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At least 19 recordsLinked to original sources
PubMed1996

Crude protein degradability, protein digestible in the intestine and net energy for lactation of whole crop barley in various vegetative stages.

Crude protein degradability (CP deg), protein digestible in the intestine (PDI) and net energy of lactation (NEL) content of whole crop spring barley were estimated by means of the in situ method using three steers fitted with rumen cannulas and fed near the maintenance level of nutrition. Barley was cut in five consecutive vegetative stages (end of the earing, S1, end of the flowering, S2, middle of the milky stage of the grain, S3, beginning of the dough stage, S4, middle of the dough stage, S5, including S5 silage). CP deg, PDI and NEL were significantly (P < 0.01) affected by the growth stage. The relationships of the CP deg (%), PDI (g/kg DM) and NEL (MJ/kg DM) to the vegetative stage of barley quantified on a decimal scale (X) were as follows: CP deg = 40.1 + 0.46X (I2yx = 82%), PDI = -330.8 + 12.86X-0.1007 X2 (I2yx = 83%), and NEL = -0.48 + 0.0802X (I2yx = 86%), resp. Compared with drying at 60 degrees C ensiling increased CP deg (P < 0.01) and NEL (P < 0.05), whereas PDI was not affected (P > 0.05).

Animals↗
PubMed2005

Signal-transduction networks and the regulation of muscle protein degradation.

Protein degradation in muscle functions in maintaining normal physiological homeostasis and adapting to new homeostatic states, and is required for muscle wasting or atrophy in various pathological states. The interplay between protein synthesis and degradation to maintain homeostasis is complex and responds to a variety of autocrine and intercellular signals from neuronal inputs, hormones, cytokines, growth factors and other regulatory molecules. The intracellular events that connect extracellular signals to the molecular control of protein degradation are incompletely understood, but likely involve interacting signal-transduction networks rather than isolated pathways. We review some examples of signal-transduction systems that regulate protein degradation, including effectors of proteolysis inducing factor (PIF), insulin and insulin-like growth factor (IGF) and their receptors, and fibroblast growth factor (FGF) and its receptors.

Animals↗
PubMed2004

Proteasome production in human muscle during nutritional inhibition of myofibrillar protein degradation.

Protein undernutrition inhibits adenosine triphosphate (ATP)-dependent muscle protein degradation-a hallmark of the proteasome system. Here we report decreased myofibrillar protein degradation during dietary protein restriction without a concomitant decrease in proteasome gene expression, proteasome protein abundance, or proteasome in vivo fractional synthesis rate. Healthy human subjects consuming the average minimum adult protein requirement (0.71 g x kg(-1) fat-free mass x d(-1)) exhibited substantially lower (68%) excretion of 3-methylhistidine, an indicator of myofibrillar protein breakdown, when compared with subjects consuming an ample, American-style protein intake (1.67 g x kg(-1) fat-free mass x d(-1)). However, they displayed no difference in the expression of mRNA for proteasome subunits C2 or C3, in the content of C2 protein, or in the rate of incorporation of stable isotopically labeled l-[1-(13)C]-leucine into proteasome proteins. The results demonstrate that nutritional inhibition of myofibrillar protein degradation does not involve suppression in vivo of proteasome production in man. This suggests that other elements of the ubiquitin-proteasome system, such as ubiquitination pathways, are more important than proteasome abundance in the nutritional regulation of skeletal muscle mass.

Adolescent↗
PubMed2006

A conserved role of Caenorhabditis elegans CDC-48 in ER-associated protein degradation.

Protein degradation mediated by the ubiquitin/proteasome system is essential for the elimination of misfolded proteins from the endoplasmic reticulum (ER) to adapt to ER stress. It has been reported that the AAA ATPase p97/VCP/CDC48 is required in this pathway for protein dislocation across the ER membrane and subsequent ubiquitin dependent degradation by the 26S proteasome in the cytosol. Throughout ER-associated protein degradation, p97 cooperates with a binary Ufd1/Npl4-complex. In Caenorhabditis elegans two homologs of p97, designated CDC-48.1 and CDC-48.2, exist. Our results indicate that both p97 homologs interact with UFD-1/NPL-4 in a similar CDC-48(UFD-1/NPL-4) complex. RNAi mediated depletion of the corresponding genes induces ER stress resulting in hypersensitivity to conditions which induce increased levels of unfolded proteins in the ER lumen. Together, these data suggest an evolutionarily conserved retro-translocation machinery at the endoplasmic reticulum.

Adenosine Triphosphatases↗
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 &#x3b2;-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↗
PubMed2003

Endoplasmic reticulum-associated protein degradation.

Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of the endoplasmatic reticulum (ER) are subjected to proteolysis by cytosolic 26 S proteasomes. This process, termed ER-associated protein degradation (ERAD), has also been implicated in the generation of some important human disorders, for example, cystic fibrosis. To become accessible to the proteasome, ERAD substrates must first be retrogradely transported from the ER into the cytosol, in a process termed dislocation. Surprisingly, protein dislocation from the ER seems to require at least some components that also mediate import into this compartment. Moreover, polyubiquitination of ERAD substrates at the ER membrane as well as the cytoplasmic Cdc48p/Npl4p/Ufd1p complex were shown to contribute to this export reaction. In this article we will summarize our current knowledge on ERAD and discuss the possible function of certain components involved in this process.

Animals↗
PubMed1991

Mechanisms of plasma membrane protein degradation: recycling proteins are degraded more rapidly than those confined to the cell surface.

Plasma membrane proteins of intact mouse 3T3 fibroblasts and H4-II-E-C3 hepatoma cells were separated into two groups based on their compartmentation between the cell surface and an intracellular compartment accessible at 20 degrees C but not at 0 degrees C. One group was derivatized at 0 degrees C with sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate but not at 20 degrees C. The second group was derivatized at 20 degrees C as well as at 0 degrees C. Derivatized proteins were isolated from 35S-labeled cells on streptavidin/agarose and resolved by two-dimensional PAGE. With few exceptions, pulse-chase experiments revealed that those proteins confined exclusively to the cell surface turned over slowly (t1/2, greater than 75 h), while those bimodally compartmentalized between the cell surface and the 20 degrees C accessible compartment were degraded more rapidly (t1/2, less than 31 h). These observations suggest a mechanism to explain the varied metabolic stability of plasma membrane proteins in which the half-life of each protein is determined by the proportion of time spent in the endocytic compartment.

Animals↗
PubMedDate not supplied

Search and destroy: ER quality control and ER-associated protein degradation.

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms. The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.

Animals↗
PubMed1987

Ruminal protein degradation and protein value of feeds.

The relationship between dry matter (DM) degradation and crude protein (CP) degradation in the dairy cow's rumen was determined with a view to defining the protein value of feeds for ruminants. The nylon bag technique was applied for these studies. For all the feeds investigate (green fodder and preserves from cocks-foot, ryegrass, alfalfa/grass and meadow grass, as well as alfalfa, extracted soybean meal) a significantly positive relationship was found to exist between the levels of DM and CP degradation (r = 0.73 to 1.0). The regression coefficient b1 (CP degradation as regressor) was found to average 0.87. The positive relationship between DM degradation and CP degradation implies that microbial protein amount and unfermented feed protein at the duodenum are negatively correlated. Model calculations show that, on account of the compensation between microbial protein and feed protein at the duodenum, in feeds with a CP concentration below 200 g/kg DM, the extent of ruminal protein degradation does not exert a marked influence on duodenal protein passage. The partial calculation of the duodenal protein supply on the basis of undegraded feed protein and microbial protein, as practiced in the new models of protein evaluation, leads to systematic errors unless the relationship between DM degradation and CP degradation is considered.

Animal Feed↗
PubMed1998

Control of neuronal size homeostasis by trophic factor-mediated coupling of protein degradation to protein synthesis.

We demonstrate that NGF couples the rate of degradation of long-lived proteins in sympathetic neurons to the rate of protein synthesis. Inhibiting protein synthesis rate by a specific percentage caused an almost equivalent percentage reduction in the degradation rate of long-lived proteins, indicating nearly 1:1 coupling between the two processes. The rate of degradation of short-lived proteins was unaffected by suppressing protein synthesis. Included in the pool of proteins that had increased half-lives when protein synthesis was inhibited were actin and tubulin. Both of these proteins, which had half-lives of several days, exhibited no degradation over a 3-d period when protein synthesis was completely suppressed. The half-lives of seven other long-lived proteins were quantified and found to increase by 84-225% when protein synthesis was completely blocked. Degradation-synthesis coupling protected cells from protein loss during periods of decreased synthesis. The rate of protein synthesis greatly decreased and coupling between degradation and synthesis was lost after removal of NGF. Uncoupling resulted in net loss of cellular protein and somatic atrophy. We propose that coupling the rate of protein degradation to that of protein synthesis is a fundamental mechanism by which neurotrophic factors maintain homeostatic control of neuronal size and perhaps growth.

Actins↗
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↗
PubMedDate not supplied

Stabilization of a degradable protein by its overexpression in Escherichia coli.

Synthesis of proteins in Escherichia coli using recombinant DNA methodology has become an important tool for isolating and studying proteins. However, the E. coli protein degradation systems can interfere with the expression of cloned genes. To examine the effect of protein degradation, we have cloned the X90 allele of the E. coli lacZ gene. The X90 allele, an ochre mutant, codes for beta-galactosidase lacking approx. 12 amino acids from the carboxyl terminus. The X90 protein is rapidly degraded in wild-type E. coli. Randomly sheared DNA fragments from lambda placZ-X90 were inserted into the EcoRI site of the plasmid pOP203-UV5-3, a derivative of pMB9 containing the lactose operator-promoter region. Recombinant plasmids that carry the lacZ-X90 gene were identified by the Lac+ phenotype of their transformants in an ochre-suppressor-containing host and the Lac- phenotype in Su degrees or supE hosts. One recombinant plasmid, p41, with an insert of 7.6 kb codes for the synthesis of the X90 promoter at a quantity equal to or greater than 50% of the total cellular protein of several strains. In contrast to the normal situation, the X90 molecules synthesized in great excess from the plasmid are stable in Su degrees hosts and can be recovered primarily from the 10 000 X g pellets of sonication lysates. The surprising stability of the overproduced X90 protein may be due to the formation of proteinaceous aggregates.

Bacterial Proteins↗
PubMed1986

Supplemental protein degradation, bacterial protein synthesis and nitrogen retention in sheep eating sodium hydroxide-treated straw.

1. Alkali (sodium hydroxide)-treated wheat straw was given to six rumen- and abomasal-cannulated sheep to study the rumen degradation of cotton-seed meal (CSM) and barley (B), and the effects of these supplements on nitrogen retention and efficiency of bacterial protein synthesis were measured. 2. N degradation, using porous synthetic (nylon) bags incubated within the rumen (P), and in vivo measurement determined from the abomasal flow of N (V), distinguished quantitatively between the two supplements. Estimates of P, corrected for fractional outflow rates/h (FOR), underestimated estimates of V when FOR of undegraded protein from the rumen (k) of 0.05 and 0.08 were used. Estimates of V for CSM and B were 70.9 and 80.8% respectively. 3. Intakes of alkali-treated straw were not affected by the supplements. Intakes of digestible organic matter (DOM) for the diets comprising alkali-treated straw alone (W), straw plus CSM (WC) and straw plus barley (WB) were 477, 575 and 590 g/d respectively (P less than 0.05) and organic matter (OM) apparently digested in the rumen (OMADR) was 339, 399 and 435 g/d respectively (P less than 0.05). 4. On W, WC and WB respectively, flows at the abomasum were 11.0, 14.0 and 13.3 g/d for bacterial N (P less than 0.05) and 0, 2.8 and 0.5 g/d for dietary supplemental N; g bacterial N/kg OMADR were 32.4, 35.6 and 30.9 (P greater than 0.05) and N balances were 2.37, 4.27 and 3.29 g/d (P less than 0.05) on the respective treatments. It was suggested that supplements increased total OM intake as a result of increased OM digested in the rumen rather than OM flow from the rumen.

Animal Feed↗
PubMed1998

Role of calpain in skeletal-muscle protein degradation.

Although protein degradation is enhanced in muscle-wasting conditions and limits the rate of muscle growth in domestic animals, the proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The goals of this study were to evaluate the roles of the calpains (calcium-activated cysteine proteases) in mediating muscle protein degradation and the extent to which these proteases participate in protein turnover in muscle. Two strategies to regulate intracellular calpain activities were developed: overexpression of dominant-negative m-calpain and overexpression of calpastatin inhibitory domain. To express these constructs, L8 myoblast cell lines were transfected with LacSwitch plasmids, which allowed for isopropyl beta-D-thiogalactoside-dependent expression of the gene of interest. Inhibition of calpain stabilized fodrin, a well characterized calpain substrate. Under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain reduced protein degradation by 30%, whereas calpastatin inhibitory domain expression reduced degradation by 63%. Inhibition of calpain also stabilized nebulin. These observations indicate that calpains play key roles in the disassembly of sarcomeric proteins. Inhibition of calpain activity may have therapeutic value in treatment of muscle-wasting conditions and may enhance muscle growth in domestic animals.

Amino Acid Sequence↗
PubMed1982

Effects of cold exposure on feed protein degradation, microbial protein synthesis and transfer of plasma urea to the rumen of sheep.

1. Three diets of barley-canola-seed (Brassica campestris), lucerne (Medicago sativa) or chopped brome-grass (Bromus inermis) were given at intervals of 3 h to closely-shorn Suffolk wethers held at a temperature of 1-5 degree (cold) or 22-24 degree (warm). Apparent digestibility of organic matter (OM) and nitrogen was reduced by 0.08-0.05 and 0.04 units respectively for lucerne and brome-grass diets given to cold-exposed sheep, but no treatment effects on digestibility were observed for the barley-CSM diet. Measurements achieved using infusion of the digesta markers 58Co-EDTA and 103Ru-phenanthroline (103Ru-P) showed that cold exposure depressed apparent OM digestion in the stomach and intestines by 33 and 42 g/d for the lucerne diet, and 13 and 35 g/d for the brome-grass diet respectively. 2. The turnover time (h) of the 103Ru-P marker in the rumen of warm sheep was 38.9 for barley-CSM, 18.4 for lucerne, and 15.6 for brome-grass. In cold-exposed sheep, 103Ru-P turnover time (h) tended to be reduced to 32.3, 12.3 and 15.3 for the three diets, respectively. OM fermentation in the stomach was highly related to 103RU-P turnover time for lucerne and brome-grass diets. 3. Cold exposure increased the escape of dietary N from the abomasum by 0.04 and 0.09 of dietary N intake for sheep given lucerne and brome-grass diets respectively. Dietary N degradation was closely related to 103Ru-P turnover time for lucerne, and to the proportion of large particles in rumen digesta for the brome-grass diet. Estimates of feed N degradation made by use of information on the rate of fermentation of the diet in nylon bags and 103Ru-P turnover time were consistently lower than those observed in vivo for barley-CSM and lucerne diets. Intestinal digestibility of non-ammonia N was not significantly changed by cold exposure. 4. Transfer of urea from plasma to the rumen was 1.4-2.5 g N/d for the barley-CSM and lucerne diets, but the value for brome-grass was 4.5-4.9 g N/d. Cold exposure did not affect urea transfer. The production of ammonia from feed and endogenous protein was approximately 0.66 and 0.47 g N/g N intake of barley-CSM and lucerne diets, with no effect of cold exposure. Cold exposure reduced the value from 0.57 to 0.38 for brome-grass. 5. The results are compared with those obtained previously with pelleted hay, and the importance of large particle breakdown in the prediction of OM and N fermentation using nylon bags is discussed.

Animal Feed↗