39 - Protein Turnover Flashcards
Major Pathways of
PROTEIN TURNOVER
- *Lyosome**
- *Vacuolar** –> Autophagy
- *Proteasome**
- *Cytoplasmic** –> Ubiquitin-Proteasome System
minor pathway:
capsases = cysteine proteases
involved in apoptosis
- *Bortezomib = Velcade**
- *Proteasome Inhibitor’s**
BINDING / MoA
Tetrahedral Adduct
occupy peptide binding pockets –> Nu- N-terminal throenine
Good Selectivity
- *REVERSIBLE BINDING**
- *Nucleophilic attack** –> binds to the B5 of PROTEOLYTIC CORE
- *BORON**
- *readily accepts electrons** & can interconvert w/ ease
Proteasome Function
3 specificities:
Chemotrypsin -Like
Trypsin -like
Caspase - like
Substrate docks @ LID/CAP where
Ub-Chains are recognized
proteasome undergoes
Major CONFORMATIONAL CHANGE
which also removes the Ub-units
THREONINE PROTEASE
activates in the proteolytic core
Degradation / breakdown @ the CORE
LYSOSOMAL STORAGE DISEASES
LSD
- *Substrate accumalation** –> chronic/progressive clinical syndromes
- *RARE**, recessively inherited
MAINLY DUE TO:
- *DEFICIENCY in_ _LYSOSOMAL HYDRALASES**
- but also some non-enzymatic proteins*
Most LSD’s are associated with severe neurdegeneration in:
childhood / mental decline / cognitive problems
Short-Lived Proteins
broken down by Proteasomes (Cytoplasmic) via Ub
Regulatory Proteins
Cell-Cycle Regulators
Damaged/Misfolded Proteins
Minutes –> Hours
Ixazomib
Ninlaro
Oral Proteasome Inhibitor
prodrug
_3 Methods of RESISTANCE
to Proteasome Inhibitors_
KNOW THIS
“M-O-A”
MUTATION
B5- results in less affinity
OVEREXPRESSION
B5–> compensate by adding more binding sites
Activation of AUTOPHAGY
Histone deacytylase 6 -> promote cell survival
Ubiquination Steps
- E2 + E3 BIND to the DENATURED PROTEIN
-
E1 grabs / activates / binds Ub
- –> brings it to the E2
-
E2 conjugates Ub COVALENTLY
- –> brings it to E3
-
•E3 ligase
- Ub –> Denatured protein
-
REPEATED
- The NEXT/2nd Ub –> is bound to the FIRST Ub
- And so forth….
- Creating the Ubiquitin code to be read by
- the UBD proteins
- Creating the Ubiquitin code to be read by
- And so forth….
- The NEXT/2nd Ub –> is bound to the FIRST Ub
What type of Therapeutic Strategy for LSD?
Help Proteins fold in their functioning conformation
many mutations –> abnormal folding of lysosomal enzymes
CHAPERONE THERAPY
Migalstat = Galafold
indicated for
Fabry Disease
Pharmacologic Chaperone Therapy
for LSDs
ADV / DISADV
Migalstat = Galafold –> Fabry Disease
ORALLY / no immune rxns
BBB crossing
DISADV:
Drugs are mutation-specific –> some mutations are unresponsive
PC bind to catalytic sites –> dosing is difficult
What DECIPHERS the Ubiquitin Code
&
Links the substrates to downstream processes?
like
proteasomal degradation / lysosomal degradation
or
Non-proteolytic processes
UBD PROTEINS
Ub-Binding Domain Proteins
Glycocalyx
Polysaccharide-based coating
on the INNER SIDE of the
Lysosomal membrane
that PROTECTS it from self-digestion by the
lyosomal enzymes (acid hydrolases)
Chloroquine
Indication // Target / MoA
Cancer Treatment by targetting AUTOPHAGY
LYSOSOMOTROPIC DRUG
compound that is able to penetrate Lysosome / target them
chloroquine = weak base –> protonated & trapped in lysosome
and
DECREASES LYSOSOMAL pH -> 4.5
which then inhibits autophagic degradation
–> need 5.0 for optimal fxn
Ubuitin Proteasome System
Stages
“Tag & Chew it”
- *TAG first**
- *UBIQUINATION** –> targets proteins for degradation
- *CHEW second**
- *PROTEASOME** –> degrades
What are UBD Proteins?
Ubiquitin Binding Domain Proteins:
various effector proteins that
Recognize / Read the UBIQUITIN CODE
&
link the Ub-tagged protein to different processes such as:
Proteolysis = Proteosome / autophagy
or
non-proteolytic processes
Bortezomib
Brand Name / Target / Indication
Velcade
Throenine Proteasome inhibitor
for
Cancer
Multiple Myeloma // Mantle Cell Lymphome
3 APPROVED PROTEASOME INHIBITORS
know this
“BCI-VKN”
Bortezomib - Velcade
Carfilzomib - Kyprolis
Ixazomib - Ninlaro
What type of Therapeutic Strategy for LSD?
Good strategy because LSD’s are
Well-Characterized / Single-Gene Disorders
Still Investigational
GENE THERAPY
still investigational
Carfilzomib
Kyprolis
Proteosome Inhibitor
Long-Lived Proteins
Broken down by LYSOSOMES(vacuolar) via Autophagy
HEMOGLOBIN
>100 days
ALBUMIN
>20 days
Structural Proteins = Collagen
> Years
What TYPE of Autophagy?
Autophagy INDUCED by STARVATION
Is NON-Selective
BULK AUTOPHAGY
Bulk starvation
What is the UBIQUITIN CODE?
& what does it recognize?
After the substrate is UBIQUINATED
there are different types of
UB LInkages & Chain Lengths
that are read by UBD Proteins
( Ub-Binding Domain )
specificity for distinc linkage/lengths
What is the Ub-Proteasome System
essential for?
Regulatory Mechanisms:
Cell-Cycle Progression
Signal Transduction
APOPTOSIS / Oncogenesis (cancer)
Ubiquitin
Function / Mechanism
TAGS protein for protesomal degradation
terminal Glycine + multiple Lysines
“ACL”
- *E1**
- *ACTIVATION** of Ub w/ ATP (adenylation
- *E2**
- *CONJUGATION** –> transfer
- *E3**
- *LIGASE** = brings teh specificity
Physiological roles of
Autophagy
Maintainance of cellular homeostasis
Body’s response to nutritional stress or starvation
Cellular Remodeling
including adaptation to changes in availble nutrients
Containing Intracellular microbial infections
TB
Other Investigational Targets
in Ub-Proteasome System
Target E3 Ub Enzyme
instead of the whole proteasome
since it is the most specific –> less toxic
Cross-talk
between autophagy & proteasome
Also for
- *neurogenerative diseases &**
- *Immune-mediated disorders**
- *Lyosome**
- *Structure / Components**
Vacuolar System of Protein Turnover
HYDROLASES
for the degradation of specific substrates:
Proteins / Carbs / NA’s / Lipids
GLYCOLYX
a polysacchride-based coating on the INNER side of the lyosomal membrane that protects the organelle from self-degradation
PROTON PUMP
which maintains a ACIDIC LUMEN, pH 5.0
SRT = Substrate Reduction Therapy
for LSDs
ADV / DISADV
Eliglustat = Cerdelga for Gaugcher disease
ORALLY!
does NOT generate immune reactions
may cross the BBB
Disadvantages:
Currently only available for 2 LSDs
FABRY DISEASE
how do we treat?
LSD cause by mutations in GLA gene
- *Enzyme Replacement Therapy**
- *Agalsidase = Fabrazyme**
CHAPERONE THERAPY
helps enzyme properly fold
Migalstat = Galafold
Autophagy CYCLE
-
Isolation Membrane
- Can be a Piece off of the ER (induction)
- –>ENGULFS CARGO = mitocondria etc
-
Autophagosome
- Formed once the isolation membrane is ENCLOSED
-
AutophagoLYSOSOME
-
LYSOSOME + Autophagosome
- Lysosome contains HYDROLASES
- Degrades material & recycles
-
LYSOSOME + Autophagosome
Which Enzyme in the Ub-Proteasome System
brings SPECIFICITY?
E3
they have different specificities
recognize different substrate AA sequences“motifs” @ N/C terminus
Proteasome
Cytoplasmic pathway of Protein Turnover
ATP-Dependent proteolytic machine
Degrades proteins with HIGH SPECIFICITY from
tagging system = UBIQUITIN
CROSSTALK
Between Autophagy / Proteasome
Another topic of research:
After UBD Proteins recognize the UB code
they TRANSFER the tagged proteins to EITHER:
Proteasome or Autophagosome
impairment of one can lead /direct to the other
Autophagy & Neurodegeneration
ALZ / Parkingson’s / Huntington’s disease
pathologies are possibly implicated by
Toxic Protein Aggregates
which can be degraded via AUTOPHAGY
Autophagy Defects –> neurodegeneration
so Autophagy can play a PROTECTIVE role against these diseases
Enzyme Replacement Therapy
(used for LSD’s)
Advantages / Disadvantages
GAUCHER DISEASE
Imiglucerase / Cerezyme
ERT has been shown to be effective in
Reducing Storage Materials
disadvantages:
IV administration // Immune RXN
Difficulty Targetting to tissues that are not reached through circulation
bone / cartilage / brain
Proteasome Inhibitor
(threonine proteasome)
Drug name / Indication
Bortezomib = VELCADE
Indicated for CANCER:
Multiple Myeloma & Mantle Cell Lymphoma
LYSOSOME
FUNCTIONS
Degrade & Recycle BOTH:
Intracellular Material
obsolete components of the cell ITSELF
via Autophagy
EXTRAcellular Material
degrade material that is taken from OUTSIDE of the cell
via Endocytosis
What TYPE of Autophagy?
Autophagy that uses UBIQUITIN to tag the protein
todegrade:
Protein Aggregates // Dysfunctional Organells // Invading Pathogens
SELECTIVE AUTOPHAGY
Uses UB –> tag protein to degrade:
Protein Aggregates // Dysfunctional Organells // Invading Pathogens
Autophagy
“Self-Eating”
A function of Vacuolar Protein Turnover
LYSOSOMAL degradation / recycling
of the
cell’s OWN materials/molecules
What DRUG targets AUTOPHAGY
for CANCER TREATMENT
clinical trials still
CHLOROQUINE & Hydroxychloroquine
as Lysosome Inhibitors
Autophagy Inhibition can be BOTH Bad & Good
since it is used to:
keep cancer cells alive
as well as
immune response to kill cancer cells
What type of Therapeutic Strategy for LSD?
- *Compensate** for metabolic defects through
- *infusions of RECOMBINANT ENZYMES**
ERT** = **Enzyme Replacement Therapy
USED FOR BOTH DISEASES
- *Gaucher Disease**
- *Cerezyme** = Imiglucerase
- *Fabry Disease**
- *Agalsidase = Fabrazyme**
Therpeutic Strategies for LSD’s
Lysosomal Storage Diseases
Goal is to restore balnce between substrate influx & degradation
ORPHAN DRUGS –> developed to treat a rare condition
Do NOT CURE,
but just stabilize organ fxn & slow progression of disorder
ERT = Enzyme replacement Therapy
SRT = Substrate Replacement Therapy
Chaperone Therapy
Gene Therapy
26S Proteasome
Structure
2 Halves + 7 proteolytic sites
that form a Barrel / Cylinder
2 Outer Regulatory Caps
2 Inner Proteolytic Cores
Protein Turnover / Recycling
BALANCE
between Protein Synthesis & Degradation
crucial for cellular activity
Most protein are synthesized from AA’s
are from degradation of pre-existing proteins
300-500g proteins / day vs 60-80g digested
Implications of AUTOPHAGY DYSREGULATION
CANCER
Good & Bad Autophagy –> survival vs immune response
NEURODEGENERATION
Autophagy degrades Toxic-protein-aggregates which are implicated in
ALZ / Parkinson’s / Huntington’s disease
Some MYOPATHIES
primary symptom is muscle weakness do to dysfunction in muscle fiber
–> cardiac diseases
Gaucher Disease
how do we treat?
Lysosomal Storage Disease = LSD
Enzyme Replacement Therapy
for type 1 –> replaces the enzyme
Eliglustat = Cerdelga
Substrate Replacement Therapy
was later approved –> inhibits the enzyme
Imiglucerase = Cerezyme
What type of Therapeutic Strategy for LSD?
Slows the production of storage material
instead of degrading it
reduce the substrate from getting into the lysosome first
Biosynthesis Inhibitor
SUBSTRATE REDUCTION THERAPY
for LSD
Eliglustat = Cerdelga
inhibits ceramide GT
for Gaucher Disease
Threonine protease = COVALENT CATALYSIS
- The goal is to CLEAVE / HYDROLYZE an amide bond
-
Difficult to do because of delocalization
- Done through COVALENT catalysis only
-
Difficult to do because of delocalization
- Forms a Tetrahedral intermediate = forms an ester (easier to cleave than an amide)
-
•–> Acyl –Enzyme intermediate
-
–> HYDROLYSIS by the acyl enzyme
- Uses WATER
-
–> HYDROLYSIS by the acyl enzyme
-
•–> Acyl –Enzyme intermediate
- *26S Proteosome**
- *Facts**
COVALENT CATALYSIS
- *Proteolytic Sites** can CLEAVE almost ANY sequence
- but they are*
- *VERY SPECIFIC** –> will Only target Ub-TAGGED Proteins
- does not harm untagged proteins*
Proteolytic activity is only at the CENTER and access is
Sterically Controlled
