Aplikovaná chemie a biochemie
Přednáška č. 3
Proteinové techniky
(2)
Proteiny (2):
• funkce jednotlivých domén;
• posttranslační modifikace (fosforylace,
acetylace, glykosylace);
• regulace odborávání proteinu (ubikvitinace;
proteazomální degradace);
• manipulace s proteinem (overexprese;
dominant negative constructs, antisense
oligonuclotides, siRNA).
Proteiny se skládají z domén, definovaných na základě
struktury a funkce:
Primární a sekundární struktura proteinu může naznačit
jeho funkci:
P53 domain structure
Proteinové domény často charakterizují celou
rodinu proteinů:
Families of DNA-binding domains
(A) Zinc finger domains consist of
loops in which an α helix and a β
sheet coordinately bind a zinc ion.
(B) Helix-turn-helix domains consist
of three (or in some cases four)
helical regions. One helix (helix 3)
makes most of the contacts with
DNA, while helices 1 and 2 lie on
top and stabilize the interaction.
(C) The DNA-binding domains of
leucine zipper proteins are formed
from two distinct polypeptide
chains. Interactions between the
hydrophobic side chains of leucine
residues exposed on one side of a
helical region (the leucine zipper)
are responsible for dimerization.
Immediately following the leucine
zipper is a DNA-binding helix,
which is rich in basic amino acids.
(D) Helix-loop-helix domains are
similar to leucine zippers, except
that the dimerization domains of
these proteins each consist of two
helical regions separated by a loop.
Různé strukturní
domény mohou
plnit stejnou
funkci:
Příklad – funkce domén v interakci proteinů:
Identification of the region
responsible for the nuclear
localization of ARNT. Various
portions of ARNT were
synthesized using PCR, and the
resulting fragments were fused
to the modified -Gal control
vector. An expression vector of
-Gal/ARNT-(1–789) fusion
gene was delivered into the
indicated cells by means of
electroporation. After a 48-h
incubation at 37 °C, the cells
were fixed and stained with 5bromo-4-chloro-3-indolyl
b-Dgalactopyranoside
solution. The
subcellular localization of the
fusion proteins were
examined by microscopy.
Regulace hladiny, aktivity a lokalizace
proteinu – dynamický proces:
• posttranslační modifikace;
• vazba ligandu;
• interakce protein-protein;
• štěpení inaktivní formy proteinu;
• degradace proteinů (lysozóm,
proteazóm)
Overview of sorting
of nuclear-encoded
proteins in
eukaryotic cells.
All nuclear-encoded
mRNAs are
translated on
cytosolic ribosomes.
Ribosomes are
directed to the rough
endoplasmic
reticulum (ER) by an
ER signal sequence
And these proteins
move to the Golgi
complex, from
whence they are
further sorted to
several destinations
After synthesis of proteins lacking an ER signal sequence is completed on free
ribosomes, the proteins are released into the cytosol and those with an organellespecific
uptake-targeting sequence are imported into the mitochondrion,
peroxisome, or nucleus.
Control of cell
cycle
CyclinUbiquitinUbiquitination
Blood-clot
formation
Fibrinogen3′-
Phosphoadenosine-
5′-phosphosulfate
Sulfation
Blood clottingThrombinHCO3
-γ-Carboxylation
Signal transductionRasFarnesyl
pyrophosphate
Farnesylation
TranscriptionRNA polymeraseNADADP-ribosylation
Signal transductionSrcMyristoyl CoAMyristoylation
DNA packing;
transcription
HistonesAcetyl CoAAcetylation
Glucose
homeostasis;
energy
transduction
Glycogen
phosphorylase
ATPPhosphorylation
Protein functionExample of
modified protein
Donor moleculeModification
Posttranslační modifikace:
Protein kinases and phosphatases. Protein kinases catalyze the
transfer of a phosphate group from ATP to the side chains of serine and
threonine (protein-serine/threonine kinases) or tyrosine (proteintyrosine
kinases) residues. Protein phosphatases catalyze the removal of
phosphate groups from the same amino acids by hydrolysis.
Příklady PT modifikací – detekce specifickými
protilátkami
Příklady PT modifikací – detekce pomocí specifické
protilátky
Příklady PT modifikací – detekce podle MW a pI
Příklady PT modifikací – detekce aktivity
modifikujících enzymů
Příklad proteomové
analýzy – karbonylace
proteinů v průběhu
apoptózy
Proteinová degradace:
The levels of proteins within cells are determined not only by
rates of synthesis, but also by rates of degradation. The halflives
of proteins within cells vary widely, from minutes to several
days, and differential rates of protein degradation are an
important aspect of cell regulation. Many rapidly degraded
proteins function as regulatory molecules, such as transcription
factors. The rapid turnover of these proteins is necessary to
allow their levels to change quickly in response to external stimuli.
Other proteins are rapidly degraded in response to specific
signals, providing another mechanism for the regulation of
intracellular enzyme activity. In addition, faulty or damaged
proteins are recognized and rapidly degraded within cells, thereby
eliminating the consequences of mistakes made during protein
synthesis. In eukaryotic cells, two major pathways—the ubiquitinproteasome
pathway and lysosomal proteolysis—mediate protein
degradation.
The ubiquitin-proteasome pathway Proteins are marked for rapid degradation by
the covalent attachment of several molecules of ubiquitin. Ubiquitin is first
activated by the enzyme E1. Activated ubiquitin is then transferred to one of
several different ubiquitin-conjugating enzymes (E2). In most cases, the ubiquitin
is then transferred to a ubiquitin ligase (E3) and then to a specific target protein.
Multiple ubiquitins are then added, and the polyubiquinated proteins are degraded
by a protease complex (the proteasome).
Cyclin degradation
during the cell cycle.
The progression of
eukaryotic cells through
the division cycle is
controlled in part by the
synthesis and
degradation of cyclin B,
which is a regulatory
subunit of the Cdc2
protein kinase. Synthesis
of cyclin B during
interphase leads to the
formation of an active
cyclin B–Cdc2 complex,
which induces entry into
mitosis. Rapid
degradation of cyclin B
then leads to
inactivation of the Cdc2
kinase, allowing the cell
to exit mitosis and
return to interphase of
the next cell cycle.
Další typy modifikací – SUMO, sentrin, NEDD
Detekce ubikvitinace – shift assay:
Detekce neddylace – rekombinantní protein:
The other major pathway of protein
degradation in eukaryotic cells involves the
uptake of proteins by lysosomes.
Lysosomes are membrane-enclosed
organelles that contain an array of
digestive enzymes, including several
proteases. They have several roles in cell
metabolism, including the digestion of
extracellular proteins taken up by
endocytosis as well as the gradual turnover
of cytoplasmic organelles and cytosolic
proteins. The containment of proteases
and other digestive enzymes within
lysosomes prevents uncontrolled
degradation of the contents of the cell.
Therefore, in order to be degraded by
lysosomal proteolysis, cellular proteins
must first be taken up by lysosomes. One
pathway for this uptake of cellular
proteins, autophagy, involves the formation
of vesicles (autophagosomes) in which small
areas of cytoplasm or cytoplasmic
organelles are enclosed in membranes
derived from the endoplasmic reticulum.
Lysosomes are able to degrade cytosolic
proteins in a selective manner. The proteins
degraded by lysosomal proteases under these
conditions contain amino acid sequences similar
to the broad consensus sequence Lys-Phe-Glu-
Arg-Gln.
Životní cyklus konexinů:
Dráhy regulující „protein trafficking“ lze
studovat pomocí specifických inhibitorů:
Práce s DNA a RNA:
• manipulace s proteinem (overexprese;
dominant negative constructs, antisense
oligonuclotides, siRNA);
• detekce exprese mRNA
• příprava a izolace plazmidů, izolace
genomové DNA, transfekce živočišných buněk