de degradação proteica

Autophagy-Lysosome pathway (ALP)

Ubiquitin-Proteasome System (UPS)
Autophagy-Lysosome pathway (ALP)

A autofagia actua como um mecanismo protector em

condições de stress. É essencial para a manutenção da
viabilidade celular através da reciclagem de organelos
danificados, proteínas missfolded, nutrientes e remoção de
metabolitos tóxicos.
No entanto, a degradação excessiva de componentes
citosólicos leve à morte celular
Importância fisiológica da autofagia
Approximately 1%–1.5% of cellular proteins are catabolized per hour by autophagy.

Basal autophagy acts as the quality-control machinery for cytoplasmic components,

and it is crucial for homeostasis of various postmitotic cells, such as neurons.

Cell 147, November 11, 2011

Both UPS and ALP activities decline with aging and such decline may
play a pivotal role in the initiation and propagation of neurodegenerative
diseases, where protein aggregation is a crucial factor
Tipos de autofagia
 Starvation was recognized as one of the best stimuli of macroautophagy

Cell Metabolism 13, May 4, 2011

Protein breakdowns: to utilize amino acids for cellular fueling and to replenish the intracellular
pool of amino acids required to maintain protein synthesis.
 Macroautofagia

Microbes, Damaged organelles, Protein aggregates, cell

homeostasis
 Formação do autofagossoma (macroautofagia)

autophagy-related
proteins or Atg

Cell 147, November 11, 2011

 PROBLEMA DE EXAME !!!!

Cell Death DCF fluorescence (ROS

production)
40 200
* *
* 180

DCF fluorescence [% of
30

corresponding control]

corresponding control]
Lethal cells [% of
160
*
20
* 140
* * *
10
120

ATG5 is important for 0 100

ATG 5 siRNA
ATG5+/+
ATG 5 siRNA

SIPS
ATG5+/+
SIPS

ATG5-/-
ATG5-/-

ATG6V0A1 siRNA
ATG6V0A1 siRNA
phagosomal extension and
closure
ATG5 siRNA
ATG5-/-

Macroautophagy as a survival mechanism!

Höhn, A. et al. Free Rad. Biol. Med. 2014
Chaperones-mediated auphagy (CMA)

The CMA targeting motif is present in about 30% of all cytosolic proteins

M. Xilouri, L. Stefanis / Molecular and Cellular Neuroscience 2015

 CMA and neurodegeneative disorders

M. Xilouri, L. Stefanis / Molecular and Cellular Neuroscience 2015

Lewi body (a-syn aggregates)
in substantia nigra

Mutations on LRRK2 and a-synuclein cause autosomal dominant PD

 Mitophagy and Parkinson disorders (PD)

European Journal of Neuroscience, pp. 1–13, 2015

(Mitochondrial clearance)

Mutations on PINK1, parkin, and DJ-1 cause autosomal recessive PD

Ubiquitin-Proteasome System

ATP and ubiquitin 2004 Nobel Prize - Chemistry

dependent for 26S
recognition and
degradation:
 Pathways controlled by regulated proteolysis

Desempenha funções importantes numa variedade de processos celulares

fundamentais tais como a regulação do ciclo, divisão, desenvolvimento e
diferenciação celular; apoptose; tráfego celular e modulação das respostas imunes e
inflamatórias.

Potencial alvo terapêutico para o tratamento do cancro e doenças neurodegenetaivas

O Sistema Ubiquitina-Proteassoma- Ubiquitina, polypéptido de 76 aminoácidos denominada de “Kiss of Death”

Required ATP for activity

 Ubiquitin is not the only small peptide to be
covalently attached to proteins and or lipids

SUMO 1/2

Nedd8

ISG15

ATG8

FAT10

Not thought to target proteins for destruction

Each is thought to have its own conjugation and
deconjugation system
The UPS is enormous!

The genes of the UPS

constitutes ~5% of
the genome

E1’s- 1-2 activating

enzymes

E2’s- 10-20 conjugating
enzymes

E3’s- 500-800 ubiquitin
ligase- drives specificity

DUBs- 100 ubiquitin specific
proteases- regulators of
pathway
O PROTEASSOMA
Vídeo
 19S Regulatory particle (RP)

Recognition and binding of ubiquitinated proteins

Unfolding of ubiquitinated substrate to enter 20S mediated by
AAA ATPases (ATP dependent)

Removal of ubiquitin side chains to allow entry into 20S (
lumen ~1.3 nm) by deubiquitinating enzymes

Activation/opening of 20S lumen
 20S Core Particle (CP)

Contains the endopeptidase activity

The alpha subunits function is to control the opening
and closing of the 20S gate (interacts with 19S)

The beta subunits β1, β2 and β5 contain the
endopeptidase activity of the proteasome.

Proteins are not degraded into amino acids but into
short peptides ( very important for immune
surveillance).
 The UPS, mechanism of surveillance

20S proteasome: Immunoproteasome: Hybrid-Proteasome: 26S Proteasome:

41 ± 5 % 20 ± 9 % 24 ± 9 % 15 ± 3 %

Oxidized proteins

Generation of
antigenic peptides
and others Still under
investigation
Polyubiquitinated
proteins
 20S: avoid protein aggregates

Proteasome behavior upon stress

20S proteasome:
41 ± 5 %

Oxidized proteins

Oxidized proteins are mainly degraded by the 20S, why?

-- Lys carbonylation, Poly(ub) impaired

-- hydrophobic exposure, appealing substrate (trigger signal)
-- No ATP/ub involved faster, cheaper and lesser risk
-- 20S more resistant than the 26S
Oxidized proteins degradation
 Interplay between ALP and UPS

The UPS degrades 80–90% of proteins including shortlived, native, misfolded, or

damaged proteins in the cell.

Autophagy primarily degrades most long-lived proteins that likely reside in the
membrane structure of cellular organelles or exist as aberrant protein
aggregates.

The UPS and autophagy were generally assumed as

completely parallel pathways and serving distinct
functions. However, emerging studies strongly suggest
functional interactions between the two degradation
systems. The UPS and autophagy appear to be
concertedly regulated for maintaining proteostasis
in both healthy and disease states.
 ALP and UPS as protective compensatory
mechanisms

When misfolded proteins escaped from the UPS surveillance, they tend to form aggregates
which are inaccessible by the proteasome, however, activate the ALP and are thereby degraded
by the lysosome.

Proteasome inhibition or impairment activates autophagy and conversely. Hence, alleviate the
burden of increased misfolded proteins on the UPS
UPR crosstalk with autophagy

Trends in Biochemical Sciences March 2015, Vol. 40, No. 3

And don´t forget interplay between Autophagy and UPS !!!!
Protective mechanisms upon stress STRESS

Aggregates Misfolded Proteins

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