Nitrogen Metabolism & Protein Turnover (MDSC 1404) - 2023/2024 PDF

MDSC 1404 NITROGEN METABOLISM PROTEIN TURNOVER Nikita Sahadeo (Ph.D.) Department of PreClinical Sciences, FMS The U.W.I., St. Augustine Campus 2023/2024 RECALL A certain amount of dietary protein is required to synthesize endogenous proteins The basis for the dietary requirement of protein is the bo...

MDSC 1404 NITROGEN METABOLISM PROTEIN TURNOVER Nikita Sahadeo (Ph.D.) Department of PreClinical Sciences, FMS The U.W.I., St. Augustine Campus 2023/2024 RECALL A certain amount of dietary protein is required to synthesize endogenous proteins The basis for the dietary requirement of protein is the bodies inability to synthesize certain amino acids 9 essential amino acids: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine PROTEIN TURNOVER Refers to the continual renewal or replacement of protein Defined by the balance between protein synthesis and protein degradation The net result of continuous synthesis and breakdown of body proteins and ensures maintenance of optimally functioning proteins 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 THE PROTEOSTASIS NETWORK PROTEIN DEGRADATION PATHWAYS THE 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 (UB) 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 (polyubiquitylation) 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 THE UPS – WHAT ACTUALLY HAPPENS? Ub binding to proteins or ubiquitination is preceded by a series of stages. These include: 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. PROTEASOME The proteasome is a large protein complex responsible for degradation of intracellular proteins ­ requires metabolic energy 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 The 26S proteasome is a 2.5-MDa multicatalytic degradation machine that contains a 20S CP and one or two 19S RPs, which associate with the termini of the barrel-shaped central particle The 19S RP serves to recognize ubiquitylated client proteins and is thought to play a role in their unfolding and translocation into the interior of the 20S CP β rings are associated with catalytic activity 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 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 AUTOPHAGY There are three types of proteins in cells that are degraded by autophagy: ­ Cytoplasmic proteins ­ Misfolded proteins ­ Insoluble misfolded protein complexes CHAPERONE-MEDIATED AUTOPHAGY (CMA) Degrades soluble or unfoldable proteins in a molecule-by-molecule fashion Can be activated by prolonged starvation to provide amino acids for essential protein synthesis Heat shock protein 70 (HSC70) and lysosome membrane protein type 2A (LAMP2A) are two key factors that are involved in this process The criterion for the protein to be a putative CMA substrate is the presence of a peptide sequence that is biochemically related to the KFERQ motif THE 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 Disruptions in protein turnover and its regulation associated with many diseases: ­ 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 NITROGEN BALANCE (NB) 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 DO WE CALCULATE NB? à 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 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 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 THAT’S IT FOR THIS SESSION