1404 Biochemistry- Nitrogen Metabolism-Protein Turnover

What are the 9 essential amino acids?

• histidine

• isoleucine

• leucine

• lysine

• methionine

• phenylalanine

• threonine

• tryptophan

• valine

what is protein turnover?

• it is the continual renewal or replacement of protein

• The rates of turnover vary from tissue to tissue, and

  the relative contributions of different tissues to total

  protein turnover change with age and adaptation

  to various levels of protein intake

Proteostasis Network

The proteostasis network (PN) is a complex system of molecular pathways that maintains protein homeostasis (proteostasis)—ensuring that proteins are correctly synthesized, folded, trafficked, and degraded in the cell. It is essential for cellular function and preventing diseases related to protein misfolding or aggregation.

What are the protein degradation pathways?

• Ups

• Macroautophagy

• CMA

What is ubiquitin/Proteasome system (UPS)?

• The major pathway of selective protein degradation in eukaryotic

cells

• Uses ubiquitin as a marker that targets cytosolic and nuclear proteins

for rapid proteolysis

• The first ubiquitin attaches the amino group of the side chain of a

lysine residue on the protein substrate (isopeptide bond). Additional

ubiquitins are then added to form a multiubiquitin chain.

­ -Mono-ubiquitylation

­ -Multiple mono-ubiquitylation

• Polyubiquinated proteins are recognized and degraded by a large, multisubunit protease complex; aka the proteasome ­ Chains of 4 or more ubiquitin molecules target protein for destruction

• Ubiquitin is released in the process, so it can be reused in another

cycle

• Process requires ATP (attachment and degradation)

Ubiquitin

• A 76-amino-acid polypeptide that is highly

conserved in all eukaryotes

Adopts a compact globular fold, the β-grasp

or ubiquitin-like fold

• Covalent attachment of ubiquitin to target

proteins is mediated by an elaborate

enzymatic conjugation cascade; in most cases,

lysine residues serve as acceptors for ubiquitin

• Can also be attached to the α-amino group

of the N-terminal residue of a protein or to

serine or threonine residues via the formation

of a peptide bond or ester bond, respectively

• Can serve as its own substrate resulting in the

formation of ubiquitin chains on target proteins (poly- ubiquitylation)

• Contains 7 lysine residues and each of these as well as

its N-terminal residue can serve as an attachment site

for ubiquitin resulting in 8 homotypic, numerous

heterotypic chains and branched chains

What happnes in the UPS?

1. Ub activation – A thioester is formed between the

carboxyl of the terminal glycine residue of Ub and a

cysteine residue of the activating enzyme, or E1. The

energy that fuels this reaction is provided by

hydrolysis of ATP.

2. Conjugation to E2 – Activated Ub is transferred to

a cysteine residue of a conjugating enzyme, or E2.

3. Binding of Ub to E3 – A new transesterification

attaches Ub to the enzyme ubiquitin ligase, or E3. Most cells have one type of E1, but different forms of

E2 and E3. Different kinds of E2 and E3 recognize

different proteins as substrate.

what is proteasome?

• The proteasome is a large protein

complex responsible for degradation of

intracellular proteins

­ requires metabolic energy

what is proteasome made up of?

The proteasome is made up of two subcomplexes: a catalytic core particle (CP; also

known as the 20S proteasome) and one or two terminal 19S regulatory particle(s)

(RP) that serves as a proteasome activator.

What is autophagy?

• Autophagy is a cellular process in which cytoplasmic contents are degraded within the

lysosome/vacuole, and the resulting macromolecular constituents are recycled

• Autophagy complements the ubiquitin-proteasome system in mediating protein turnover

• Whereas the proteasome degrades individual proteins modified with ubiquitin chains,

autophagy degrades many proteins and organelles en masse

How is macromolecules destined for autophagic degradation selected?

• Through sequestration

within a specialized double-membrane compartment termed the phagophore, the precursor to

an autophagosome, and then are hydrolyzed in a lysosome- or vacuole-dependent manner

• A pair of distinctive ubiquitin-like proteins (UBLs), Atg8 and Atg12, regulate degradation by

autophagy in unique ways by controlling autophagosome biogenesis and recruitment of

specific cargos during selective autophagy

what are the proteins in cells that are degraded by autophagy?

­- Cytoplasmic proteins

­ - Misfolded proteins

­ - Insoluble misfolded protein complexes

(polymers, aggregates and

aggresomes)

what is chaperone-mediated autophagy (CMA)?

Degrades soluble or unfoldable proteins in

a molecule-by-molecule fashion.

How can chaperone-mediated autophagy be activated?

Can be activated by prolonged starvation

to provide amino acids for essential protein

synthesis

What are the key factors involved in the CMA process?

Heat shock protein 70 (HSC70) and

lysosome membrane protein type 2A

(LAMP2A) are two key factors that are

involved in this process.

Proteasome network and disease

Changes in hormone secretion, tissue perfusion, oxygen availability, energy-protein

intake, free amino acid pattern, hydration state, acid-base balance as well as

activation of the systemic inflammatory response may affect protein synthesis and

degradation.

What diseases are associated with the disruption of protien turnover?

­ - Angelman syndrome is caused by maternal deficiency of the E6-AP ubiquitin E3 ligase (UBE3A)

­ - Other disorders of E3 ligases have been identified, including autosomal recessive juvenile Parkinson

disease, the APECED form of autoimmune polyendocrinopathy syndrome, von Hippel-Lindau syndrome,

and congenital polycythemia

­ - Disorders that disturb ubiquitin regulatory signaling include at least two subtypes of Fanconi anemia,

the BRCA1 and BRCA2 forms of breast and ovarian cancer susceptibility, incontinentia pigmenti, and

cylindromatosis

­ - Many disorders affect ubiquitin pathways secondarily

What is nitrogen balance?

• Reflects the equilibrium between protein intake and losses

• Occurs when nitrogen intake equals nitrogen output (NB = 0), and is also referred to

as nitrogen equilibrium

• A positive NB or anabolic state exists when nitrogen intake exceeds nitrogen output

­ -A net 24-hour positive NB of 2 to 4g is optimal for anabolism

• When nitrogen excretion is greater than nitrogen intake,

a negative NB or catabolic state exists

How to calculate NB?

• Subtract the total nitrogen output from total nitrogen intake

The total nitrogen intake is determined by dividing the daily protein intake (grams)

from both enteral and parenteral sources by 6.25

• Nitrogen output consists primarily as urine urea nitrogen (UUN)

­ An aliquot of a 24-hour urine collection is assayed for its urea nitrogen content by a standard

enzymatic laboratory technique

• The above value, plus 4 (the constant used for nitrogen losses from the skin and feces),

is subtracted from the grams of nitrogen intake during the same 24-hour period to

calculate the NB

What are the factors affecting NB?

• Dietary intake

­ The recommended protein intake is 0.8g/kg body wt/day

. This amounts to about 58 g protein/day for a 72-kg

(160-lb) man and about 44 g/day for a 55-kg (120-lb)

woman

• Dietary Content of Carbohydrate and Fat

• Growth

• Pregnancy

• Illness and major trauma

What is the clinical significance of NB?

• Protein-Energy Malnutrition (PEM)

­ The most common form of malnutrition in the world

While the symptoms vary widely from case to case, it is common to classify most cases

as either marasmus or kwashiorkor

• Marasmus

­ Caused by inadequate intake of both protein and energy

• Kwashiorkor

­ Caused by inadequate intake of protein with adequate energy intake