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At least 19 recordsLinked to original sources
PubMed2026

Oncogene activation mechanism determines the limits of targeted protein degradation.

Protein degrader drugs such as PROTACs are being advanced as therapeutics targeted against oncogenic proteins. During tumorigenesis, oncogenic proteins can become constitutively activated via mechanisms including gene amplification, which increases protein production, and point mutations, which can extend protein half-life. Few experimental studies have addressed how disease-associated changes in target protein homeostasis influence PROTAC activity. We developed orthogonal methods to increase production or enhance stability of β-catenin, an important oncoprotein and target for degrader therapeutics, and used the dTAG system to evaluate the consequences for PROTAC activity. Stabilizing oncogenic missense mutations increase protein expression up to 5-fold but do not alter the PROTAC-imposed minimal steady-state level. In contrast, transcriptional upregulation increases both pre- and post-treatment target levels, revealing a synthesis-dependent ceiling on achievable depletion. Our results highlight distinct constraints on PROTAC activity arising from different mechanisms of oncogene activation, with potential implications for preclinical modeling, drug resistance and personalized medicine.

Humans↗
PubMed1979

Application of liver perfusion as an in vitro model in studies of intracellular protein degradation.

Amino acids appear to be prime regulators of autophagy and proteolysis in liver. They both attain a maximum rapidly when livers from fed rats are perfused in the single-pass mode without amino acids and are suppressed to basal levels by amino acid additions. The fact that their greatest responsiveness to amino acids occurs slightly below normal plasma levels suggests that these cellular processes could play a role in regulating plasma amino acid concentrations in vivo. Autophagy and proteolysis are also inhibited by insulin and stimulated by glucagon. In the latter instance the hormonal action is not direct but mediated indirectly by depletion of intracellular glutamine, probably as a consequence of enhanced gluconeogenesis. Close correlations among (1) rates of intracellular proteolysis, (2) the aggregate volume of lysosomal elements, and (3) estimates of degradable protein internalized within lysosomes indicate that lysosomal function can explain total intracellular protein degradation (with the possible exception of rapidly turning over fractions) over the full range of proteolysis from maximum down to and including the basal state. Since ratios of degradable intralysosomal protein to corresponding rates of proteolysis in intact liver are constant over this range, protein internalization may be the rate-limiting step in lysosomal proteolysis.

Amino Acids↗
PubMed2026

Post-translational chemical modification of E3 ligase for efficient target protein degradation.

Targeted protein degradation (TPD) has emerged as a powerful therapeutic strategy, with proteolysis-targeting chimeras (PROTACs) leading efforts to address previously undruggable targets. However, PROTACs face challenges such as low bioavailability and poor pharmacokinetic properties which limit their biological applications. Here, we report a strategy termed post-translational chemical modification targeting chimera (PTcM-TAC), which integrates ligand-directed chemistry into the PROTAC framework to achieve sustained target protein degradation through covalent modification of E3 ligases. PTcM-TAC incorporates an electrophilic dibromophenyl benzoate warhead into the linker connecting the E3 ligase ligand and the protein-of-interest (POI) ligand, enabling selective transfer of the POI ligand onto the recruited E3 ligase while releasing the E3-binding moiety. Mechanistic studies, including LC-MS/MS peptide mapping, pull-down assays, and structural modeling, demonstrated site-selective modification of CRBN by the PTcM-TAC. The resulting ligand-labeled E3 ligase enables sustained pseudo-catalytic target recognition through a simplified binary interaction, thereby maintaining degradation activity even after compound washout. Furthermore, we successfully applied the PTcM-TAC strategy to another representative E3 ligase, von Hippel-Lindau (VHL), which exhibited substantially sustained degradation activity compared with conventional PROTACs. To our knowledge, PTcM-TAC represents the first ligand-directed chemical strategy that converts transient PROTAC-mediated ternary complex formation into binary target recognition via post-translational chemical modification of an E3 ligase. We believe that PTcM-TAC could provide a platform for next-generation targeted protein degraders to overcome the current limitation of PROTAC approach.

Ubiquitin-Protein Ligases↗
PubMed1979

Protein degradation and the regulation of protein balance in muscle.

The rate of of protein degradation in muscle changes in many states but the nature of these changes is often paradoxical. Thus there can be increases during growth (anabolic increases) as well as during wasting (catabolic increases). Decreases can occur during growth (anabolic decreases) as well as during wasting (catabolic decreases). These changes are observed in man (as judged by changes in 3-methylhistidine excretion) and in experimental animals. The nature of the changes is not understood but it is possible that muscle growth induces increased degradation as part of the accompanying myofibre remodelling. The rate of protein degradation can also be influenced by thyroid status, since in thyroid deficiency degradation is reduced and can be stimulated by triiodothyronine. This response is independent of changes in muscle growth. Finally, acute exercise suppresses protein degradation in vivo in man as well as suppressing protein synthesis (in vivo in rats). When protein degradation rates change, acid proteinase activities also change in muscle. The anabolic increase in degradation appears to involve increases in mainly cathepsin D whereas catabolic increases in degradation are associated with an increase mainly in pepstatin-insensitive acid autolytic activity.

Adult↗
PubMed1976

Role of changes in protein degradation in the growth of regenerating livers.

The significance of changes in rates of synthesis, export, and degradation of proteins during liver regeneration was assessed. (a) Proteins were pulse labeled by the intravenous injection of radioactive leucine and, 5 min later, pactamycin (an inhibitor of the initiation of protein synthesis). One-half of the protein radioactivity was lost from the normal liver within 3 hours. From the radioactivity of the plasma proteins at that time and a study of the disappearance of these proteins from the circulation, it was calculated that 28% of the newly synthesized proteins were exported. Serum albumin accounted for a third of the exported proteins. Thirty-six hours after partial hepatectomy the proportion of albumin to total protein synthesis remained constant, while that of the other plasma proteins increased by 50%. The fraction of the newly synthesized proteins retained by the liver after 3 hours decreased by 20%. (b) During the first 36 hours of liver regeneration the average rates of protein degradation slowed down to one-half the normal values. This was determined either by the loss of radioactivity from total protein (or the guanidino-C of protein-bound arginine) in livers labeled with [14C]bicarbonate, or calculated as the balance between protein synthesis and net protein gain. (c) From these results, and those of our previous study of the protein synthetic machinery of normal and regenerating livers (Scornik, O.A. (1974)J. Biol. Chem. 249, 3876-3883), we conclude that changes in the rate of protein degradation are the single most important factor determining the increase in protein content during liver compensatory growth.

Animals↗
PubMed1991

The ubiquitin pathway for protein degradation.

Cellular proteins are marked for selective degradation by their ligation to the polypeptide ubiquitin. Recent studies have revealed information on the mechanisms involved in the selection of proteins for ligation to ubiquitin and on the mode of degradation of ubiquitinated proteins. Much remains to be learned about the high selectivity of this degradation pathway. Recent evidence that the cell-cycle regulatory proteins, cyclins, are degraded by the ubiquitin pathway points the way to future challenges in ubiquitin research.

Animals↗
PubMed1991

The effect of protein degradability on milk composition and production of early lactation, somatotropin-injected cows.

Twenty multiparous Holstein cows in early lactation that received 500 mg bST injected every 2 wk were assigned to one of two treatments to examine the influence of diets that varied in degradability of protein. Effects of degradability were determined on milk production and components and on nutrient digestibility. Treatments consisted of a basal ration (control) containing soybean meal as its primary degradable protein source and a ration (treatment) containing corn gluten and meat and bone meals as the primary undegradable protein source, representing 33% undegradable protein in CP. The undegradability of protein sources did not influence DMI and BW. Milk yield, 3.5% FCM, and production efficiency of bST-treated cows were not affected by increased undegradable protein in the diet. Milk fat and SNF were not significantly increased by treatment. Lactose was significantly higher for the control diet (5.0 vs. 4.9%) but was not biologically significant. Increasing undegradability of protein significantly increased total protein in milk and casein percentage in milk protein (3.14 vs. 2.86% and 62.11 vs. 58.24%, respectively). Total tract digestibility of nutrients was unaffected by treatment; however, CP digestibility tended to be higher as undegradability increased (67.85 vs. 62.83%).

Animal Feed↗
PubMed1991

Dietary fat, protein degradability, and calving season: effects on nutrient use and performance of early lactation cows.

Twelve multiparous Holstein cows calving in fall and 12 calving in summer were blocked into four groups and used in a 2 x 2 x 2 factorial to determine the effects of season of calving, dietary fat, and protein degradability on milk production and efficiency of NEL utilization in a 16-wk study. Blocks were assigned randomly to one of four dietary treatment combinations: 1) control concentrate plus soybean meal (high degradability protein supplement); 2) control concentrate plus a mixture of heated soybean meal and corn gluten meal (low degradability protein supplement); 3) a blend of the control concentrate and a concentrate containing 12.1% fat to provide 1 kg d-1 fat, plus soybean meal; and 4) concentrate as in diet 3 plus heated soybean meal and corn gluten meal. Nutrient intake, milk yield and composition, BW changes, and daily ambient temperature were monitored. Intake of DM appeared to be related to NDF intake but was not affected by fat, protein degradability, or calving season. Intake of NEL was increased by feeding fat. Digestabilities of DM and CP were increased and fiber was decreased by feeding fat. Percentage and yields of milk fat, SNF, and protein and 4% FCM production were higher in cows calving in fall. Milk fat percentage was low in all cows in the study. Efficiency of energy utilization for milk production was decreased in cows fed fat and calving in the summer and by low protein degradability during wk 5 to 8 of lactation. At high concentrate intake, calving season had more effect on milk production than level of fat or protein degradability.

Animal Feed↗
PubMed1992

Regulation of protein degradation in normal and transformed human bronchial epithelial cells in culture.

Protein degradation rates are decreased in some transformed cells of mesenchymal origin. We have tested the generality of this phenomenon and evaluated the role of the lysosomes in this down-regulation. To this end we have compared the induction of lysosomal protein degradation among normal, transformed (BEAS-2B), and transformed tumorigenic (BZR, Calu-1) human bronchial epithelial cells in culture. Serum and/or nutrient deprivation, cell confluency, and Ca2+ were used to modulate lysosomal protein degradation. Protein degradation and synthesis were determined by the release or incorporation of [14C]valine in the cells. Autophagic degradation of cytoplasm by lysosomes was evaluated by ultrastructural morphometry. Basal protein degradation was lower (27%) in two of the transformed cell lines (BEAS-2B and BZR). Incorporation of [14C]valine label was raised approximately 4-fold in the transformed cells. Nutrient deprivation stimulated protein degradation equally (2-fold) in transformed and normal cells. Postconfluency increased (1.5-fold) basal protein degradation in Calu-1 cells and a marked enhancement (4-fold) of degradation occurred during nutrient deprivation. Culture of normal human bronchial epithelial cells in high Ca2+ caused phenotypic changes and increased (30%) the degradation of protein induced by nutrient deprivation. In Calu-1, high Ca2+ caused only phenotypic changes. The volume density (Vd) of autophagic vacuoles and dense bodies in the transformed cells was lower under basal conditions but increased markedly during nutrient deprivation. A marked accumulation of lysosomes also occurred in transformed cells during postconfluency. We conclude that cell transformation lowers basal protein degradation in some human epithelial cells. Lysosomal proteolysis of transformed cells is not down-regulated and can be markedly enhanced during nutritional deprivation by the autophagic degradation pathway.

Analysis of Variance↗
PubMed1992

Alterations of protein degradation and 2-D protein pattern in muscle cells of MDX and DMD origin.

Intracellular protein turnover of MDX, DMD and normal muscle was determined by [35S]methionine pulse-chase experiments and subsequent high resolution 2-D gel electrophoresis. In MDX myotubes intracellular degradation of short-lived and long-lived proteins was markedly increased by a factor of 1,4-2,1. In wildtype the rate of degradation of short-lived proteins was approximately 2.6%/h, whereas in MDX these proteins were degraded by 5.7%/h. Long-lived proteins were degraded in wildtype at a rate of 1.8%/h, and in MDX at a rate of 2.5%/h. Furthermore, we have described a 51.000 Da protein with an IEP of 5.1 (p51/5.1), whose net content is highly and specifically reduced in cultured MDX and DMD muscle cells as well as in isolated MDX muscle fibers. Treatment with calcium-channel blockers Dantrolene and Verapamil inhibited the degradation of p51/5.1 in MDX myotubes by more than 90% in contrast to controls.

Actins↗
PubMed1979

Protein metabolism in lung: use of isolated perfused lung to study protein degradation.

This study investigates the use of the isolated perfused lung to study protein degradation. Proteins were labeled in vivo for 10 min or for 5 h using L-[U-14C]phenylalanine. When prelabeled lungs were perfused in vitro virtually all of the acid-soluble and acid-insoluble radioactivity in the tissue and perfusate remained as phenylalanine. Protein degradation was measured as the accumulation of free [14C]phenylalanine in ther perfusate; during the time this accumulated the amount of intracellular free phenylalanine and the free phenylalanine space remained constant. Proteins labeled during 10 min had a constant rate of degradation between 45 and 90 min of perfusion (about 11%.h-1); those labeled during 5 h had a constant rate of degradation for 90 (about 3%.h-1). The percent dry lung weight did not change during the perfusion. We conclude that measurable rates of proteolysis of "rapid" and "slowly" turning over proteins can be obtained while the lung is virtually free of edema. This system should allow studies on the modulation of proteolysis in intact lung under defined conditions.

Animals↗
PubMed1979

Protein metabolism in lung. II. Influence of amino acids and glucose on protein degradation.

We used the isolated perfused lung to study protein degradation. Proteins were labeled in vivo during 10 min (fast) or 5 h (slow). The absence of exogenous amino acids lowered the rate of proteolysis of fast but not of slowly turning over proteins. Addition of normal rat plasma levels of amino acids, after 45 min of perfusion without amino acids, returned the rate of proteolysis to control levels. The absence of exogenous glucose increased the rate of degration of rapidly turning over proteins but decreased the degradation rate of slowly turning over proteins. These changes took place in the absence of any measurable effect of amino acids or glucose on the amount of lung water, the rate of perfusate flow, the lung concentration of ATP or the intracellular concentration of free phenylalanine. We conclude that these substrates influence proteolysis in our system and that the degradation of rapidly and slowly turning over proteins are regulated independently in the isolated perfused lung.

Adenosine Triphosphate↗
PubMed1978

The effect of protein degradation on cellular growth characteristics.

The role of protein degradation in cellular proliferation was investigated by measurements of the rates of degradation of labile and stable proteins for a number of cell types under various growth conditions. The rate of protein degradation was found to be a relatively invariant parameter in that it did not change after strong inhibition of protein synthesis with cychloheximide or histidinol, it was the same in both exponential and stationary phase, and it did not correlate with the presence or absence of malignant transformation. Using three different cell types with widely differing division rates, the rate of cell division and DNA synthesis (in %/hr) was found to be precisely equal to the rate of protein accumulation (in %/hr) , i.e., to the rate of protein synthesis minus the rate of protein degradation. Division rates between the different cell types appeared to be determined chiefly by the rate of protein synthesis though, especially at low division rates, the rate of protein degradation could represent a large component of the protein accumulation rate.

Cell Division↗
PubMed1992

Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation.

Ubiquitin-dependent selective protein degradation serves to eliminate abnormal proteins and provides controlled short half-lives to certain cellular proteins, including proteins of regulatory function such as phytochrome, yeast MAT alpha 2 repressor, p53 and cyclin. Moreover, ubiquitin-dependent proteolysis is thought to play an essential role during development and in programmed cell death. We have cloned a gene from Drosophila melanogaster, UbcD1, coding for a protein with striking sequence similarity to the yeast ubiquitin-conjugating enzymes UBC4 and UBC5. These closely related yeast enzymes are known to be central components of a major proteolytic pathway of Saccharomyces cerevisiae. By doing a precise open reading frame replacement in the yeast genome we could show that the Drosophila UbcD1 enzyme can functionally substitute for yeast UBC4. UbcD1 driven by the UBC4 promoter rescues growth defects and temperature sensitivity of yeast ubc4 ubc5 double mutant cells. Moreover, expression of UbcD1 restores proteolysis proficiency in the ubc4 ubc5 double mutant, indicating that the Drosophila enzyme also mediates protein degradation. This structural and functional conservation suggests that the UbcD1-UBC4-UBC5 class of enzymes defines a major proteolytic pathway in probably all eukaryotes.

Amino Acid Sequence↗
PubMed1979

Muscle protein degradation in premature human infants.

1. Myofibrillar protein degradation has been measured by the rate of 3-methylhistidine excretion in premature infants weighing between 635 g and 1295 g. Analyses were made in conjunction with 1--3 day nitrogen balance studies. 2. In 56 balance studies in 36 infants, total muscle protein breakdown varied between 0.70 and 2.58 (mean 1.05) g day-1 kg-1 body weight while the percentage of total muscle protein degraded each day was between 3.3 and 8.3 (mean 4.8). 3. Both total and fractional rates of protein breakdown showed highly significant negative correlations with nitrogen retention but no relationship to total energy input. 4. Protein degradation was higher than average in infants who were losing weight at the time of the balance study, lower in infants who were gaining weight and higher in those who died within 2 weeks of the analysis. 5. Myofibrillar protein breakdown was not different between infants fed orally and those receiving total parenteral nutrition. 6. Generally the effects of nitrogen and evergy status on muscle protein degradation in the premature infants are different from changes reported in adult human beings or adult rats. We suggest that this difference may be a consequence of the very limited energy reserves of the premature infant.

Aging↗
PubMed1979

Intracellular protein degradation in Neurospora crassa.

In exponentially growing cultures of Neurospora crassa, the basal rate of protein degradation increases as the constant of the rate of growth decreases, so that in slow growing cells (mu = 0.13) the rate of protein degradation is about 25% of the rate of protein accumulation. During glucose starvation and shift-down transition of growth, the rate of protein degradation is greatly enhanced, and a moderate reduction (about 30%) of the ATP level is observed. Treatment of glucose-starved cells with 2-deoxyglucose reduces the ATP content by 70% and blocks protein degradation. The addition of cycloheximide, given at the onset of glucose starvation, prevents the enhancement of protein degradation; instead cycloheximide is without effect if added when proteolysis has already started. At a supraoptimal temperature (42 degrees C) the basal rate of protein degradation is not stimulated, contrary to the behavior observed in bacteria. Guanosine nucleotides, which appear to have a regulatory role for protein degradation in bacteria, are not found in N. crassa.

Amino Acids↗