4/1 Protein turnover

• when are amino acids obtained from diet?

  when proteins are digested
• why are cellular proteins degraded to aa?

  bc of damage, misfiling, or changing metabolic damands . once proteins are degraded to aa, aa are absorbed by the intestine and transported in blood

  
  

  essential amino acids = amino acids that cant be synthesized and must be from diet
• excess amino acids cannot be stored or excreted, so must be used as metabolic fuel.
• Why are protein production and consumption rates controlled?

  to maintain physiological levels and function . homeostatic mechanisms adjust protein production and consumption rates to achieve production=consumption
• essential amino acids in human beings: Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine
• Where does digestion of dietary proteins occur?

  begins In the stomach. completed by the intestine.

  the acidic nature of the stomach denatures proteins into random coils.
• pepsin - the primary proteolytic enzyme of the stomach.

  maximally activate @ph2
• partially digested proteins move from stomach ---> beginning of small intestine/duodenum... allowing secretion of sodium bicarbonate and proteolytic enzymes from the pancreas
• aminopeptidases in plasma membrane of intestinal cells enhance digestion
• the products of protein digestion are absorbed by the small intestine
• free amino acids, dipeptides, and tripeptides are transported into the intestinal cells

  • at least 7 different transporters exist-- and each are specific to a dif group of aa
  • absorbed aa are released into blood by Na+-aa transporters
• true or false?cellular proteins are degraded at different rates.

  true
• protein turnover= the degradation and resynthesis of proteins

  • takes place constantly in cells
  • essential for removing short-lived or damage proteins
• what is the life of the RBC?

  hemoglobin
• what is the life of the organism?

  the lens protein crystallin
• what catalyzes the synthesis of polyamines, and its half life?

  ornithine decarboxylase. 11 min
• protein turnover is TIGHTLY regulated
• ubiquitin - small (76aa) protein that tags proteins for destruction

  • small compact protein with 7 lysines
  • has an extended carboxyl terminus thats activated and linked to proteins targeted for destruction
  • all euk cells have it
  • highly conserved
  • attaches by its carboxyl terminal gly residue to the amino groups of 1+ Lys residues on target protein
  • needs ATP hydrolysis
• how many enzymes participate in the attachment of ubiquitin to a protein?

  
  

  3.
• Ubiquitin-activating enzyme (E1) = adenylates ubiquitin and transfers it to a sulfhydryl group of a Cys residue of E1– requires ATP
• Ubiquitin-conjugating enzyme (E2) = transfers ubiquitin to one of its own sulfhydryl groups
• Ubiquitin–protein ligase (E3) = transfers ubiquitin from E2 to an ε-amino group on the target protein– brings E2 and the target protein together– ubiquitin be transferred directly or be passed to a Cys residue of E3 first

  E3 can remain bound to the target protein and generate a chain of ubiquitin molecules.

  • E3 can dissociate after the first ubiquitin addition, and a chain can be extended by another E2/E3 pair.
• where can ubiquitin be added onto?

  any of the 7 lys or the N-terminus of previous ubiquitin

  • A chain of 4+ ubiquitin molecules linked via Lys 48 is an especially effective signal for protein degradation.
• In a tetraubiquitin chain, 4 ubiquitin molecules are linked by what type of bonds?

  isopeptide bonds

  
  

  • The ε-amino group of a Lys residue of one ubiquitin is linked to the terminal carboxylate of another.

  • This unit is the primary signal for degradation when linked to a target protein
• Degrons - aa seq that control protein t1/2, specific aa seq that indicates protein should be degraded

  (N degron = importnat signal for E3 enzymes)

  • may only be exposed after proteolytic cleavage
  • – may be added after protein synthesis
  • – may require other modifications (e.g., N-terminal acetylation)

  other ex. cyclin destruction boxes & PEST Seq
• if defective E3...what happens?

  • protein might not be degraded and might accumulate
  • disease of protein aggregation
  • **angelman syndrome - severe neurological disorder due to unusually happy disposition, cognitive disability, absence of speech, uncoordinated movement, and hyperactivity
  • – caused a defect in a member of the E3 family
• If too much E3...what happens?

  • can cause autism
  • inappropriate protein turnover can lead to CANCER
• Ubiquitination regulates proteins involved in:

  – DNArepair

  – chromatin remodeling

  – innate immunity

  – membrane trafficking

  – autophagy
• what does the proteasome do?

  • digests ubiquitin tagged proteins
  • large , ATP driven protease complex that digests ubiquitinated proteins
  • 26S proteasome has 2 components
  • 1 20S catalytic units (arranged as a barrel, made up of 28 homologous subunits)
  • 2 19S regulatory units that control access to interior of 20S catalytic subunit

  
  

  imagine a sandwich (19 bun 20 meat 19 bottom bun)
• 19S regulatory unit functions

  • contain ubiquitin receptors that bind specifically to polyubiquitin chains
  • useATP to unfold polyubiquitinated chains and direct them into the catalytic core
  • contain an isopeptidase that cleaves off intact ubiquitin molecules so that they can be reused

  
  

  Key components of the 19S complex are six ATPases of the AAA+ class

  • A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes
• The proteolytic active sites of the 20S barrel?

  • There are three types of active sites in the β subunits (ALL HAVE different specificity)

  • All active sites have a N-terminal Thr residue.

  – The hydroxyl group of the Thr residue attacks the carbonyl groups of peptide bonds, forming acyl-enzyme intermediates.

  – Substrates degraded without intermediate release.

  – Substrates reduced to peptides( 7-9 residues before release)
• proteasome and protease generate free amino acids.

  • ubiquitinated proteins are processed to peptide fragments
  • ubiquitin is removed and recycled prior to protein degradation
  • peptide fragments are further digested to yield free amino acids, used for biosynthetic reactions-notably protein synthesis.
  • amino group removed, processed to urea and carbon skeleton used to synthesize carbs or fats or used as fuel for cellular respiration
• ubiquitin pathway and the proteasome appear to be present in all eukaryotes. homologs of the proteasome also found in some prokaryotes. proteasome of some archaea are similar to eukaryotic proteasomes.
• 14 alpha outer ring subunits

  14 beta inner ring subunits

  THESE ARE ALL IDENTICAL
• In eukaryotes, each α or β subunit is one of the seven different isoforms

  – gene duplication and specialization has led to 7 distinct subunits of each type.

  – provides distinct substrate specificity

  – the overall architecture of the proteasome is conserved
• many biological processes are controlled by protein degradation via the ubiquitin-proteasome pathway.
• Processes regulated by protein degradation

  • Gene transcription
  • Cell-cycle progression
  • Organ formation
  • Circadian rhythms
  • Inflammatory response
  • Tumor suppression
  • Cholesterol metabolism
  • Antigen processing
• bortezomib (Velcade) = a dipeptidyl boronic acid inhibitor of the proteasome. used as a therapy for multiple myeloma.
• degrons - used as regulatory mechanisms for protein expression
• HT1171 = a suicide inhibitor of the proteasome of M. tuberculosis

  – has no effect on human proteasomes