Monitoring newly synthesized proteins over the adult life span of Caenorhabditis elegans.

Little is known regarding how the synthesis and degradation of individual proteins change during the life of an organism. Such knowledge is vital to understanding the aging process. To fill this knowledge gap, we monitored newly synthesized proteins on a proteome scale in Caenorhabditis elegans over time during adulthood using a stable-isotope labeling by amino acids in cell culture (SILAC)-based label-chase approach. For most proteins, the rate of appearance of newly synthesized protein was high during the first 5 days of adulthood, slowed down between the fifth and the 11th days, and then increased again after the 11th day. However, the magnitude of appearance rate differed significantly from protein to protein. For example, the appearance of newly synthesized protein was fast for proteins involved in embryonic development, transcription regulation, and lipid binding/transport, with >70% of these proteins newly synthesized by day 5 of adulthood, whereas it was slow for proteins involved in cellular assembly and motility, such as actin and myosin, with <70% of these proteins newly synthesized even on day 16. The late-life increase of newly synthesized protein was especially high for ribosomal proteins and ATP synthases. We also investigated the effect of RNAi-mediated knockdown of the rpl-9 (ribosomal protein), atp-3 (ATP synthase), and ril-1 (RNAi-induced longevity-1) genes and found that inhibiting the expression of atp-3 and ril-1 beginning in late adulthood is still effective to extend the life span of C. elegans.

Global fold of human cannabinoid type 2 receptor probed by solid-state 13C-, 15N-MAS NMR and molecular dynamics simulations.

The global fold of human cannabinoid type 2 (CB2 ) receptor in the agonist-bound active state in lipid bilayers was investigated by solid-state (13)C- and (15)N magic-angle spinning (MAS) NMR, in combination with chemical-shift prediction from a structural model of the receptor obtained by microsecond-long molecular dynamics (MD) simulations. Uniformly (13)C- and (15)N-labeled CB2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. (13)C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of Cα, Cß, and C=O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the (13) C chemical shift distribution of Cα resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB2. Thus the shape of the Cα band can be used as an indicator of CB2 global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥ 1.5 ppm for carbons and ≥ 5.0 ppm for nitrogens). Simulated two-dimensional (13) Cα(i)-(13)C=O(i) and (13)C=O(i)-(15)NH(i + 1) dipolar-interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB2 by solid-state MAS NMR.

Conformational flexibility in the catalytic triad revealed by the high-resolution crystal structure of Streptomyces erythraeus trypsin in an unliganded state.

With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.

Recombinant cannabinoid type 2 receptor in liposome model activates g protein in response to anionic lipid constituents.

Human cannabinoid type 2 (CB(2)) receptor expressed in Escherichia coli was purified and successfully reconstituted in the functional form into lipid bilayers composed of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), and cholesteryl hemisuccinate (CHS). Reconstitution was performed by detergent removal from the protein/lipid/detergent mixed micelles either on an adsorbent column, or by rapid dilution to below the critical micelle concentration of detergent followed by removal of detergent monomers on a concentrator. Proteoliposomes prepared at a protein/phospholipid/CHS molar ratio of 1/620-650/210-220 are free of detergent as shown by (1)H NMR, have a homogeneous protein/lipid ratio shown by isopycnic gradient ultracentrifugation, and are small in size with a mean diameter of 150-200 nm as measured by dynamic light scattering. Functional integrity of the reconstituted receptor was confirmed by quantitative binding of (2)H-labeled agonist CP-55,940-d(6) measured by (2)H magic angle spinning NMR, as well as by activation of G protein. The efficiency of G protein activation by agonist-bound CB(2) receptor was affected by negative electric surface potentials of proteoliposomes controlled by the content of anionic CHS or POPS. The activation was highest at an anionic lipid content of about 50 mol %. There was no correlation between the efficiency of G protein activation and an increase of hydrocarbon chain order induced by CHS or cholesterol. The results suggest the importance of anionic lipids in regulating signal transduction by CB(2) receptor and other class A GPCR. The successful reconstitution of milligram quantities of pure, functional CB(2) receptor enables a wide variety of structural studies.

Rapid and effective removal of perfluorooctanoic acid from proteomics samples.

We recently demonstrated that perfluorooctanoic acid (PFOA), a volatile surfactant, is as effective as sodium dodecyl sulfate at solubilizing the membrane proteins. PFOA can be removed by repeated evaporation prior to mass spectrometry analysis. However, the removal of PFOA by evaporation is a lengthy process that takes approximately 6 h. Toward the goal of decreasing the length of time required to remove PFOA from protein digests, we tested the efficiency of PFOA removal and subsequent peptide recovery using strong cation exchange (SCX) chromatography, hydrophilic interaction chromatography (HILIC), fluorous solid phase extraction (FSPE), and anion exchange (ANX) chromatography. We found that all these chromatographic techniques except ANX chromatography remove PFOA thoroughly from protein digest. Peptide recovery rates from the SCX chromatography varied widely; nonacidic peptides were recovered at a rate of up to 95%, while acidic peptides were recovered at a rate of less than 10%. On the other hand, acidic peptides were recovered well from HILIC, while peptides whose pIs are greater than 6 were recovered poorly. Peptide recovery using FSPE was considerably lower, less than 10% for most of the peptides. These results indicate that the SCX and HILIC chromatography provide a more rapid alternative to the evaporation method for applications in which recovery of entire set of peptides is not required.

Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay.

Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min(-1) × 10(-3)), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail.

Stabilization of functional recombinant cannabinoid receptor CB(2) in detergent micelles and lipid bilayers.

Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB(2), and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB(2), here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB(2) were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of (2)H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB(2) in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB(2) reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.

Streptomyces erythraeus trypsin inactivates α1-antitrypsin.

Streptomyces erythraeus trypsin (SET) is a serine protease that is secreted extracellularly by S. erythraeus. We investigated the inhibitory effect of α(1)-antitrypsin on the catalytic activity of SET. Intriguingly, we found that SET is not inhibited by α(1)-antitrypsin. Our investigations into the molecular mechanism underlying this observation revealed that SET hydrolyzes the Met-Ser bond in the reaction center loop of α(1)-antitrypsin. However, SET somehow avoids entrapment by α(1)-antitrypsin. We also confirmed that α(1)-antitrypsin loses its inhibitory activity after incubation with SET. Thus, our study demonstrates that SET is not only resistant to α(1)-antitrypsin but also inactivates α(1)-antitrypsin.

Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the alpha subunit of GS protein.

The serotonin type 6 (5-HT(6)) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (G(S)). To identify the structural basis for the interaction of the 5-HT(6) receptor with the G(S) protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT(6) receptor and the alpha subunit of G(S) (Galpha(S)). The direct interaction of iL3 and Galpha(S) was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and Galpha(S) were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10(-6)M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to Galpha(S). The critical residues within the iL3 region for the interaction with Galpha(S) were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT(6) receptor mutants.

Purification and characterization of human nucleolar phosphoprotein 140 expressed in Escherichia coli.

Human nucleolar phosphoprotein 140, hNopp140, is one of the most highly phosphorylated mammalian proteins, which is involved in the biogenesis of nucleolus. It regulates the transcription of rDNA and has a tendency to bind to doxorubicin, which is widely used as an anti-cancer drug. The biochemical and biophysical property of hNopp140 has not been reported due to the fact that it is rather difficult to obtain protein in large enough quantity. In this paper, we report the cloning and overexpression of the soluble form of hNopp140 in Escherichia coli. The protein was purified to more than 90% homogeneity using hydroxyapatite and ion exchange chromatography. The purified protein can be extensively phosphorylated by casein kinase II and oligomerized into an insoluble aggregate in the presence of magnesium, carbonate, and fluoride ions.