Food protein fragments are regulatory oligopeptides

Until recently food proteins were considered to be an energy source and a source of essential and nonessential amino acids required for protein synthesis and precursors of many vital biomolecules. However, we assumed earlier that food protein fragments might perform some regulatory functions. The theoretical justification for this assumption is advanced in this work. In the present work, the primary structures of protein fragments were compared with amino acid sequences of known natural regulatory oligopeptides in silico. It is shown that fragments formed as a result of animal food protein cleavage by proteolytic enzymes can exist in the gastrointestinal tract for a long time. Many of them are enzyme inhibitors, regulators of nervous, endocrine, and immune system, and possess antimicrobial and other activities. It has also been shown that the lifetime of fragments before their cleavage in the gastrointestinal tract could be enough for performing corrective functions. Thus, as a result of food protein fragmentation a dynamic pool of exogenous regulatory oligopeptides with functions changing as shorter fragments are generated may form. The detection of an endogenous-exogenous pool of regulatory molecules expands the significance and content of the Ashmarin-Obukhova hypothesis on a functional continuum of natural oligopeptides. The possible practical importance of these results is noted.     

Estimating protein turnover with a [15N,13C]leucine tracer: a study using simulated data.

We explore the use of [15N,13C]leucine tracer to estimate whole-body fractional rates of a fast-turning-over protein pool employing synthetic data. The kinetics of [15N,13C]leucine tracer are simplified compared with those of traditional leucine tracers and benefit from irreversible transamination to [13C]alpha-ketoisocaproaic acid (KIC) resulting in a simplified model structure. A three-compartment model of [15N,13C]leucine kinetics was proposed and evaluated using data generated by a Reference Model (based on a model by Cobelli et al.). The results suggest that fractional turnover rates of a fast-turning-over protein pool can be estimated with a low but acceptable precision during a six-hour constant intravenous infusion of [15N,13C]leucine with frequent sampling of plasma tracer-to-tracee ratio (TTR) of [15N,13C]leucine. We conclude that [15N,13C]leucine may be useful for the measurement of protein kinetics and its full potential should be explored in clinical studies with compartmental data analysis.     

A plasma membrane pool of cholesteryl esters that may mediate the selective uptake of cholesteryl esters from high-density lipoproteins

Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL particles occurs by a nonendocytotic pathway that requires no specific apolipoprotein and results in the net delivery of cholesteryl esters to cells. Here we examine a reversibly cell-associated pool of cholesteryl ester tracer and its relationship to selective uptake. A fraction of cholesteryl ester tracer selectively taken up from HDL by rat primary or mouse Y1-BS1 adrenocortical cells was chased from the cells by subsequent incubation with unlabeled HDL. This pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL particles bound to the cell surface. In response to various metabolic effectors, cholesteryl ester tracer in this reversibly cell-associated pool of Y1-BS1 cells correlated linearly with irreversible selective uptake. Both reversibly and irreversibly cell-associated pools of cholesteryl ester tracer displayed similar saturation kinetics for uptake from HDL, and both pools correlated inversely with cell-free cholesterol levels. Cholesteryl ester tracer in the reversible pool was shown to serve as a precursor for irreversible selective uptake. A pool with properties similar to the reversibly cell-associated pool was identified in plasma membrane fractions; enough tracer was incorporated into this pool to account for the reversibly cell-associated pool of intact cells. The data suggest that a pool of cholesteryl esters in the plasma membrane is involved in selective uptake at a step prior to irreversible internalization.     

Loss of AS160 Akt substrate causes Glut4 protein to accumulate in compartments that are primed for fusion in basal adipocytes

The Akt substrate AS160 (TCB1D4) regulates Glut4 exocytosis; shRNA knockdown of AS160 increases surface Glut4 in basal adipocytes. AS160 knockdown is only partially insulin-mimetic; insulin further stimulates Glut4 translocation in these cells. Insulin regulates translocation as follows: 1) by releasing Glut4 from retention in a slowly cycling/noncycling storage pool, increasing the actively cycling Glut4 pool, and 2) by increasing the intrinsic rate constant for exocytosis of the actively cycling pool (k(ex)). Kinetic studies were performed in 3T3-L1 adipocytes to measure the effects of AS160 knockdown on the rate constants of exocytosis (k(ex)), endocytosis (k(en)), and release from retention into the cycling pool. AS160 knockdown released Glut4 into the actively cycling pool without affecting k(ex) or k(en). Insulin increased k(ex) in the knockdown cells, further increasing cell surface Glut4. Inhibition of phosphatidylinositol 3-kinase or Akt affected both k(ex) and release from retention in control cells but only k(ex) in AS160 knockdown cells. Glut4 vesicles accumulate in a primed pre-fusion pool in basal AS160 knockdown cells. Akt regulates the rate of exocytosis of the primed vesicles through an AS160-independent mechanism. Therefore, there is an additional Akt substrate that regulates the fusion of Glut4 vesicles that remain to be identified. Mathematical modeling was used to test the hypothesis that this substrate regulates vesicle priming (release from retention), whereas AS160 regulates the reverse step by stimulating GTP turnover of a Rab protein required for vesicle tethering/docking/fusion. Our analysis indicates that fusion of the primed vesicles with the plasma membrane is an additional non-Akt-dependent insulin-regulated step.     

Germination of wheat embryos and the transport of amino acids into a protein synthesis precursor pool

Wheat (Triticum aestivum L. var. Lew) embryonic axes take up externally supplied radioactive amino acid (from a solution greater than 2 millimolar) such that the specific radioactivity of the total internal amino acid rapidly reaches that of the external solution. Nevertheless, incorporation of radioactive amino acid into protein increases steadily as the concentration of external amino acid is increased, indicating that the amino acid that is precursor to protein synthesis is not in equilibrium with the total internal amino acid pool. When the external source of amino acid is removed, incorporation of radiolabeled amino acid into protein continues at a rate comparable to that of embryos maintained in the radioactive solution. In explanation of these data, it is suggested that there are two separate cytoplasmic pools of amino acids, one a protein synthesis precursor pool, and the second, an expandable pool into which exogenous radioactive amino acids are taken up. The protein synthesis pool is fed at a limited rate from the expandable pool and at a far greater rate from an endogenous source. As a consequence, the specific activity of the amino acid that is the precursor for protein synthesis is considerably below that of the total internal pool and is determined by the rate of movement into the protein synthesis pool from the expanded radioactive cytoplasmic pool.

The rate of movement of amino acids from the expandable pool into the protein synthesis pool increases approximately 5-fold during the initial 4.5 hours of embryo germination. When this change is considered in analyzing the relative rates of protein synthesis, there is probably no more than a 2-fold increase in protein synthetic capacity between embryos germinated for 1.5 and 4.5 hours. The leveling off of the change in transport capacity after 4.5 hours suggests that the earlier increase in the rate of this process may be a necessary step before the embryos can begin to accelerate their growth rate.

     

Measurement of myo-inositol turnover in phosphatidylinositol: description of a model and mass spectrometric method for cultured cortical neurons

Rates of myo-inositol (Ins) incorporation and turnover in phosphatidylinositol (PtdIns) were determined in cultured mouse cortical neurons. Cells were incubated with deuterium-labeled myo-inositol (Ins) in culture medium free of unlabeled Ins. The time-dependent changes in the specific activity of cytosolic Ins and membrane PtdIns were measured by mass spectrometry. PtdIns turnover was modeled incorporating values for Ins flux, cytosolic dilution, PtdIns concentration, and rate of incorporation into PtdIns. Recycled Ins diluted the labeled precursor pool, and a time course was obtained for this cytosolic process. The specific activity of the precursor pool at the plateau of the time-course curve was 0.43 +/- 0.04 (mean +/- SD). The incorporation of the tracer into PtdIns was linear between 4 and 10 h incubation of the neurons. After factoring in the extent of dilution of the tracer in the precursor pool, the rate of Ins incorporation into PtdIns was found to be 315 +/- 51 nmol (g of protein)(-1) x h(-1). The half-life of Ins in PtdIns was calculated for each point on the linear incorporation curve and then corrected for the tracer reincorporation. The half-life of Ins in PtdIns was 6.7 +/- 0.2 h, which translates into a basal turnover rate of 10.3%/h in this in vitro system. The mathematical model and the stable isotope method described here should allow assessment of the dynamics of PtdIns signaling altered in certain diseases or by agents.     

Characterization of stress and protein turnover from protein overexpression in fed-batch E. coli cultures.

A structured kinetic model that accounts for proteolytic degradation due to recombinant protein overexpression is introduced and its performance evaluated by comparison with previously reported fed-batch experimental data. This mathematical model contains an additional pool for a generic key precursor (in our case phenylalanine), an improved IPTG transport term, a phenylalanine transport term, and a variable protein turnover expression that accounts for proteolytic activity. The model predictions concerning proteolytic activity, glucose level, and cell growth are in very good agreement with an amino acid depletion hypothesis. Cultures exposed to greater stress showed higher and/or longer proteolysis, whereas less overall proteolytic activity was observed when the effect of induction was somewhat ameliorated.     

Identification of protein domains on topological basis

A theoretical method is proposed to identify structural domains in proteins of known structures. It is based on the distribution of the local axes of the polypeptide chain. In particular, a statistical analysis is applied to the contributions of the local axes to the absolute writhing number, a topological property of a space curve resulting from the number of self-crossings in the curve projections onto a unit sphere. This finding supports the hypothesis that topological requirements should be satisfied in the process of protein folding and in the final organization of the tertiary structures.     

Measuring VLDL-triglyceride turnover in humans using ex vivo-prepared VLDL tracer

There has been more interest in VLDL-triglyceride (TG) kinetics during the last decade. Unfortunately, robust measurement methods are elaborate and not readily available. Here, we describe a method using unique, ex vivo labeling of the fatty acid moiety of VLDL-TG followed by intravenous bolus infusion in the same person. We found that plasma disappearance of ex vivo-labeled VLDL-TG was comparable to that of in vivo-labeled VLDL-TG and that turnover rates can be safely estimated from the log linear decay of VLDL-TG specific activity. We found minor labeling of the plasma FFA (oleate) pool, which was largely attributable to coinfusion of free [14C]triolein; VLDL-TG did not contribute substantially to the plasma FFA pool. The plasma decay curve of VLDL-TG was not affected by the presence of tracer in the FFA pool, provided that the data from 2 h after the VLDL tracer bolus infusion was used. The FFA contamination problem was circumvented by minor modification of the VLDL-TG tracer preparation. The approach we describe should expand the opportunity to study processes that cannot be assessed if the FFA precursor pool is labeled. This method for VLDL-TG tracer preparation can allow measurement of VLDL turnover, tissue uptake of VLDL-TG, and oxidation of VLDL-TG.     

The effect of protein a cycle number on the performance and lifetime of an anion exchange polishing step

Most mAb platform purification processes consist of an affinity capture step followed by one or two polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak‐partitioning anion‐exchange chromatography polishing step used in a mAb purification process was characterized through high‐throughput screening (HTS) experiments, small‐scale experiments including a cycling study performed on qualified scale‐down models, and large‐scale manufacturing runs. When material from a Protein A column that had been cycled <10× was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness, and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in‐process control parameter for monitoring the performance of the AEX step. Biotechnol. Bioeng. 2013; 110: 1142–1152. © 2012 Wiley Periodicals, Inc.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

ATP-coupled transport of vesicular stomatitis virus G protein between the endoplasmic reticulum and the Golgi

The temperature and ATP dependence of transport of the vesicular stomatitis virus strain ts045 G protein from the endoplasmic reticulum (ER) to an early Golgi compartment containing mannosidase I was studied in the mutant Chinese hamster ovary cell clone 15B. Appearance of G protein containing the Man5GlcNAc2 oligosaccharide species occurred after a shift to the permissive temperature with a lag period of 5 min and without detectable formation of the intermediate Man7GlcNAc2 and Man6GlcNAc2 species. Two biochemically distinct transport steps were detected during transport from the ER to the Golgi. An initial step is temperature sensitive, thermoreversible, and requires a high threshold of cellular ATP for maximal rate of transport (80% of the normal cellular ATP pool). Export from the ER is inhibited at 65% of the normal cellular ATP pool. Prolonged incubation at reduced levels of cellular ATP or at the restrictive temperature resulted in the accumulation of G protein in either the Man8GlcNAc2 species or the Man7GlcNAc2 and Man6GlcNAc2 species, respectively. Reversal of the temperature-sensitive block is ATP coupled. A second step is insensitive to incubation at the restrictive temperature and proceeds efficiently when the cellular ATP pool is reduced to 20% of the control. G protein accumulates at this intermediate step during prolonged incubation at 15 degrees C. The data suggest a functional division of processes required for transport of protein between the ER and Golgi compartments. The two steps may reflect the export (budding) and delivery (fusion) of proteins through vesicular trafficking between the ER and Golgi.     

Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process.

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).     

The initiation of eukaryotic and prokaryotic protein synthesis: a selective accessibility and multisubstrate enzyme reaction

An extension of our unique accessibility hypothesis for the initiation of protein synthesis is proposed following a review of the initiation of protein synthesis. The E. coli model initiation sequence generated by computer from 68 initiation sequences and the eukaryotic consensus initiation sequence derived by non-computer analysis of 211 initiation sequences do not contain a specific base in any position; they are only assigned preferred bases. The initiation site, in other words, is a varied sequence of preferred bases and its sequence is non-unique. This indicates that the ribosomal recognition of the initiation site may be the result of multiple interactions that are cooperative and cumulative and typical of multisubstrate enzymes. Because of this characteristic, the model of multisubstrate enzymes with broad substrate specificity is proposed as a paradigm for the initiation of protein synthesis. As predicted by this model, changes in the leader and downstream sequences that improve the agreement with the preferred base sequence do indeed enhance the rate of protein synthesis. The eukaryotic/prokaryotic hybrid studies show a considerable overlap in the specificities of the two groups of ribosomes. The scanning of the mRNA from the 5'-end postulated by the scanning hypothesis is not a necessary step since eukaryotic ribosomes are able to bind to internal mRNA sites and initiate synthesis. Our unique accessibility hypothesis, which is extended by coupling cooperative and cumulative specificity in ribosomal function, is referred to for brevity as the cumulative specificity hypothesis. The hypothesis actually postulates a selective accessibility and cooperative-cumulative specificity mechanism; it is able to account for the behavior of both eukaryotic and prokaryotic initiation of protein synthesis. From another perspective, the hypothesis can be regarded as providing a mechanism that enables ribosomes to recognize the IS in the absence of a unique initiation sequence.     

Functional and morphological studies of protein transcytosis in continuous endothelia

Continuous microvascular endothelium constitutively transfers protein from vessel lumen to interstitial space. Compelling recent biochemical, ultrastructural, and physiological evidence reviewed herein demonstrates that protein transport is not the result of barrier "leakiness" but, rather, is an active process occurring primarily in a transendothelial vesicular pathway. Protein accesses the vesicular pathway by means of caveolae open to the vessel lumen. Vascular tracer proteins appear in free cytoplasmic vesicles within minutes; contents of transport vesicles are rapidly deposited into the subendothelial matrix by exocytosis. Caveolin-1 deficiency eliminates caveolae and abolishes vesicular protein transport; interestingly, exchange vessels develop a compensatory transport mode through the opening of a paracellular permeability pathway. The evidence supports the transcytosis hypothesis and the concept that transcytosis is a fundamental component of transendothelial permeability of macromolecules.     

Steady-state and time-resolved fluorescence studies of the intestinal fatty acid binding protein

The intestinal fatty acid binding protein contains two tryptophan residues (Trp6 and Trp82) both of which have been shown by X-ray and NMR methods to be buried in hydrophobic clusters. By using a combination of steady-state and time-resolved fluorescence experiments, we have deconvoluted the lifetime weighted contribution of each of the tryptophans to the steady-state fluorescence quantum yield. While Trp82 has been implicated in an intermediate that appears at relatively high denaturant concentrations, the variation of the lifetime weighted contribution of Trp6 with urea or guanidium hydrochloride shows formation of an intermediate state at low concentrations of the denaturant before the actual unfolding starts. Trp82 did not show similar behavior. Fluorescence quenching experiments by acrylamide show that while Trp6 in the native protein is less solvent-exposed, its accessibility is increased significantly at low urea concentration indicating that the early intermediate state is partially unfolded. Time-resolved anisotropy experiments indicate that the volume of the partially unfolded intermediates is larger than the native protein and lead to the speculation that the last step of the protein folding might be the removal of solvent molecules from the protein.     

Fluorescent tracer method for protein SH groups. II

An improved fluorescent tracer technique for protein SH groups is described using a fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide. The direct measurement with a scanning fluorometer of the fluorophore-labeled proteins separated by SDS polyacrylamide gel electrophoresis achieved a simple, precise, and sensitive determination of the SH group of the order of picomoles per band. In addition, the direct recording of peaks enabled us to analyze more complex protein systems, compared with our previously reported method using an extraction step from gel slices. As an application, we compared the reactivity of the SH groups of proteins in glycerinated muscle fibers under two conditions, in rigor and in contraction.     

Total-body creatine pool size and skeletal muscle mass determination by creatine-(methyl-d3) dilution in rats

There is currently no direct, facile method to determine total-body skeletal muscle mass for the diagnosis and treatment of skeletal muscle wasting conditions such as sarcopenia, cachexia, and disuse. We tested in rats the hypothesis that the enrichment of creatinine-(methyl-d(3)) (D(3)-creatinine) in urine after a defined oral tracer dose of D(3)-creatine can be used to determine creatine pool size and skeletal muscle mass. We determined 1) an oral tracer dose of D(3)-creatine that was completely bioavailable with minimal urinary spillage and sufficient enrichment in the body creatine pool for detection of D(3)-creatine in muscle and D(3)-creatinine in urine, and 2) the time to isotopic steady state. We used cross-sectional studies to compare total creatine pool size determined by the D(3)-creatine dilution method to lean body mass determined by independent methods. The tracer dose of D(3)-creatine (<1 mg/rat) was >99% bioavailable with 0.2-1.2% urinary spillage. Isotopic steady state was achieved within 24-48 h. Creatine pool size calculated from urinary D(3)-creatinine enrichment at 72 h significantly increased with muscle accrual in rat growth, significantly decreased with dexamethasone-induced skeletal muscle atrophy, was correlated with lean body mass (r = 0.9590; P < 0.0001), and corresponded to predicted total muscle mass. Total-body creatine pool size and skeletal muscle mass can thus be accurately and precisely determined by an orally delivered dose of D(3)-creatine followed by the measurement of D(3)-creatinine enrichment in a single urine sample and is promising as a noninvasive tool for the clinical determination of skeletal muscle mass.     

What determines the intracellular ATP concentration

Analysis is made of the mechanisms that control the intracellular ATP level. The balance between energy production and expenditure determines the energy charge of the cell and the ratio of [ATP] to the adenylate pool. The absolute ATP concentration is determined by the adenylate pool, which, in its turn, depends on the balance between the rates of AMP synthesis and degradation. Experimental data are discussed that demonstrate an increase in the adenylate pool in response to activation of energy-consuming processes. A hypothesis is proposed according to which variation in the adenylate pool and absolute ATP concentration affords a cell the possibility of additional control over processes fulfilling useful work. A mechanism involved in this regulation is described using human erythrocytes as an example. The hypothesis explains why different metabolic pathways (protein and DNA syntheses, polysaccharide synthesis, and lipid synthesis) use different trinucleotides (GTP, UTP, and CTP, respectively) as an energy source. This allows the cell to independently control these metabolic processes by varying the individual nucleotide pools.     

Effect of ouabain,amiloride, and antidiuretic hormone on the sodium-transport pool in isolated epithelia from frog skin (Rana temporaria)

Summary When tracer Na⁺ is added to the solution bathing the apical side of isolated epithelia the observed transepithelial tracer influx increases with time until a steady state is reached. The build-up of the tracer flux follows a single exponential course. The halftime for this build-up under control conditions was 0.92 ±0.06 min, and in the presence of ouabain 4.51±0.7 min. It is shown that the calculated Na⁺-transport pool is located in the cells. The Na⁺-transport pool under control conditions was 35.6 ±3.4 nmol/cm², which corresponds to an intracellular Na⁺ concentration of 7.9mm. Activation of the active Na⁺ transport by addition of antidiuretic hormone resulted in a highly significant increase in the Na⁺ transport pool, and inhibition of the transcellular Na⁺ transport with amiloride resulted in a decrease in the Na⁺-transport pool.Furthermore, the active Na⁺ transport increased along anS-shaped curve with increasing intracellular Na⁺ concentration (Na⁺-transport pool). The Na⁺ pump was found to be half saturated at an intracellular Na⁺ concentration of 12.5mm.