Lecture VIII Flashcards
What kind of diseases may be affected by protein trafficking?
kidney diseases
What is Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD)?
it is an autosomal dominant disease that has a phenotype characterized by tubular damage and interstitial fluids
Why is ADTKD undetectable in terms of lab findings?
urine analysis is not very informative (no protein urea, which is a sign that glomerulus is not filtering correctly)
kidneys have a normal or slightly smaller size
What are some functional defects that can be seen in patients with ADTKD?
patient has urinary concentration defects, which leads to polyurea and polydipsia
kidneys lose function
What are the therapies available for ADTKD patients?
none, the only option is to undergo dialysis or have a kidney transplant
What kind of disease is ADTKD?
genetically heterogenous disease
What are the 2 most frequent mutations associated with ADTKD?
UMOD (uromodulin) and MUC1 (Mucin 1)
What is the most common human kidney disease in the world?
ADTKD-UMOD
What is a TF related to ADTKD?
HNF1β
What are some ultra rare ADTKD diseases?
ADTKD-REN and ADTKD-SEC61A1
What can this flowchart be useful for?
help nephrologists correctly diagnose patients with ADTKD
What is another name for Uromodulin?
Tamm-Horsfall protein
What is uromodulin?
protein with immunomodulatory function that is abundant in urine
it has a high MW polymer, which forms large filaments once it is secreted in urine
Where is uromodulin expressed?
ONLY in the kidneys
ONLY in the Thick Ascending Limb (TAL) of Henle’s loop: it is most likely localized on the membrane of TAL cels
Describe uromodulin:
large amount of cysteins involved in SS-bond formation
EGF is involved in protein-protein interactions
D8C is a domain of non-function
ZP domain is involved in the formation of extracellular polymers
internal and external hydrophobic patches are involved in keeping the ZP domain in closed conformation
What happens after uromodulin reaches the membrane?
hepsin cleaves at the end of ZP and seperates the interactions of the 2 domainsand changes the conformation of uromodulin → allows other uromodulins to interact and form filaments
What does uromodulin do in the kidney?
regulates ion transport in the TAL segment
Why is the loop of Henle important?
it is where important molcules are reabsorbed from urine (ex: 20% of Na+)
What happens to the urine as it progresses through the loop of Henle?
it is progressively diluted as it progresses via the tubules
What does the TAL segment do?
implements the mechanism where we have the counter current gradient in kidneys where most of the water gets reabsorbed thanks to the osmotic gradient
If one of the 1st symptoms of ADTKD-UMOD is urine concentration defects, what can be implied?
uromodulin defects cause damage in TAL causing urinalysis
What makes uromoduline a great defense factor?
it is a great antibacterial agent as it is kept in a high mannose conformation (not modified in golgi) and it keeps the original conformation that the glycans acquire in the ER
it is able to sequestrate bacteria from the urinary tract
What can be used instead of antibiotics to treat urinary tract infections?
uromoduline
WHat else can uromodulin function as?
immunoregulatory agent
acts as a DAMP (damage-associated molecular pattern), so when the tubule is damaged, the protein extravates into the interstitium and activated inflammatory cells
*acts as a guardian of the integrity of the tubules once it is secreted within the nephron
What kind of mutations occur in ADTKD patients?
4% are in-frame deletions
rest are missense mutations
What is the mechanism of action of ADTKD?
mutation causing a gain of function probably due to the misfolding of uromodulin because if a cysteine is mutated, we lose a SS-bond
Analyze the biopsies: what is formed in ADTKD patients?
there is the formation of huge intracellular aggregates, so this suggests there is something wrong in the trafficking of the protein on its way to the plasma membrane
What is the primary effect of uromodulin mutations?
UMOD (red) and calnexin (green) colocalize and leads to the protein accumulating in the ER (yellow) because the mutations are interfering with protein folding
What is the function of the deglycosylating enzyme, EndoH?
it digests glycosylated enzymes only when they are in high mannose conformation (ex: uromodulin)
What is the primary effect of the mutated UMOD?
ER retention
What is the unfolded protein response (UPR)?
pathway triggered by the cell when there is an accumulation of misfolded proteins in the ER
*activates the expression of chaperones, decreases protein translation, and increases protein degradation associated with the ER
What does it mean if a pathway is an acute adaptive mechanism? What is an example of an acute adaptive mechanism?
cell switches it on and off when needed
the unfolded protein response is an example
What happens if the trigger for the acute adaptive mechanism is a mutated protein?
the cell cannot switch it off because the pathway is constantly being activated, which can lead to apoptosis or active inflammation
What activates the unfolded protein response?
the activation of the 3 receptors:
ATF6
PERK
IRE1
What happens when we have an increased presence of unfolded proteins in the ER?
protein sequesters BiP (main ER chaperone)
BiP sequestration frees 3 sensors that now signal downstream
What are the effects due to the downstream signaling caused by the freeing of the 3 sensors?
the adaptive pathway becomes maladaptive and is chronically induced in cells
What is IRE1?
RNAse whose main job is to cleave and splice the transcriptome of XBP1, which becomes in-frame when spliced and can translate the XBP1 protein
What is the XBP1 protein?
TF which regulates the synthesis of chaperones, lipid synthesis, and ERAD proteins
What is PERK?
activates IF2 and the main function is to quench translation, but the genes of interest are still actived by ATF4
translation of XBP1 is active and so are its functions
IF2 can also be activated by other kinases that are part of different pathways including stress response
What is ATF6?
TF which is cleaved in the golgi
it migrates to the nucleus and activates target genes mostly involved in chaperone synthesis and expansion of the ER membrane to accommodate the presence of misfolded proteins
What is the TF, Atf3, an indication of?
ER stress
What 2 things were increased in ADTKD patients’ biopsies?
BiP
CRELD2: upregulated downstream on the unfolded protein response (can be used as a biomarker to study the disease progression of patients)
Give the general pathway of uromodulin:
misfolded protein begins to accumulate in the ER
ER stress and UPR are activated
triggers the release of chemokines and activates inflammation (activates resident macrophages and then recruits other macrophages)
deposition of fibrotic tissue begins
What is renin>
aspartyl protease that is synthesized in the kidney and other organs
What does renin do?
cleaves angiotensinogen to form angiotensin I
What does the formation of angiotensin I cause>
cascade of renin angiotensin, which regulates blood vessels (vasoconstriction and blood pressure)
What part of renin is responsible for the protein’s activities?
mature part
Where is mature renin synthesized?
only in a very specific type of cell that is located near the macula densa
What can a mutation of renin be associated to?
recessive type of disease called Renal Tubular Dysgenesis
What does Renal Tubular Dysgenesis effect?
all of the genes associated with the renin angiotensin system
*all mutations are a loss of function so if 2 copies are carried of the mutate renin, a very severe disease associated with premature death is developed
What happens with a dominant mutation of renin?
ADTKD
How many renin mutations cause a gain of function?
20
What kind of mutations are in renin?
missense mutations
Where are the missense mutation in renin located?
leader peptide
pro sequence
mature part
How can a cell know if a protein is a cytosolic protein or a secretory protein?
N-terminus starts to exit the ribosomes and once it enters the cytosol, a protein complex with an RNA component called SRP (signal recognition protein) recognizes if a protein must go in the secretory pathway
SRP binds to the signal that exits the ribosomes and a bond is formed because of the recognition on the leader peptide
SRP bound to receptor is associated with a translocon, which allows the protein to enter into the ER
SRP is released and the leader peptide is inserted into the membrane and the rest of the protein is synthesized and enters the ER
What happens if a protein has a defective leader peptide?
it does not bind to SRP and the protein is synthesized in the cytosol
What happens when there is a mutant renin (L16R and delL16)?
there is a mutation in the leader peptide which leads to a defective secretion of the protein that is accumulated in the cytosol
What does a mutation in the leader peptide or in the mature part of renin lead to?
induction of the UPR
What kind of disease is associated to a mutation in the leader protein of mutation in mature renin?
autosomal dominant tubulointerstitial kidney disease
What happens if there is a mutation in the leader peptide?
protein accumulates in the cytosol because it cannot be inserted into the ER
What happens if there is a mutation in the mature protein?
protein is retained in the ER
What is a pro-renin mutation?
when there is clustering/aggregation of renin in the ERGIC compartment between the ER and golgi (where there is an exchange of vesicles moving forward and backwards along the secretory pathway)
What 2 classes of mutation are associated with severe form of disease?
leader peptide and prosegment mutations
What is a phenotype associated with leader peptide and prosegment mutations?
anemia during childhood
When does the phenotype associated with mature protein mutations occur?
in adulthood
Where is uromodulin and uromodulin predicted to be localized?
extracellularly
What happens to the mutants in the leader peptide and prosequence of renin?
they either relocate fully or partially to the mitochondria which leads to a defect in the mitochondria that can be measures by fragmentation of the mitochondria and the defect of import in the mitochondria
How can we make renin enter and fragment mitochondria?
we take the sequence of mutated renin and fuse it to GFP
What mutations lead to a more severe form of disease?
mutations in the leader peptide and prosequence (they both have mitochondrial relocalization)
How stable is the mutated protein inside the mitochondria?
not stable, which makes us think that renin reaching the mitochondria is degraded
What happens if we have a mutation in Sec61 subunit alpha?
renin is misloacted
What is the most common type of ADYKD after uromodulin?
MUC1
What is MUC1?
big protein, mostly expressed on the membrane (mainly extracellular), and it forms a barrier preventing pathogens’ interaction with the epithelial cells
it is heavily glycosylated
it is mutated in ADTKD patients
What do all patients of ADTKD have in common?
the same effect of the mutation, even if the mutations are different
they all lead to the generation of the frameshift of MAK1 protein
What was a molecule scientists found that is able to induce the degradation of the protein?
BRD4780, which targets TMED9 and promotes lysosomal degradation to reverse proteinopathy
Why was the BRD4780 molecule so effective?
protein accumulated in ERGIC because mutant MAK1 interacts with a chaperon TMED9 and exhorts a QC downstream of the ER in the ERGIC
TMED9 binds the mutant protein and stops the trafficking of the protein in the ERGIC
- the small molecule discovered (a drug) is able to specifically bind TMED( and transport the mutatnt protein to the lysosomes to be fully degraded
What could be a possible 1st treatment for ADTKD patients?
BRD4780 molecule
*drug also works for uromodulin and on other conformational diseases due to ER retention of mutant proteins
List the 4 types of ADTKD mutations:
uromodulin
renin
MAK1
SEC61
What does SEC61 do?
encodes for the subunit 𝛼 of the SEC61 translocon (SEC61A1)
it forms the prore of the ER and the protein that is newly synthesized enters the ER and is folded
it also regulates the passive eflux of calcium from the ER
What can be observed in patients with expressed mutant SEC61A1?
the protein can colocalize to the golgi
*this implies the protein can exhort its function in the ER and also move to another compartment (loss and gain of function compartments)
Why can there be a gain of function when there is an expressed mutant SEC61A1?
mutations in SEC61 is associated with a defect in inflammatory cells
What can the mutations in the 5 different genes described lead to?
ER stress
mitochondrial stress
unfolded protein response (UPR)
What is the final endpoint of the 5 mutations?
tubulointerstial fibrosis
What happens if there are defects in the renin gene?
the protein cannot localize in the ER
What do different mutations activate?
same downstream response
What happens when a protein enters the secretory pathway?
SRP interacts with the ribosome as soon as the signal peptide is synthesized
ribosomal translation is blocked
SRP receptor binds to the ribosome
leads to the binding of the ribosome to SEC61
leader peptide is cleaved
rest of the protein is co-translationally inserted into the ER
What happens if there is a mutation in the leader peptide?
the recognition of the signal peptide is affected and the protein will NOT enter the secretory pathway and potentially be relocalized in the mitochondria
What is believed to be the downstream mechanism of the kidney disease?
inflammation and fibrosis as a consequence of inflammation
