Pulm/Renal - Biochemistry - The Golgi Apparatus: Mucolipidoses; Glycobiology: Mucopolysaccharidoses & Sphingolipidoses Flashcards
Describe the hypothesized signalling mechanism by which proteins that are being synthesized end up in the rough endoplasmic reticulum.
(1) Translation begins in the cytosol; an RER-targeting sequence on the partially synthesized protein is bound by a signal recognition particle (SRP).
(2) Upon binding to an SRP receptor in the RER, GTP hydrolysis opens an associated translocon protein (the RER channel opens, and the SRP dissociates from the ribosome).
(3) Translation continues directly through the translocon and into the RER lumen; signal peptidase cleaves the signalling sequence on the synthesized protein.
In the signal hypothesis of RER synthesis, what binds to signalling sequences on partially synthesized proteins?
Where are these proteins when this occurs?
Where are they taken next?
Signal receptor particles (SRPs);
the cytosol;
SRP receptors on the RER
Secretory protein synthesis begins in the cytosol. How are these proteins targeted to the RER?
What effect does this targeting have on translation?
By signal receptor proteins (SRPs) that bind SRP receptors on the RER surface;
translation is blocked when SRPs bind the partially synthesized proteins, but it is restored when the SRP dissociates and the ribosome is bound to the SRP receptor
What must occur for both signal receptor proteins to dissociate from SRP receptors on the RER and also the associated translocons to open?
GTP hydrolysis
Secretory proteins begin their translation in the ________, where an N’ signalling sequence targets them towards the RER.
Upon entering the RER, what cleaves this signalling sequence from the newly synthesized proteins?
Cytosol;
signal peptidase (in the RER membrane)
Signal receptor particles (SRPs) bind what to bring partially synthesized secretory proteins to the RER?
An N’ signalling sequence on the partially synthesized protein and the 60s ribosomal subunit
What are the two main purposes of protein glycosylation?
What are some other purposes?
Folding, targeting;
E-leaflet protein stability, enhanced cell-to-cell contact
________ are carbohydrate-binding proteins, and can interact with other glycosylated proteins.
Lectins
Lectins are ___________-binding proteins, and can interact with other ___________ proteins.
Carbohydrate;
glycosylated
True/False.
Glycosylation is typically the addition of single glucose monomers to amino acid side chains.
False;
it is usually the addition of glycans (either single oligosaccharides or complexes) to amino acid side chains.
What are the two types of protein glycosylation?
O-linked;
N-linked
N-linked glycosylation occurs in the _____________.
O-linked glycosylation occurs in the _____________.
RER;
Golgi apparatus
Describe O-linked glycosylation (in terms of starting substrate and any involved amino acids or enzymes).
Give an example.
Individual carbohydrates are sequentially added to –OH groups of serine or threonine by glycosyl transferases;
the formation of ABO blood antigens
Describe N-linked glycosylation (in terms of starting substrate and any involved amino acids or enzymes).
What amino acid sequence is required?
Preformed oligosaccharide complexes are added to asparagine residues as the peptide is translocated through the RER membrane (translocation process shown in attached image);
Asn - X - (Ser/Thr)
__-linked glycosylation is characterized by the addition of preformed oligosaccharide complexes to __________ residues.
__-linked glycosylation is characterized by the sequential addition of individual carbohydrates to ___________ residues.
N, Asparagine (Asn-X(Ser/Thr);
O, Serine/Threonine
Describe the steps of synthesis of the oligosaccharide complex used in N-linked glycosylation.
(1) UDP-linked sugars are added to dolichol phosphate from the cytosolic side.
(2) This complex then ‘flips’ from the cyotosolic to the RER lumen side.
(3) The completed oligosaccharide complex can then be cleaved and added to asparagine residues by oligosaccharyl transferase.
In synthesis of the oligosaccharide complexes used in N-linked glycosylation, _______-linked sugars are first added to _____________ _____________ on the cytosolic side of the RER membrane.
UDP;
dolichol phosphate
What must occur for the oligosaccharide complexes used in N-linked glycosylation to enter the RER lumen?
It ‘flips’ from the cytosolic side to the RER lumen
What is the first substrate for synthesis of the oligosaccharide complexes involved in N-linked glycosylation?
It is attached to what substance in the RER membrane?
What substance can be used in the lab to inhibit this process, thus disrupting proper protein folding?
UDP-GluNAc;
dolichol phosphate;
tunicamycin
N-linked glycosylation is accomplished via what enzyme?
Oligosaccharyl transferase
What effect does tunicamycin have on cells?
What is its clinical use?
Inhibition of first step of N-linked glycosylation
–> inhibition of proper protein folding in eukaryotic cells;
none (laboratory use only)
True/False.
Disulfide bonds form in many proteins synthesized both in the cytosol and the RER.
False.
Disulfide bonds form mainly in secretory proteins and proteins of the E-leaflet;
they are only made in RER-synthesized proteins
Why do disulfide bonds only form in the RER lumen?
Via what enzyme?
It is an oxidative environment;
protein disulfide isomerase
What is the most common genetic cause of liver disease (jaundice, cirrhosis) in children?
What is the most common disorder necessitating pediatric liver transplants?
α-1 antitrypsin deficiency;
α-1 antitrypsin deficiency
What percentage of emphysema cases are due to an α-1 antitrypsin deficiency?
1 - 3%
What enzyme(s) are inhibited by α-1 antitrypsin?
From what organ(s) is this protein secreted?
Elastase, trypsin;
the liver
What is the main molecular cause of an α-1 antitrypsin deficiency?
What tissues does this damage?
A point mutation inhibiting proper folding of the α-1 antitrypsin protein in the RER;
pulmonary (increased elastase activity),
hepatic (α-1 antitrypsin crystalline aggregate buildups)
GI (increased trypsin activity)
What cell type(s) secrete elastase?
Neutrophils
How does an α-1 antitrypsin deficiency damage the lungs?
How does an α-1 antitrypsin deficiency damage the GI tract?
How does an α-1 antitrypsin deficiency damage the liver?
Decreased elastase inhibition;
decreased trypsin inhibition;
misfolded α-1 antitrypsin aggregates
What are the signs/symptoms of an α-1 antitrypsin deficiency?
[Pulmonary] Shortness of breath, fatigue;
[hepatic] jaundice, cirrhosis;
[GI] bleeding
What are the two main pillars of α-1 antitrypsin deficiency treatment?
Mainly the following:
(1) prophylaxis against pulmonary issues (e.g. vaccinations, bronchodilators),
(2) liver transplants
(Note: α-1 antitrypsin IV infusions may or may not be beneficial.)
What are the three primary locations to which the Golgi sends synthesized proteins?
(1) Secretory vesicles;
(2) endosomes/lysosomes;
(3) the plasma membrane
What are the three main sections (cisternae) of the Golgi apparatus?
The cis-Golgi, medial-Golgi, and trans-Golgi
What are the two forms of substrate movement through the Golgi apparatus?
Vesicular (anterograde, retrograde);
cisternal maturation (cis to medial to trans)
The cisternae of the Golgi apparatus facing the RER is the ___-Golgi.
The cisternae of the Golgi apparatus between the other two cisternae is the ___-Golgi.
The cisternae of the Golgi apparatus facing the plasma membrane is the ___-Golgi.
- Cis*;
- medial*;
- trans*
What protein is involved in anterograde Golgi budding and vesicular movement?
What protein is involved in retrograde Golgi budding and vesicular movement?
COP-II (RER to cis-Golgi);
COP-I (cis-Golgi to RER)
What is an important reason why retrograde Golgi transport is needed?
As cisternal maturation occurs (cis- turning into medial- turning into trans-Golgi), the necessary proteins are shuttled back from the maturing cisternae to the cisternae before them
The COP-I protein in the Golgi apparatus facilitates what event?
The COP-II protein in the Golgi apparatus facilitates what event?
Retrograde vesicular transport
either from the cis-Golgi to the ER
or from cisternae to earlier cisternae;
anterograde vesicular transport from the ER to the cis-Golgi only
What protein mediates vesicular transport from the Golgi apparatus out to some other location (e.g. endosomes, lysosomes, melanosomes, platelet vesicles)?
Clathrin
What is the role of coat proteins in the Golgi apparatus?
What are some clathrin-associated examples?
To form the curvature of transport vesicles;
AP1, AP2, AP3
What GTPase is associated with the COP-I protein in Golgi vesicular transport?
What GTPase is associated with the COP-II protein in Golgi vesicular transport?
What GTPase is associated with clathrin in Golgi vesicular transport?
ARF;
SAR1;
ARF
What proteins allow for vesicular interactions with target locations?
SNARE proteins
- (v-SNARE = vesicular SNARE;*
- t-SNARE = target SNARE)*
What are some of the major players involved in vesicular transport formation, coating, de-coating, docking, etc.?
Coat proteins;
GTPases (ARF or SAR1);
secretory product receptors;
SNARE proteins
True/False.
GTPases are important in Golgi vesicular coating when bound to GTP, and they are important in vesicular decoating after hydrolyzing that GTP to GDP.
True.
Where does high mannose glycosylation occur (addition of GlcNAc and mannose to synthesized cellular products)?
Where does complex glycosylation occur (addition of galactose and neuraminic acid to synthesized cellular products)?
The cis-Golgi;
the trans-Golgi
What sugars can be added to a product in the cis-Golgi?
What sugars can be added to a product in the medial-Golgi?
What sugars can be added to a product in the trans-Golgi?
Mannose + GlcNAc
Fucose + Mannose + GlcNAc
Galactose + N-acetylneuraminic acid
What signalling sugar targets Golgi products to the lysosome?
What vesicle type will be used (COP-I, COP-II, or clathrin)?
What coat proteins are involved?
What GTPase is involved?
Mannose-6-phosphate;
clathrin;
AP3;
ARF
Coat protein AP_ aids in delivery of Golgi products to the lysosomes.
Coat protein AP_ aids in delivery of Golgi products to the endosomes.
Coat protein AP_ is involved in receptor-mediated endocytosis.
3;
1;
2
Golgi products lacking mannose-6-phosphate are destined for ____________.
Golgi products with attached mannose-6-phosphate go to the ____________.
Secretion;
lysosomes
What is the molecular cause of I-Cell disease?
Failure of mannose-6-phosphate addition to lyosomal proteins
(lysosomal proteins secreted)
What disease results as a failure of mannose-6-phosphate addition to lysosomal proteins?
What effect does this have on cellular activity?
I-Cell disease;
buildup of material-filled endosomes with no acid hydrolases to break any of it down
(hence, I-Cell disease means inclusion-Cell disease)
What are some of the signs and symptoms of I-Cell disease?
When does it manifest?
Coarse facial features and craniofacial abnormalities,
low birth weight and slowed growth rate,
psychomotor retardation;
at birth
What enzyme is blocked in I-Cell disease?
GlcNAc phosphotransferase
(normally, this adds the phosphate with GlcNAc to mannose chains and then cleaves the GlcNAc to leave just the phosphate)
How is I-Cell disease different from mucopolysaccharidoses or sphingolipidoses?
Instead of being an enzyme deficiency that prevents breakdown of a certain sugar (i.e. a mucopolysaccharidosis) or a certain fat (i.e. a sphingolipidosis),
there is an inability to break down any product brought into the cell for digestion
What is a mucopolysaccharidosis?
(examples?)
What is a sphingolipidosis?
(examples?)
What is a mucolipidosis?
(examples?)
Enzyme deficiency –> inability to breakdown a specific sugar
(e.g. Hurler, Hunter, or Sanfilippo syndromes)
Enzyme deficiency –> inability to breakdown a specific lipid
(e.g. Niemann-Pick, Fabry, Krabbe, Gaucher, or Tay-Sachs, or metachromatic leukodystrophy syndromes)
A disease with features of both of the above categories
(e.g. I-Cell disease, pseudo-Hurler syndrome, or sialidase deficiency)
What are some example mucopolysaccharidoses?
What is the general defect in these disorders?
Hurler, Hunter, or Sanfilippo syndromes;
an enzyme deficiency leads to an inability to breakdown a specific sugar
What are some example sphingolipidoses?
What is the general defect in these disorders?
Niemann-Pick, Fabry, Krabbe, Gaucher, or Tay-Sachs, or metachromatic leukodystrophy syndromes
an enzyme deficiency leads to an inability to breakdown a specific lipid
What are some example mucolipidoses?
What is the general defect in these disorders?
I-Cell disease (ML 2), pseudo-Hurler polydystrophy (ML 3), sialidase deficiency (ML 1);
a disease with features of both a mucopolysaccharidosis and a sphingolipidosis (errors in both fat and sugar breakdown)
What enzyme is deficient in sialidosis (mucolipidosis type I) disease?
What enzyme is deficient in I-Cell (mucolipidosis type II) disease?
What enzyme is deficient in pseudo-Hurler (mucolipidosis type III) disease?
Sialidase
GlcNAc phosphotransferase (leads to exocytosis of all lysosomal enzymes)
GlcNAc phosphotransferase (though less severe than I-Cell disease)
What two mucolipidoses occur as a result of a deficiency in the GlcNAc phosphotransferase enzyme?
What is the result?
I-Cell disease (ML 2),
pseudo-Hurler polydystrophy (ML 3);
a lack of all lysosomal enzymes in the lysosome
How does an endosome become a lysosome?
It must be acidified (proton pump –> pH 5.0 - 6.0) and gain lysosomal enzymes (M6P –> acid hydrolases)
What structure becomes a lysosome?
What two structures can fuse with this structure to form lysosomes?
Endosome (early, then late);
phagosomes, autophagosomes
Endocytosis is different from phagocytosis in that endocytosis is an __vagination of the membrane while phagocytosis is an __vagination of the membrane.
in;
e
What substrate types can be broken down by lysosomal acid hydrolases?
Just about anything
(proteases, lipases, glycosidases, nucleases, sulfatases, phosphatases)
When does cholesterol dissociate from LDL upon being taken up by LDL receptors?
When does iron dissociate from apotransferrin upon being taken up by ferrotransferrin receptors?
The acidity of the lysosome causes them to dissociate;
the acidity of the lysosome turns Fe3+ into Fe2+, and it dissociates from apotransferrnin
What protein system normally allows for formation of vesicles in the lysosome but can be hijacked by HIV as a means of making extracellular vesicles for HIV ‘budding’ and spread?
The HRS-ESCRT system is hijacked by mono-ubiquinated HIV Gag proteins
Why is the HIV ‘Gag’ protein a target for HIV therapies?
Inhibiting it prevents HIV budding and spread
Are sugars in the human body typically in the D- or L-configuration?
What does it mean for them to be α or β?
D
(alcohol group ‘up’ at carbon 5, hydrogen ‘down’ at carbon 5);
whether the carbon 1 hydroxyl is ‘up’ (β) or ‘down’ (α)
(Note: the above α/β rule is correct for D-sugars. It is reversed for L-sugars.)
Mannose is the __ epimer of glucose.
Galactose is the __ epimer of glucose.
C2;
C4
Describe the Hayworth projection for glucose (α or β).
How can this D-glucose monomer be changed into N-acetylglucosamine?
How can this D-glucose monomer be changed into N-glucuronic acid?
What is the C2 epimer for D-glucose?
Mannose
What is the C4 epimer for D-glucose?
Galactose
Glucuronic acid is a(n) __-sugar.
Iduronic acid is a(n) __-sugar.
D;
L
What is another name for neuraminidase?
What is another name for N-acetylneuraminic acid?
Sialidase;
sialic acid
Glucose is a 6-carbon sugar.
N-acetylneuraminic (sialic) acid is a __-carbon sugar.
9
Lactose is a disaccharide made of what two monosacharrides connected by what type of linkage?
Glucose + galactose;
1,4 β-galactosidic (glycoside) linkage
True/False.
In order to combine glucose and galactose to make lactose, the glucose must be activated (often UDP-bound).
False.
In order to combine glucose and galactose to make lactose, the galactose must be activated (often UDP-bound).
When is lactose made in the body?
Only in females during lactation
True/False.
In order for monosacharrides to be added together, one of the sugars is usually nucleotide-activated (e.g. UDP-bound).
True.