Lecture #8 Flashcards
what is ADKTD?
autosomal dominant tubulointerstitial kidney disease
what symptoms are associated with a loss of kidney function?
- progressive loss of kidney function
- bland urine sediment
- no proteinuria
- small kidney
- urinary concentrating defect
what are the main functional defects seen in patients with ADKTD?
urinary concentrating defects - polyuria and polydipsia
what physical attributes are seen in the kidney of patients with ADKTD?
- interstitial fibrosis
- tubular atrophy
- thickening / lamellation of the tubular base membrane
- tubular dilations
- progressice fibrotic phenotype
as of now, it is known that ADKTD is caused by mutations in how many different genes?
5
what are the two most frequently mutated genes?
UMOD (uromodulin) and MUC1 (mucin 1)
what is unique about HNFβ?
it is a transcription factor so it is associated with many diseases besides ADKTD
what are the most rare versions of ADKTD?
ADKTD-REN and ADKTD-SEC61A1
what are the three main diagnostic methods?
urinalysis, imaging, inheritance
what is the other name for uromodulin?
Tamm-Horsfall protein
describe the normal occurrence of uromodulin:
the most abundant protein in human urine in physiological conditions
where is uromodulin expressed in the kidney?
only expressed in the thick ascending limb (TAL) of Henle’s loop where there are specific cells of the nephron
describe the structural features of uromodulin:
there are a huge amount of cysteines which are involved in the formation of S-S bonds and there is the zona pellucida domain which is found in a lot of extracellular proteins that are all involved in the formation extracellular polymers
what protein cleaves uromodulin once it reaches the membrane, cleaving the ends of the ZP and separating the interactions of the two domains?
hepsin
what does cleavage by hepsin allow for?
interactions with other uromodulin forming filaments
what is the function of uromodilin in the TAL segment?
regulates ion transport
what is the function of the loop of Henle?
the portion where important molecules are reabsorbed from the urine → in general urine comes in the loop super concentrated and then gets progressively diluted implementing the mechanism in which we have the counter current gradient in kidneys where most of the water gets reabsorbed thanks to the osmotic gradient
what is the function of uromodulin once it is secreted?
acts as a defense factor against infectious and calcium oxalate crystal formation
describe the immunoregulatory function of uromodulin:
this protein acts as a DAMP → when the tubule is damaged the protein extravasates into the interstitium and activates inflammatory cells acting as a guardian of the integrity of the tubules once its secreted in the nephron
what generally causes disease in uromodulin mutations?
not a loss of function but instead a gain of function
describe the general cause of mutations in uromodulin:
majority of mutations affect cysteine → gof is most likely due to a misfolding of uromodulin causing the loss of a S-S bond
where does uromodulin accumulate in these mutations?
in the ER → mutations are likely interfering with protein folding
when uromodulin is mutated what does it colocalize with?
calnexin - fully or partially retained in the ER
what is the function of EndoH?
digests glycosylated enzymes only when they are in high mannose conformation
under what condition is EndoH able to be used?
can be used to detach glycans from a protein only when they are in the ER - if they get modified in the golgi it is no longer possible
what was demonstrated to be the primary effect of mutated UMOD?
ER retention - different mutations show a different extent and therefore a more or less functional protein
how many classes are UMOD mutations divided into?
4
what does a class 1 UMOD mutation represent?
a milder mutation associated with a better kidney survival than other classes
what is the UPR?
an adaptive pathway triggered when the cell tries to counteract the accumulation of misfolded proteins inside the cell itself
what does the UPR activate?
the expression of chaperones, decreases protein translation, and increases protein degradation associated with the ER
if the UPR is mutant and is unable to be switched off, what occurs?
it can be associated with cell damage eventually leading to transdifferentiation and activation of inflammatory pathways and apoptosis
what three pathways does the UPR activate?
ATF6, PERK, IRE1
when there is an increase in unfolded proteins in the ER what protein is sequestered?
BiP - the main chaperone in the ER
what is IRE and what is its main function?
an RNAse → cleaves and splices the transcriptome of XBP1 which becomes in frame and can translate the XBP1 protein which regulates the synthesis of chaperones, lipid synthesis, and ERAD proteins
what is the function of PERK?
activates IF2 and functions to quench translation
what is ATF6 and what is its function?
a transcription factor → cleaved in the golgi and translocates to the nucleus where it activates target genes mostly involved in chaperone synthesis and expansion of the Er membranes to accommodate the presence misfolded proteins
what was shown in mouse models with UMOD mutations?
they were very similar to humans → showed protein aggregation of the mutant protein in the ER and developed disease with the feature that was see in patients, as well as a change in the conformation of the ER
what was seen in ADTDK patients with increased BiP levels?
there was also an increase in a protein called CRELD2 which is known to be unregulated downstream on the unfolded protein response → could be used in the future to study the disease progression of patients
what is the proposed progression of disease as seen in mouse models?
misfolded proteins start accumulating in the ER activating ER stress and UPR, and these events trigger the release of chemokine activating inflammation which very likely activates resident macrophages first, then the recruitment of other macrophages and other inflammatory cells leading to the deposition of fibrotic tissue
what is renin?
an aspartyl protease
where is renin synthesized?
in the kidneys and other organs
what is the function of renin?
it cleaves angiotensinogen to form angiotensin I and it starts a cascade of renin angiotensin which regulates blood vessels ( vasoconstriction - blood pressure in general)
what cells are responsible for renin expression?
glomeruli cells within the vessels close to the macula densa
what is special about the renin synthesized in the kidneys vs that from other tissues?
only in the kidney is it synthesized in the mature form
what is the macula densa?
a region of the tubule that is proximal to the glomeruli where there are the afferent and efferent arteries
what can mutations in renin be associated with?
ADTKD or renal tubular dysgenesis
describe renal tubular dysgenesis:
loss of function mutation affecting virtually all genes of the renin angiotensin system → if two copies are carried severe disease is developed and associated with a very premature death
describe the mutations in ADTKD:
misses mutations suggesting a gain of function localized in all three parts of the gene
when scientists conducted experiments involving the import of proteins into the ER, what mutation was shown to not be associated with any proteins?
Mut L16R
when scientists conducted experiments involving the import of proteins into the ER, wha mutation was shown to be more associate with proteins in the medium?
Del L16R
what recognizes that a protein must enter the secretory pathway?
SRP - signal recognition peptide →protein complex with an RNA component
when SRP binds to the protein, what occurs?
it binds to a receptor that is associated to a translocon which allows for the protein to be inserted into the ER and synthesis to continue while SRP is released
what does a mutation in the leader peptide lead to?
SRP is unable to bind and leads to a defective secretion of the protein that leads to accumulation in the cytosol
what do mutations in the mature part of renin cause?
ADTKD → associated with misfolding and ER retention of mutant renin causing a milder disease
where does the misfolded proteins accumulate when there is a mutation in the leader peptide?
in the cytosol
where do proteins associate in a pro-renin mutations?
proteins cluster / aggregate in the ERGIC
which two mutations cause the most severe phenotype of disease?
mutations in the leader peptide and pro-segment mutations - associated with anemia during childhood
describe the mature protein mutations:
associated with a late set of disease that do not show up during childhood or adolescence
what did they discover when using PSORT to study mutations in the leader peptide?
single mutations in the leader provide convert the signal peptide for the ER into a mitochondrial localization signal
what is one of the things that can happen to the mitochondria due to this localization, from either leader peptide mutations or from pro-segment mutations?
fragmentation of the mitochondrial network
what other effect can these mutations have on mitochondria besides network fragmentation?
affects mitochondrial import
where is renin localized in the mitochondria when it is mutated?
matrix
what do dominant mutations in renin show in zebrafish?
defective pronephros development not observed in the lof mutations (D104N)
what is seen if the same mutations are produced in UMOD?
it does not relocate to the mitochondria
what is the difference between UMOD and renin that cause this difference in localization upon mutation?
the leader peptide of renin already has a week signal for ER localization which can be more easily converted to mitochondrial localization
what happens to renin upon a mutation in the ⍺ subunit of SEC61?
renin is also mislocated
behind uromodulin, what is the second most common type of ADTDK?
mutations in mucin I
what is the function of mucin I?
a large protein expressed on the membrane (extracellular) and forms a barrier preventing pathogen interaction on the apical membrane of epithelial cells
what channels does MUCI stabilize on the cell surface?
TRPV5 calcium channels
describe the repeated sequences in the MUCI mutations and its effect:
VNTR is about 60bb repeated 20220 copies in tandem as well as a stretch of 7 cytosines → in 90% of patients there is the insertion of an additional cysteine
the repeated sequences code for a series of amino acids, so if there is an extra nucleotide inserted there is a frameshift the remainder of the repeats will code for different proteins and we encounter a stop codon
when an additional cysteine or aa is added, what protein is created?
MAK1 protein
scientist generated cell lines with the MAK1 mutation in order to look at the presence of the protein in cells and developed an antibody with this specific frameshift to detect these protein - where were these aberrant proteins accumulating?
in the ERGIC compartment leading to the activation of the UPR similar to UMOD and some renin mutations
scientists then searched for a molecule to induce the degradation of these proteins, which one did they select?
BRD4780 → targets TMED9 and promotes lysosomal degradation to reverse proteinopathy
was BRD4780 successful?
yes - in cell lines the mutant protein was completely removed
what did scientists discover to be the cause of accumulation in the ERGIC?
MAK1 interacts with chaperone TMED9 which stops the trafficking of the protein in the ERGIC so it does not continue downstream
how was the BRD4780 molecule able to rid the cell of the aberrant protein?
it binds to TMED9 and transports it to the lysosome so the protein is degraded
does BRD4780 only have potential usage for ADTKD?
no - this drug can also work for uromodulin and other conformational diseases due to ER retention of mutant proteins
what is SEC61 and what are its functions?
a heterotrimeric complex that forms a pore in the ER allowing entry of newly synthesized proteins that enter the ER to be folded
also regulates the passive efflux of calcium from the ER (calcium concentration)
what are the two mutations related to ADTKD in SEC61?
partial loss of function or gain of function
describe what was seen when scientists expressed mutant SEC61A1?
the protein can also co-localize in the Golgi - exerts its function in the ER but is also able to move to other compartments
what is the endpoint of any of the 5 genes leading to ER stress and the activation of the UPR?
tubulointersitial fibrosis
describe the life cycle of a protein that enters the secretory pathway:
as soon as the signal peptide is synthesized, SRP interacts with the ribosomes and blocks translation, leading to the binding of the ribosome to the SEC61 complex
then the leader peptide is cleaved and th rest of the protein can co-translationally insert into the ER
what do we see when there is a mutation in the leader peptide of the protein?
the recognition of the signal peptide is affected and the protein fails to enter the secretory pathway and we can see localization in the mitochondria
what effect do we see when there is a defect in the SEC61 function?
once the ribosome binds to the ER membrane the protein enters but fails to fold
