Protein Sorting and Post-Translational Modifications

Why do we have compartments within the cell?

1. establish a physical boundary

2. generate specific micro-environments

3. establish specific locations where certain processes occur

describe the process in which specific proteins have to be within specific organelles

• Synthesis begins in cytosol

• Elongation continues on ER

• Synthesized polypeptide is released inside of ER

what are the AA sequences for the mitochondria and nucleus called?

Nuclear proteins – nuclear localization signal
Mitochondrial proteins – mitochondria entry signal

What are secretory vessicles? what are some examples of molecules that needs secretory vessicles?

What are extracellular vesicles and what are their clinical importance?

• Secretory vesicles contain molecules
  destined for secretion outside the cell:

  • Gastric acid

  • Digestive enzymes

  • Lung surfactants’

  • Sebum to lubricate skin and hair

• carry proteins and nucleic acids

  • linked to metastatic cancers

describe the process in a protein translocating to the mitochondria.

• A precursor of mitochondrial protein contains a signal sequence that binds to the receptor on mitochondrial outer membrane.

• With the assistance of a TOM/TIM23 complexes a precursor translocates into mitochondrial matrix.

• The signal sequence is cleaved producing a mature protein.

list out the covalent modifications and where they attach to.

• Phosphorylation and dephosphorylation of Ser, Thr, and Tyr.

• Ubiquitination is the attachment of ubiquitin to Lys

• Acetylation of Lys residues (acetylation of histones is involved in the regulation of transcription).

• Methylation of Lys and Arg

• Pro and Lys are hydroxylated (stabilization of collagen).

• Carboxylation – blood coagulation (clot formation).

• Attachment of fatty acids to anchor protein in membrane.

• Glycosylation of Ser, Thr, or Asn residues of membrane or secreted proteins.

• Glycation is a nonenzymatic attachment of a sugar to Arg or Lys

List out some protein kinases.

tyrosine kinases, serine-threonine kinases

What is Chronic myelogenous leukaemia? Describe its pathology.

slow-growing cancer of the blood- forming tissue (bone marrow) leading to the production of too many white blood cells.

Fusion of ABL1 (chromosome 9) gene with BCR gene (chromosome 22) = BCR-ABL1 (aka, philadelphia) —> protooncogene tyrosine protein kinase.

This protein phosphorylates multiple targets in leukemia cells → uncontrolled proliferation

where does ubiquitin attach? What are the two types? What are the official names of E1, E2, E3?

• amino group of lysine

• Monoubiquitination is involved in DNA repair, regulation of transcription and internalization of
  transmembrane proteins.

• Polyubiquitination is involved in proteosomal degradation of modified protein

• ubiquitin-activating (E1),

• ubiquitin-conjugating (Ubc),

• ubiquitin ligase (E3)

what is the VHL gene and what happens if it is defective?

• Encodes E3 ubiquitin ligase; target hypoxia-inducible transcription family factor (HIF) for destruction.
  

• Mutations in VHL gene impair its function and
  promote angiogenesis and renal tumor.

How is BRCA1 responsible for breast cancer?

• human tumor suppressor gene and is
  responsible for DNA repair.
  

• It has E3 ubiquitin ligase activity.
  

• Mutation that affects the ubiquitin ligase function are
  found in various cancers.

Where does acetylation happen? Give examples of acetylation

• most common = lysin; at n-terminum

Examples:

• Acetylation of COX1/COX2 by aspirin.

• Histone acetylation by histone acetyltransferase (HAT)

  • promotes transcription

  • Deacetylation by histone deacetylase (HDAC) suppresses transcription.

Describe the relationship between p53 and acetylation

Acetylation of tumor suppressor p53 is necessary for
its activation.

• A suppression of p53 acetylation can:

  • Prevent it from activation of p23

  • Lead to the loss of cell growth control.

  • Prevent p53 from performing its proapoptotic functions.

  • Increases the incidence of cancer

Describe where and how methylation occurs. What are some of the effects of methylation?

• catalyzed by methyltransferases and
  demethylases.

• Addition usually occurs on the nitrogen side-chain of lysine or arginine.

Some functions:

1. Regulation of transcription

2. Modulation of enzymatic activity

3. Protein trafficking

4. Signal transduction

What two enzymes are active in methylation of histones? How does this affect transcription?

1. histone methyltransferase

2. SAM (methyl donor)

Affects transcription by activation or repression

1. Trimethylation of histone H3 at
   Lys 4 (H3K4me3) is an active
   mark for transcription.

2. Dimethylation of histone H3 at
   Lys 9 (H3K9me2), a signal for
   transcriptional silencing.

Which AA does hydroxylation take place? How does this relate to collagen? Describe this hydroxylation reaction

at proline

• stabilizes the secondary structure of collagen due to the strong electronegative effects of oxygen

• catalyzed by a multi-subunit enzyme: prolyl 4-hydroxylase.

  • requires iron and α-ketoglutarate for oxidation and to return iron to its oxidized state
    

Describe the pathology of scurvy. Symptoms?

cause by vitamin C deficiency

Collagen needs Vitamin C for its stability as vitamin C return the iron used in prolyl 4-hydroxylase to its oxidized state;

lacking thereof results in:

• Decreased red blood cells

• Gum disease

• Poor wound healing

What is carboxylation? Where does it occur? How is it catalyzed? What is its cofactor?

• Carboxylation is a posttranslational modification of glutamate (Glu) residues, to γ-carboxyglutamate, in proteins.

• Carboxylation occurs in the liver and is performed by γ-glutamyl carboxylase.
  

• Requires vitamin K as a cofactor.

What types of protein does carboxylation primarilly affect? Describe these proteins’ functions

• carboxylation primarily affect proteins in the blood clotting cascade: specifically factors II, VII, IX, and X

Function:

• γ-carboxyglutamate binds calcium, which is essential for its function.
  

• In prothrombin (factor II):

  • calcium binding —> protein association with plasma membrane of platelets

  • Bringing it closer to protein that cleaves prothrombin to thrombin

Describe how GPI-linked proteins are produced? What are some examples of this protein?

• Glycosylphosphatidylinositol-anchored (GPI-linked) protein

• produced in the ER

How it is produced:

1. Protein co-translationally inserted into ER membrane.

2. hydrophobic C-terminus cleaved off and replaced with GPI-anchor by
   GPI-transamidase.

3. Then, translocated to Golgi and then to the plasma membrane.

Examples:

• Antigens; human carcinoembryonic antigen (CEA) is used as a cancer marker.

• Enzymes; alkaline phosphatases and acetylcholinesterase

Describe the relationship between GPI-anchored proteins and RBC

GPI-anchored proteins on the surface of RBCs protect them from destruction by the complement system.

What is PNH? Describe its pathology

• paroxysmal nocturnal hemoglobinuria

• somatic mutation in X-chromosomal gene PIGA

  • this is an important component for GPI-transamidase.

Consequences:

1. destruction of RBC by compliment system

2. 50% of individuals die through thrombotic complications

   1. increased risk of leukemia

What is glycoslyation? Which types of proteins are modified this way? what is glycosylation important for?

• covalent attachment of carbohydrate to the
  target molecule.

• Most soluble and membrane-bound proteins expressed in the endoplasmic reticulum are glycosylated:

  • Secreted protein

  • Surface receptors

Glycosylation is important for:

1. Protein folding

2. Cell-cell adhesion

3. ABO blood group

4. Glycosylation is used by viruses to shield from immune recognition

Compare and contrast N and O glycosylation. What determines whetehr a protein has N or O glycosylation?

N: Glycan bind to the amino group of Asn in the ER.

O: Monosaccharides bind to the hydroxyl group of
Ser or Thr in the ER, Golgi, cytosol and nucleus

Type depends on

1. enzyme availability

2. Amino acid sequence: Asn for N-linked or Ser/Thr for O- linked.

Occurs in ER and Golgi

What is glycation? Describe this reaction.

non-enzymatic covalent attachment of carbohydrate
to Arg or Lys of the target molecule.

Reactions are:

1. very slow and occur mainly in the bloodstream.

2. They result in the formation of advanced glycation endproducts (AGEs). AGEs form in hyperglycemic conditions and/or the natural process of aging.

What is the clinical importance of AGEs? How does diet impact presence of AGEs

AGEs implicated in:

1. Cardiovascular

2. Alzheimer’s

3. Deafness

4. Cancers

AGEs causes altercations to these organs:

1. Heart and vasculature – vessel stiffness

2. Vitreous body – affect vision

3. Skin – skin stiffness

4. Lung collagen – affect respiration

5. Intervertebral discs – decreases flexibility

We measure AGEs via hbA1c for diabetes

A significant generation of AGEs occurs when sugars are cooked with proteins.
It has been reported that, in renal failure patients, there was a 29% increase in glycation levels in the blood of those subjected to an AGE-rich diet