Protein and aminoacid metabolism'

1
Protein and amino-acid metabolism.
Amino-acid oxidation.
Fate of amino nitrogen
Metabolism of carbon skeletons
Protein breakdown
Lysosomal pathway
Proteasome mediated Caspases and apoptosis
Protein turnover under clinical conditions
2
BEFORE WE START SOME NECESSARY REVISION!
General amino acid structure
Some simple, neutral amino acids Glycine R
H Alanine R CH3
COO-

H
H3N
C
H
COO-
COO-

H
O
H3N
C
C
CH3
CH3
IMPORTANT METABOLIC INTERMEDIATE!
?
3
BEFORE WE START SOME NECESSARY REVISION!
General amino acid structure
Acidic amino acids
COO-
COO-

H
H3N
C
C
O
CH2
CH2
CH2
CH2
COO-
COO-
Glutamate is related to the citric acid cycle
intermediate
?-ketoglutarate
4
BEFORE WE START SOME NECESSARY REVISION!
General amino acid structure
Acidic amino acids
COO-
COO-

H
H3N
C
C
O
CH2
CH2
CH2
CH2
COO-
COO-
Glutamate is related to the citric acid cycle
intermediate
?-ketoglutarate
THESE ARE IMPORTANT METABOLIC INTERMEDIATES!
5
BEFORE WE START SOME NECESSARY REVISION!
General amino acid structure
Acidic amino acids
COO-
COO-
COO-

H
H3N
C
C
H
C
O
CH2
CH2
CH
COO-
COO-
COO-
Aspartate is related to the citric acid cycle
intermediates oxaloacetate and fumarate
THESE ARE IMPORTANT METABOLIC INTERMEDIATES!
6
BEFORE WE START SOME NECESSARY REVISION!
General amino acid structure
Amino acids with hydroxyl groups
COO-
COO-
COO-



C
H
H
H3N
H
H3N
C
H3N
C
CH2
CH
CH2OH
CH3
?
serine
OH
tyrosine
threonine
OH
Within some proteins, hydroxyl groups on one or a
few, specific Ser, Thr or Tyr residues are
targets for phosphorylation. The additional
negative charge on the protein from the added
phosphate leads to changes in its conformation
which can regulate activity
7
Basic amino acids


NH2
N
N
H3
IMPORTANT METABOLIC INTERMEDIATES!
arginine
ornithine



N
H3
CH2
Epsilon (?) amino group Site of covalent linkage
(by peptide bond) by cofactor (e.g. biotin) or
modifying protein (e.g. ubiquitin)
CH2
lysine
CH2
CH2

8
BRANCHED CHAIN AMINO ACIDS
COO-
COO-
COO-



H3N
C
H
H3N
C
H
H3N
C
H
CH2
CH
CH
CH3
CH3
CH3
CH
CH2
valine
isoleucine
CH3
CH3
CH3
leucine
IMPORTANT METABOLIC INTERMEDIATES! Can
temporarily substitute for fatty acids as a fuel
for oxidation in muscle during short-term
starvation. Very hydrophobic important for
protein structure.
9
AMINO ACID METABOLISM
No special storage form analogous to glycogen or
fatty acids.
Oxidation can provide energy or carbon for
gluconeogenesis.
10
glucose
POOL OF FREE AMINO ACIDS
CELLULARPROTEINS
extracellular amino acids
CO2 H2O
11
AMINO ACID METABOLISM
No special storage form analogous to glycogen or
fatty acids.
Oxidation can provide energy or carbon for
gluconeogenesis.
During metabolic stress proteins are degraded
yielding amino acids for energy.
There is much traffic of amino-acid between
tissues, within cells and into and out of protein.
12
AMINO ACID METABOLISM
No special storage form analogous to glycogen or
fatty acids.
Oxidation can provide energy or carbon for
gluconeogenesis.
During metabolic stress proteins are degraded
yielding amino acids for energy.
There is much traffic of amino-acid between
tissues, within cells and into and out of protein.
Each amino acid has its own metabolic
relationships.
Some amino acids are essential in the mammalian
diet.
13
OXIDATION OF AMINO ACIDS
cell proteins
glucose
urea
citric acid cycle

H2O
14
OXIDATION OF AMINO ACIDS
Individual mechanisms remove the NH2 group from
each amino acid. More general sequence of
mechanisms transfers the released amino groups to
urea
glucose
urea
citric acid cycle

H2O
15
THE UREA CYCLE A DISPOSAL SYSTEM FOR AMINO
GROUPS IN MAMMALS
Incorporates nitrogen into the non-toxic urea,
which can then be excreted. Cycle only occurs in
liver and kidney.
NH4 CO2 3ATP aspartate
Urea fumarate 2ADP AMP 4Pi
16
THE UREA CYCLE

2ATP
2ATP
aspartate
citrulline
carbamoyl phosphate
ATP
AMP PPi
argininosuccinate
ornithine
arginine
fumarate
NH2
urea
C
O
NH2
17
THE UREA CYCLE

2ATP
2ADP

carbamoyl phosphate
carbamoyl phosphate synthetase
18
THE UREA CYCLE
2ATP
citrulline
O
N
H

N
H3

ornithine

Ornithine transcarbamoylase
19
THE UREA CYCLE
COO-

H3N
C
H
citrulline
2ATP
aspartate
CH2
ATP
O
N
H
COO-
AMP PPi

N
H3
N
H
N

ornithine
argininosuccinate


20
THE UREA CYCLE
COO-

H3N
C
H
citrulline
2ATP
aspartate
CH2
ATP
O
N
H
COO-
AMP PPi

N
H3
N
H
N

ornithine
argininosuccinate



NH2
N
COO-
C
H
CH

COO-
arginine
fumarate
21
THE UREA CYCLE
COO-

H3N
C
H
citrulline
2ATP
aspartate
CH2
ATP
O
N
H
COO-
AMP PPi

N
H3
N
H
N

ornithine
argininosuccinate



NH2
N
COO-
C
H
NH2
urea
CH
C
O

COO-
NH2
arginine
fumarate
22
THE UREA CYCLE

2ATP
2ATP
aspartate
citrulline
ATP
carbamoyl phosphate
AMP PPi
argininosuccinate
ornithine
arginine
fumarate
NH2
urea
C
O
NH2
23
THE UREA CYCLE

2ATP
2ATP
aspartate
citrulline
ATP
carbamoyl phosphate
AMP PPi
argininosuccinate
ornithine
arginine
fumarate
NH2
urea
Two amino groups enter at different points NH4
ion via aspartate.
C
O
NH2
24
ENTRY OF AMINO GROUPS INTO THE UREA CYCLE.

• Two amino groups enter at different points
• NH4 ion
• via aspartate

Immediate source of both of these is
GLUTAMATE. 1. Glutamate dehydrogenase liberates
ammonium ion. 2. Aspartate formed by
transamination from glutamate.
25
ENTRY OF AMINO GROUPS INTO THE UREA CYCLE.

• Two amino groups enter at different points
• NH4 ion
• via aspartate

Immediate source of both of these is
GLUTAMATE. 1. Glutamate dehydrogenase liberates
ammonium ion.
COO-
COO-



H
C
H3N
C
O
NH4
Glutamate dehydrogenase
CH2
CH2
NADH
NAD
Carbamoyl phosphate
CH2
CH2
COO-
COO-
?-ketoglutarate
glutamate
Urea cycle
26
ENTRY OF AMINO GROUPS INTO THE UREA CYCLE.

• Two amino groups enter at different points
• NH4 ion
• via aspartate

Immediate source of both of these is
GLUTAMATE. 2. Aspartate formed by transamination
from glutamate.
COO-
COO-
COO-
COO-


H
C
O
C
H3N

H3N
C
H
C
O
CH2
CH2
CH2
CH2
CH2
CH2
COO-
COO-
COO-
COO-
aspartate
oxaloacetate
?-ketoglutarate
glutamate
Urea cycle
27
TRANSAMINATION INTERCHANGE OF AMINO GROUPS
BETWEEN GLUTAMATE AND ANOTHER AMINO-ACID.
Catalysed by transaminases (or aminotransferases).
Uses the cofactor pyridoxal phosphate.
Aspartate is formed by transamination from
glutamate, catalysed by the enzyme aspartate
aminotransferase. There are high levels of this
enzyme in liver to allow the transfer of amino
groups into the urea cycle.
COO-
COO-
COO-
COO-


H
C
O
C
H3N

H3N
C
H
C
O
CH2
CH2
CH2
CH2
CH2
CH2
COO-
COO-
COO-
COO-
aspartate
oxaloacetate
?-ketoglutarate
glutamate
Urea cycle
28
oxaloacetate
?-ketoglutarate
aspartate
glutamate
glutamate
citrulline

?keto- glutarate
citrulline
ATP
carbamoyl phosphate
ornithine
AMP PPi
ornithine
argininosuccinate
NH2
urea
C
O
arginine
fumarate
NH2
29
Amino acid X
NH4
OR
oxaloacetate
?-ketoglutarate
aspartate
glutamate
glutamate
citrulline

?keto- glutarate
citrulline
ATP
carbamoyl phosphate
ornithine
AMP PPi
ornithine
argininosuccinate
NH2
urea
C
O
arginine
fumarate
NH2
30
Amino acid X
THE CENTRAL ROLE OF GLUTAMATE IN AMINO ACID
METABOLISM
NH4
OR
oxaloacetate
?-ketoglutarate
aspartate
glutamate
glutamate
citrulline

?keto- glutarate
citrulline
ATP
carbamoyl phosphate
ornithine
AMP PPi
ornithine
argininosuccinate
HIGH LEVELS IN LIVER OF GLUTAMATE DEHYDROGENASE
(IN BOTH CYTOSOL AND MITOS) AND ASPARTATE
AMINOTRANSFERASE ALLOW GLUTAMATE TO TRANSFER BOTH
AMINO GROUPS INTO THE UREA CYCLE
NH2
C
O
arginine
fumarate
NH2
urea
31
Amino acid X
THE CENTRAL ROLE OF GLUTAMATE IN AMINO ACID
METABOLISM
NH4
OR
oxaloacetate
?-ketoglutarate
aspartate
glutamate
glutamate
citrulline

?keto- glutarate
citrulline
ATP
carbamoyl phosphate
ornithine
AMP PPi
ornithine
argininosuccinate

• TRANSFER OF AMINO GROUPS FROM OTHER AA TO
  GLUTAMATE
• TRANSAMINATION
• VARIOUS MECHANISMS SPECIFIC TO DIFFERENT AA

NH2
C
O
arginine
fumarate
NH2
urea
32

• TRANSFER OF AMINO GROUPS FROM OTHER AA TO
  GLUTAMATE
• TRANSAMINATION
• VARIOUS MECHANISMS SPECIFIC TO DIFFERENT AA

COO-
COO-

C
O

H3N
C
H
aspartate aminotransferase
CH2
CH2
CH2
COO-
COO-
oxaloacetate
?-ketoglutarate
aspartate
33

• TRANSFER OF AMINO GROUPS FROM OTHER AA TO
  GLUTAMATE
• TRANSAMINATION
• VARIOUS MECHANISMS SPECIFIC TO DIFFERENT AA

COO-
COO-
COO-
COO-


H
C
H3N
C
O


H3N
C
H
C
O
CH2
CH2
CH2
CH3
CH2
CH2
COO-
COO-
?
glutamate
?-ketoglutarate
?
34

• TRANSFER OF AMINO GROUPS FROM OTHER AA TO
  GLUTAMATE
• TRANSAMINATION
• VARIOUS MECHANISMS SPECIFIC TO DIFFERENT AA

COO-
COO-
COO-
COO-


H
C
H3N
C
O

H3N
C
H
C
O
alanine aminotransferase
CH2
CH2
CH2
CH3
CH2
CH2
COO-
COO-
pyruvate
glutamate
?-ketoglutarate
alanine
Oxidation or gluconeogenesis, depending on
nutritional state
35

• TRANSFER OF AMINO GROUPS FROM OTHER AA TO
  GLUTAMATE
• TRANSAMINATION
• VARIOUS MECHANISMS SPECIFIC TO DIFFERENT AA

COO-
COO-
COO-
COO-


H
C
H3N
C
O


H3N
C
H
C
O
? aminotransferase
CH2
CH2
CH2
CH2
CH2
CH2
CH
CH
COO-
COO-
CH3
CH3
CH3
CH3
glutamate
?-ketoglutarate
?
36

• TRANSFER OF AMINO GROUPS FROM OTHER AA TO
  GLUTAMATE
• TRANSAMINATION
• VARIOUS MECHANISMS SPECIFIC TO DIFFERENT AA

COO-
COO-
COO-
COO-


H
C
H3N
C
O


H3N
C
H
C
O
leucine aminotransferase
CH2
CH2
CH2
CH2
CH2
CH2
CH
CH
COO-
COO-
CH3
CH3
CH3
CH3
glutamate
?-ketoglutarate
leucine
metabolised to form acetyl CoA
37
?

• TRANSFER OF AMINO GROUPS FROM OTHER AA TO
  GLUTAMATE
• TRANSAMINATION
• VARIOUS MECHANISMS SPECIFIC TO DIFFERENT AA

COO-
COO-
COO-
COO-


H
C
H3N
C
O


H3N
C
H
C
O
tyrosine aminotransferase
CH2
CH2
CH2
CH2
?
CH2
CH2
COO-
COO-
glutamate
?-ketoglutarate
OH
tyrosine
38
DIRECT DEAMINATION

Amino acid
Carbon skeleton NH4
?ketoglutarate
Glutamate dehydrogenase
glutamate
39
?
DIRECT DEAMINATION

Amino acid
Carbon skeleton NH4
?ketoglutarate
Glutamate dehydrogenase
glutamate
EXAMPLE
?


NH4
CH2
histidase
CH

histidine
urocanate
40
THE KEY ROLE OF LIVER IN AMINO ACID
METABOLISM Urea cycle enzymes are expressed only
in liver and kidney Liver expresses enzymes
catalysing removal of amino groups from all
common amino acids.
41
THE KEY ROLE OF LIVER IN AMINO ACID
METABOLISM Urea cycle enzymes are expressed only
in liver and kidney Liver expresses enzymes
catalysing removal of amino groups from all
common amino acids. AMINO ACID OXIDATION IN
OTHER TISSUES Much more limited. Muscle can
utilise only a few amino acids, of which the
branched chain group is the most
important. Additional mechanisms are needed to
transport amino groups released in other tissues
to the liver and kidney. These mechanisms involve
alanine and glutamine
42
TRANSFER OF AMINO GROUPS FROM OTHER AA TO
GLUTAMATE TRANSAMINATION
COO-
COO-
COO-
COO-


H
C
H3N
C
O


H3N
C
H
C
O
leucine aminotransferase
CH2
CH2
CH2
CH2
CH2
CH2
CH
CH
COO-
COO-
CH3
CH3
CH3
CH3
glutamate
?-ketoglutarate
leucine
metabolised to form acetyl CoA
43
Amino acid X
THE CENTRAL ROLE OF GLUTAMATE IN AMINO ACID
METABOLISM
NH4
OR
oxaloacetate
?-ketoglutarate
aspartate
glutamate
glutamate
citrulline

?keto- glutarate
citrulline
ATP
carbamoyl phosphate
ornithine
AMP PPi
ornithine
argininosuccinate
HIGH LEVELS IN LIVER OF GLUTAMATE DEHYDROGENASE
(IN BOTH CYTOSOL AND MITOS) AND ASPARTATE
AMINOTRANSFERASE ALLOW GLUTAMATE TO TRANSFER BOTH
AMINO GROUPS INTO THE UREA CYCLE
NH2
C
O
arginine
fumarate
NH2
urea
44
HOW DID THE FAT LADY HELP US TO UNDERSTAND
METABOLISM?
45
ALANINE AS A VEHICLE FOR THE TRANSFER OF AMINO
GROUPS FROM MUSCLE TO LIVER
leucine
?ketoglutarate
Leucine aminotransferase
?ketoisocaproate
MUSCLE
Several steps
glutamate
pyruvate
Acetyl CoA
CO2 H2O
Alanine aminotransferase
?ketoglutarate
alanine
BLOOD
alanine
alanine
?ketoglutarate
Alanine aminotransferase
LIVER
pyruvate
gluconeogenesis
glutamate
glucose
?ketoglutarate
amino group to urea cycle
46
ALANINE AS A VEHICLE FOR THE TRANSFER OF AMINO
GROUPS FROM MUSCLE TO LIVER
leucine
?ketoglutarate
Leucine aminotransferase
?ketoisocaproate
MUSCLE
Several steps
glutamate
pyruvate
Acetyl CoA
CO2 H2O
Alanine aminotransferase
?ketoglutarate
alanine
BLOOD
alanine
alanine
?ketoglutarate
Alanine aminotransferase
LIVER
pyruvate
gluconeogenesis
glutamate
glucose
?ketoglutarate
amino group to urea cycle
47
ALANINE AS A VEHICLE FOR THE TRANSFER OF AMINO
GROUPS FROM MUSCLE TO LIVER
leucine
?ketoglutarate
Leucine aminotransferase
?ketoisocaproate
MUSCLE
Several steps
glutamate
pyruvate
Acetyl CoA
CO2 H2O
Alanine aminotransferase
?ketoglutarate
alanine
BLOOD
glucose
alanine
alanine
?ketoglutarate
Alanine aminotransferase
LIVER
pyruvate
gluconeogenesis
glutamate
glucose
?ketoglutarate
amino group to urea cycle
48
ALANINE AS A VEHICLE FOR THE TRANSFER OF AMINO
GROUPS FROM MUSCLE TO LIVER
leucine
?ketoglutarate
Leucine aminotransferase
?ketoisocaproate
MUSCLE
Several steps
glutamate
pyruvate
Acetyl CoA
CO2 H2O
Alanine aminotransferase
?ketoglutarate
alanine
BLOOD
glucose
alanine
alanine
?ketoglutarate
Alanine aminotransferase
LIVER
pyruvate
gluconeogenesis
glutamate
THIS IS KNOWN AS THE GLUCOSE-ALANINE CYCLE
glucose
?ketoglutarate
amino group to urea cycle
49
GLUTAMINE AS A VEHICLE FOR THE TRANSFER OF AMINO
GROUPS FROM MUSCLE TO LIVER
Amino acid X
EXTRAHEPATIC TISSUE
NH4 carbon skeleton
Glutamate ATP
glutamine synthetase
ADP
glutamine
BLOOD
glutamine
glutamine
glutaminase
LIVER
NH4 glutamate
amino group to urea cycle
50
BURNING THE SKELETONS.
When do we do it?
Amino acid catabolism is most active after a
high protein meal (excess oxidised, or
converted to glucose if meal low in
carbohydrate) during short-term
starvation (tissue protein broken down to make
glucose).
51
FATE OF CARBON SKELETONS
CO2
CO2
Most amino acid-derived carbon ends up in
pyruvate, acetyl CoA or citric acid cycle
intermediates.
52
FATE OF CARBON SKELETONS
Two carbon atoms enter as acetyl-CoA
CO2
Two carbon atoms leave as
CO2
CO2
In mammalian cells there is no net
gluconeogenesis from acetyl CoA
53
FATE OF CARBON SKELETONS
Two carbon atoms enter as acetyl-CoA
CO2
Two carbon atoms leave as
CO2
CO2
Use of carbon skeletons from amino acids as a
glucose supply depends on the point of entry into
central metabolic pathways.
54
FATE OF CARBON SKELETONS
55
(No Transcript)
56
AMINO ACIDS ARE PRECURSORS FOR MANY BIOLOGICALLY
IMPORTANT MOLECULES.
Arginine spermine, spermidine,
putrescine. Polyamines are synthesised when cells
are stimulated to divide.
Aspartate pyrimidines.
Glycine purines, glutathione (protects
against oxidative stress), creatine (creatine
phosphate is a high energy intermediate).
Histidine histamine (inflammatory
response).
Serine phosphatidylserine,
sphingosine (lipid components of membranes).
Phenylalanine adrenalin and noradrenalin
(hormones).
Tryptophan nicotinic acid (NAD),
serotonin (neurotransmitter).
Valine pantothenic acid (CoA)
57
A very simple principle- GROWTH SYNTHESIS -
BREAKDOWN
This applies whether one is thinking about
individual proteins single cells whole tissues
the entire body!
58
PROTEIN DEGRADATION Total protein mass can be
regulated by both synthesis and breakdown.
DNA
transcription, processing
Degradation
mRNA
translation
Degradation
Protein
59
PROTEIN DEGRADATION Total protein mass can be
regulated by both synthesis and breakdown.
physiological regulation
DNA
transcription, processing
Degradation
mRNA
translation
Degradation
Protein
60
100 -
50 -
0 -
Time (h)
5
10
15
25
20
30
chase
pulse
The half-life of a protein is the time for half
of it to be degraded in the absence of further
synthesis
61
100 -
50 -
t1/2
0 -
Time (h)
5
10
15
25
20
30
chase
pulse
The half-life of a protein is the time for half
of it to be degraded in the absence of further
synthesis. For the protein shown here the
half-life is about 6 hours.
62
HALF-LIVES OF SOME LIVER ENZYMES. Short-lived
enzymes Half Life (h) Ornithine
decarboxylase 0.2RNA polymerase
I 1.3Tyrosine aminotransferase 2.0Serine
dehydratase 4.0PEP carboxykinase 5.0 Long-li
ved (stable) enzymesAldolase 118GAPDH 13
0Cytochrome b 130LDH 130Cytochrome
c 150
63
PROTEIN BREAKDOWN (PROTEOLYSIS).
Kinetically first order. Can be expressed as
half-life.
Multiple mechanisms
Half lives of proteins differ tremendously.
Housekeeping proteins often have longer
half-lives. Regulatory proteins often have short
half-lives
Bulk protein breakdown can release amino-acids
from tissue protein into the metabolic pool
for oxidation or gluconeogenesis.
Protein degradation must be compartmentalised/
specific.
64
WHERE TO READ MORE ABOUT PROTEIN DEGRADATION Many
text-books cover this very poorly, if at all. You
can find further information in the following,
but NOTE THAT FOR REFERENCES IN RESEARCH JOURNALS
YOU SHOULD EXTRACT ONLY THE MATERIAL YOU WANT!
These papers will contain far more detail than
you need. Genes VIII, by B Lewin, Chapter 8,
sections 8.31 and 8.32 Voet, Voet Pratt (1999
edn), Chapter 20, section 1. Alberts et al (2002)
pp 358-362 Fig 17-20, p996. Pickart Cohen
(2004) Nature Reviews in Molecular Cell Biology
5, 177-187. (This is not in the Library come to
JMS 2C6 if you wish to borrow a copy). Kirschner
(1999) Trends in Cell Biology 9, M42-45. Cuervo
(2004) Trends in Cell Biology 14, 70-77.
65
glucose
POOL OF FREE AMINO ACIDS
CELLULARPROTEINS
extracellular amino acids
CO2 H2O
66
A very simple principle- GROWTH SYNTHESIS -
BREAKDOWN
This applies whether one is thinking about
individual proteins single cells whole tissues
the entire body!
67
Protein and amino-acid metabolism.
Amino-acid oxidation.
Fate of amino nitrogen
Metabolism of carbon skeletons
Protein breakdown
Lysosomal pathway
Proteasome mediated Caspases and apoptosis
Protein turnover under clinical conditions
68
THE LYSOSOMAL PATHWAY.
Involved in
Regulation of the flow of AA from protein for
metabolic use.
Internalisation of receptor molecules.
Scavenging by specialised cells.
69
LYSOSOMES.
Similar in size to mitochondria.
Contain a battery of degradative enzymes,
including cathepsins. These enzymes have a low pH
optimum.
Interior at pH 5 due to proton pump.
70
LYSOSOMES.
Similar in size to mitochondria.
Contain a battery of degradative enzymes,
including cathepsins. These enzymes have a low pH
optimum.
Interior at pH 5 due to proton pump.
Cytosol pH 7.2
71
LYSOSOMES.
Similar in size to mitochondria.
Contain a battery of degradative enzymes,
including cathepsins. These enzymes have a low pH
optimum.
Interior at pH 5 due to proton pump.Formed by
budding from Golgi.
Fuse with vacuoles containing target material.
Vacuole formation in liver is regulated by
glucagon and insulin.
72
AUTOPHAGY IN MAMMALIAN CELLS
Macroautophagy material engulfed by
non-lysosomal membrane to form autophagosome and
delivered to lysosome. Often non-selective.
Stimulated by glucagon in short-term starvation
and suppressed by insulin in the fed state.
Cuervo (2004) Trends in Cell Biology 14, 70-77
73
AUTOPHAGY IN MAMMALIAN CELLS
Microautophagy material is engulfed by lysosomal
membrane. Constitutive, i.e. does not require
stress to be up-regulated. Often non-selective.
Cuervo (2004) Trends in Cell Biology 14, 70-77
74
AUTOPHAGY IN MAMMALIAN CELLS
Chaperone-mediated autophagy Soluble cytoplasmic
proteins are selectively bound to chaperones that
interact with receptors on the lysosomal membrane
to mediate uptake.
Cuervo (2004) Trends in Cell Biology 14, 70-77
75
PROTEIN DEGRADATION BY PROTEASOMES Proteasomes
are complex structures of polypeptide chains that
include catalytic sites with protease
activity. The catalytic sites are sequestered
from cell contents on the inside of the
structure. The catalytic sites are only
accessible via a narrow channel.
From Pickart Cohen (2004) see Reference list.
76
PROTEIN DEGRADATION BY PROTEASOMES Proteasomes
are complex structures of polypeptide chains that
include catalytic sites with protease
activity. The catalytic sites are sequestered
from cell contents on the inside of the
structure. The catalytic sites are only
accessible via a narrow channel. Cellular
proteins must pass through the channel to be
degraded.
77
PROTEIN DEGRADATION BY PROTEASOMES Proteasomes
are complex structures of polypeptide chains that
include catalytic sites with protease
activity. The catalytic sites are sequestered
from cell contents on the inside of the
structure. The catalytic sites are only
accessible via a narrow channel. Cellular
proteins must pass through the channel to be
degraded. Proteins must be unfolded to pass
through the channel. Therefore proteins in their
correctly folded state are not vulnerable to
accidental attack.
78
PROTEIN DEGRADATION BY PROTEASOMES Proteasomes
are complex structures of polypeptide chains that
include catalytic sites with protease
activity. The catalytic sites are sequestered
from cell contents on the inside of the
structure. The catalytic sites are only
accessible via a narrow channel. Cellular
proteins must pass through the channel to be
degraded. Proteins must be unfolded to pass
through the channel. Therefore proteins in their
correctly folded state are not vulnerable to
accidental attack. The protease activity is not
specific. Specificity lies in the selection of
protein molecules for uptake into the proteasome.
79
MORE PICTURES OF PROTEASOMES
From Genes VIII by B. Lewin. Chapter 8, sections
8.31 and 8.32. Useful reference for this lecture.
Library has book in Short Loan section. It is
very useful for the final year course
Biochemistry of Gene Expression.
80
MORE PICTURES OF PROTEASOMES
From Kirschner (1999) see Ref list
81
The complete proteasome consists of two
parts The structure shown here is the 20S
proteasome (700 kDa) and contains the protease
activity. It consists of two types of protein
subunit, ?? and ?. The ? subunits contain the
protease activity
82
The complete proteasome consists of two
parts The structure shown here is the 20S
proteasome (700 kDa) and contains the protease
activity. It consists of two types of protein
subunit, ?? and ?. The ? subunits contain the
protease activity
From Genes VIII
83
To enter the proteasome a protein substrate must
be unfolded to allow it to pass through the
narrow channel. Unfolding is carried out by an
additional protein complex (19S) which contains
ATP-dependent unfoldase activity. This 19S
complex associates with the 20S cylinder to form
the 26S proteasome.
Pickart Cohen 2004
84
To enter the proteasome a protein substrate must
be unfolded to allow it to pass through the
narrow channel. Unfolding is carried out by an
additional protein complex (19S) which contains
ATP-dependent unfoldase activity. This 19S
complex associates with the 20S cylinder to form
the 26S proteasome.
How are proteins selected for the chop and
directed to the 19S complex for unfolding?
85
THE ROLE OF UBIQUITIN IN PROTEIN
DEGRADATION Specific proteins can be targeted for
degradation by a system that covalently attaches
a chain of ubiquitin molecules.
Ubiquitin is a small protein. It first binds to
an ubiquitin activating enzyme (E1). ATP
hydrolysis is required.
From Genes VIII
86
THE ROLE OF UBIQUITIN IN PROTEIN
DEGRADATION Specific proteins can be targeted for
degradation by a system that covalently attaches
a chain of ubiquitin molecules.
Ubiquitin is a small protein. It first binds to
an ubiquitin activating enzyme (E1). ATP
hydrolysis is required. Ubiquitin is then
transferred to a second enzyme, the
ubiquitin-conjugating enzyme E2.
87
THE ROLE OF UBIQUITIN IN PROTEIN
DEGRADATION Specific proteins can be targeted for
degradation by a system that covalently attaches
a chain of ubiquitin molecules.
Ubiquitin is a small protein. It first binds to
an ubiquitin activating enzyme (E1). ATP
hydrolysis is required. Ubiquitin is then
transferred to a second enzyme, the
ubiquitin-conjugating enzyme E2. The third
enzyme, E3 ubiquitin ligase, transfers the
ubiquitin to covalent linkage with a lysine
side-chain in the target protein.
From Genes VIII
88
UBIQUITINATION AND SELECTION E2 and E3 are not
single enzymes but occur in multiple
varieties. Both E2 and E3 enzymes can play a role
in selecting the protein substrate. In some cases
E3 selects the protein by binding it before the
process of ubiquitination.
89
The attachment of a single ubiquitin to a protein
is not enough to target it to the proteasome for
degradation. Multiple ubiquitination occurs by
addition of further ubiquitin chains attaching to
Lys46 of the previous ubiquitin.
From Genes VIII
90
THE OVERALL PROCESS LINKING UBIQUITINATION WITH
DESTRUCTION
Note that the ubiquitin is not degraded, but
released before entry of the target protein into
the barrel of the proteasome.
Pickart Cohen 2004
91
PHYSIOLOGICAL SIGNIFICANCE OF THE
UBIQUITIN-PROTEASOME SYSTEM This system is
implicated in turning on destruction
of Transcription factors and regulators c-jun,
NF?B, I ?B, yeast GCN4. Regulators of cell growth
and proliferation (cell cycle) several cyclins,
cyclin-dependent kinase inhibitors, c-mos, p53,
ornithine decarboxylase. Mis-folded or
mis-targeted proteins. Some proteins normally
present in complexes, when separated from their
partner(s).
92
FEATURES OF PROTEINS RECOGNISED BY THE
UBIQUITIN-PROTEASOME SYSTEM Motifs in the
primary sequence Regulated changes in protein
conformation covalent modification (e.g.
phosphorylation or dephosphorylation) exposure of
a motif by dissociation of an interacting
partner. Association with an ancillary protein
or chaperone.
93
APOPTOSIS or PROGRAMMED CELL DEATH Ref Simon
Morleys lectures in Introduction to Cell
Regulation
Signals from membrane receptors
Withdrawal of growth factors
DNA damage
Toxic chemicals
pro-caspases (inactive)
Active caspases
Cleavage of specific protein substrates
downstream effects leading to cell death
94
CASPASES Cysteine proteases (have a cysteine at
their active site). Cleave substrates next to an
aspartate residue. When apoptosis is activated,
a series of caspases activate each other in a
cascade.
95
PROTEIN METABOLISM NUTRITION AND DISEASE
A very simple principle- GROWTH SYNTHESIS -
BREAKDOWN
This applies whether one is thinking about
individual proteins single cells whole tissues
the entire body!
96
GAIN OR LOSS OF TISSUE OR CELL PROTEIN Free
amino acid pools in cells are very small compared
to those in protein
97
glucose
POOL OF FREE AMINO ACIDS
CELLULARPROTEINS
extracellular amino acids
CO2 H2O
98
Both protein synthesis and protein degradation
are physiologically regulated
glucose
POOL OF FREE AMINO ACIDS
CELLULARPROTEINS
extracellular amino acids
CO2 H2O
99
GAIN OR LOSS OF TISSUE OR CELL PROTEIN Free
amino acid pools in cells are very small compared
to those in protein. Protein accumulates when
synthesis exceeds breakdown. Protein mass falls
when breakdown exceeds synthesis. Muscle wasting
results when degradation exceeds synthesis for a
prolonged period.
100
PHYSIOLOGICAL STATES ASSOCIATED WITH OVERALL
POSITIVE OR NEGATIVE NITROGEN BALANCE
POSITIVE NITROGEN BALANCE NEGATIVE
NITROGEN BALANCE Response to protein intake
in Starvation the diet Childhood
growth Ageing Exercise and tissue
hypertrophy Wasting/atrophy (effects of
infection, injury, surgical trauma,
the late stages of cancer, other
physiological stresses) Response to
anabolic hormones Response to catabolic
hormones or lack of anabolic ones (e.g.
in diabetes)
101
(No Transcript)
102
GAIN OR LOSS OF TISSUE OR CELL PROTEIN Free
amino acid pools in cells are very small compared
to those in protein. Protein accumulates when
synthesis exceeds breakdown. Protein mass falls
when breakdown exceeds synthesis. Muscle wasting
results when degradation exceeds synthesis for a
prolonged period. Both protein synthesis and
protein breakdown are very fast compared to
changes in protein mass. The phenomenon of
apparently purposeless synthesis and breakdown is
called protein turnover. Protein turnover
consumes large amounts of energy.
103
PROTEIN METABOLISM AFTER A MEAL
NORMAL METABOLISM (high insulin, low glucagon)
protein
oxidation
In a normal individual most amino acids from a
protein meal will be used for protein synthesis
in peripheral tissues such as skeletal muscle.
Excess amino acids can also be used as
sources of energy, and the nitrogen derived from
their oxidation will be incorporated into urea in
the liver and excreted.
amino acids
MUSCLE
amino acids
protein
amino acids
urea
carbon skeletons
protein
CO2
glucose
fatty acids
LIVER
104
PROTEIN METABOLISM DURING STARVATION In normal
individual insulin low, glucagon high
alanine
pyruvate
protein
oxidation
-NH2
amino acids
During short-term starvation there will be a net
flow of amino acids from muscle to the liver,
with increased production of glucose and
urea. However, during long-term starvation,
tissue protein is spared because ketone bodies
replace glucose as a major energy fuel for the
brain.
MUSCLE
alanine
amino acids
amino acids
urea
carbon skeletons
protein
CO2
glucose
fatty acids
LIVER
105
REMINDER!
106
ALANINE AS A VEHICLE FOR THE TRANSFER OF AMINO
GROUPS FROM MUSCLE TO LIVER
leucine
?ketoglutarate
Leucine aminotransferase
?ketoisocaproate
MUSCLE
Several steps
glutamate
pyruvate
Acetyl CoA
CO2 H2O
Alanine aminotransferase
?ketoglutarate
alanine
BLOOD
glucose
alanine
alanine
?ketoglutarate
Alanine aminotransferase
LIVER
pyruvate
gluconeogenesis
glutamate
THIS IS KNOWN AS THE GLUCOSE-ALANINE CYCLE
glucose
?ketoglutarate
amino group to urea cycle
107
AUTOPHAGY IN MAMMALIAN CELLS
Macroautophagy material engulfed by
non-lysosomal membrane to form autophagosome and
delivered to lysosome. Often non-selective.
Stimulated by glucagon in short-term starvation
and suppressed by insulin in the fed state.
Cuervo (2004) Trends in Cell Biology 14, 70-77
108
SEVERE UNTREATED DIABETES
alanine
pyruvate
Here the negative nitrogen balance associated
with short term starvation persists even though
the subject is fed, leading to muscle
wasting. Negative nitrogen balance due to
decreased protein synthesis and/or increased
protein breakdown is also seen in conditions of
chronic infections, late stage cancer or trauma,
including that following surgery or burns
injury. Some of these effects are mediated by
cytokines.
protein
oxidation
-NH2
amino acids
MUSCLE
alanine
PROTEIN
amino acids
amino acids
urea
carbon skeletons
protein
CO2
glucose
fatty acids
LIVER