Protein interactions among Fe65, the low-density lipoprotein receptor-related protein, and the amyloid precursor protein

The adapter protein Fe65 has been proposed to be the link between the intracellular domains of the amyloid precursor protein, APP (AICD), and the low-density lipoprotein receptor-related protein (LRP-CT). Functional linkage between these two proteins has been established, and mutations within LRP-CT affect the amount of Aβ produced from APP. Previous work showed that AICD binds to protein interaction domain 2 (PID2) of Fe65. Although the structure of PID1 was determined recently, all attempts to demonstrate LRP-CT binding to this domain failed. We used biophysical experiments and binding studies to investigate the binding among these three proteins. Full-length Fe65 bound more weakly to AICD than did N-terminally truncated forms; however, the intramolecular domain-domain interactions that had been proposed to inhibit binding could not be observed using amide H-D exchange. Surprisingly, when LRP-CT is phosphorylated at Tyr4507, it bound to Fe65 PID1 despite the fact that this domain belongs to the Dab-like subclass of PIDs that are not supposed to be phosphorylation-dependent. Mutation of a critical arginine abolished binding, providing further proof of the phosphorylation dependence. Fe65 PID1 thus provides a link between the Dab-like class and the IRS-like class of PIDs and is the first Dab-like family member to show phosphorylation-dependent binding.     

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.     

Expanding Proteome Coverage with Orthogonal-specificity α-Lytic Proteases

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage through cleavage at sequences complementary to trypsin''s may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type α-lytic protease (WaLP) and an active site mutant of WaLP, M190A α-lytic protease (MaLP). We assess several relevant factors, including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. When data from separate digestions with trypsin, LysC, WaLP, and MaLP were combined, proteome coverage was increased by 101% relative to that achieved with trypsin digestion alone. To demonstrate how the gained sequence coverage can yield additional post-translational modification information, we show the identification of a number of novel phosphorylation sites in the Schizosaccharomyces pombe proteome and include an illustrative example from the protein MPD2 wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.The most powerful technique for system-scale protein measurement, or proteomics, is mass-spectrometry-based proteomics (1). Although great progress has enabled the quantification of nearly all proteins expressed in yeast (2, 3), sequence coverage is often dismal, with some proteins being identified by a single peptide sequence. Complete amino acid coverage is valuable for comprehensive profiling of post-translational modifications (e.g. phosphorylation) and for quantification of splice variants. Low observed proteome coverage can be caused by several factors, including the wide dynamic range of protein concentrations in biological samples, splice variants, and unanticipated or unconsidered post-translational modifications (PTMs).¹ Improvements to every step of the bottom-up proteomics workflow continue to increase the observable proteome.Because of length constraints that limit observable peptides, proteome coverage is ultimately limited by proteome digestion. Typically, identifiable peptides are between 7 and 35 amino acids in length, with the lower limit being determined by sequence uniqueness and the upper limit being determined by the instrument''s resolving power (4). In silico proteome digestions predict that nearly one-quarter of peptides generated from tryptic digestion of the Saccharomyces cerevisiae proteome will be only a single amino acid long. Sequences lost due to length overall result in a theoretical upper proteome coverage limit of 68.8% according to in silico predictions (supplemental Fig. S1).Recently, several groups have demonstrated that combining data from separate protease digestions improves proteome coverage (4–7). Improved peptide yield was also shown, allowing proteome analysis of small-quantity samples from laser-capture microdissection (8, 9). Swaney et al. used trypsin, Lys-C, Arg-C, Glu-C, and Asp-N to double the observed S. cerevisiae nonredundant amino acid coverage from 11.9% to 25.5% (4).Other proteases that are used in proteomics to complement trypsin mainly cleave at ionic amino acid side chains, and it would be useful to have proteases with additional, complementary specificities. Here we demonstrate the application of wild-type α-lytic protease (WaLP) (10) and an active site mutant of WaLP, M190A α-lytic protease (MaLP) (11), to proteome digestion for shotgun proteomics. Both were reported to have specificity for cleaving after aliphatic side chains, which are more common amino acids. WaLP is a serine protease secreted from the soil bacteria Lysobacter enzymogenesis (10, 12) and has been studied extensively via mutagenesis and biophysical methods (11). WaLP has been found to exhibit remarkable stability (13, 14).Non-tryptic peptides are more difficult to identify than tryptic peptides, especially when lacking defined termini (i.e. from semi-specific protease digestion or endogenous peptides) due to increased database search space and less predictable ionization and fragmentation. A lack of defined termini drastically increases database search space because more possible peptides fall within the precursor tolerance and drive up false positive rates (15). The majority of tryptic peptides have one positive charge localized at each terminus in a +2 precursor charge state upon electrospray ionization, which results in well-characterized fragmentation by collision-induced dissociation (CID) (16, 17). Non-tryptic peptides, in contrast, may lack positively charged side chains (i.e. Arg, Lys, His) altogether, making it unlikely that multiple charges will be obtained upon electrospray ionization. Those that do contain positive charges away from the C terminus produce less predictable fragmentation upon CID. Recently, additional peptide fragmentation methods have become accessible, such as electron-transfer dissociation (ETD) (18), which produces fragment ion series that are less dependent on peptide sequence, and higher-energy collisional dissociation (HCD) (19). An in-depth comparison of activation methods for non-tryptic peptide identification has been published recently by Smith''s lab. In that report the authors evaluated FT-CID, FT- ETD, and FT-HCD for sequencing peptides isolated from blood plasma (20).To enable application of the α-lytic proteases with specificity for aliphatic amino acid side chains to shotgun proteomics, we address the above issues by comparing multiple fragmentation modes in combination with the peptide identification algorithm MS-GFDB, which easily learns scoring parameters from an initial set of annotated peptide-spectrum matches for arbitrary fragmentation methods and proteases (21). We analyzed standard protein mixtures and complex Schizosaccharomyces pombe proteomes digested with trypsin, LysC, WaLP, and MaLP. Specifically, we assessed ion activation methods, observed peptide character, and biological gains due to additional digestions. The results present the pros and cons of using orthogonal proteases in proteomics.     

Production of large quantities of isotopically labeled protein in Pichia pastoris by fermentation

Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P.pastoris occurs in simple minimal defined media making this system attractive for production of labeled proteins for NMR analysis. P.pastoris is therefore the expression system of choice for NMR of proteins that cannot be refolded from inclusion bodies or that require post-translational modifications for proper folding or function. The yield of expressed proteins from P.pastoris depends critically on growth conditions, and attainment of high cell densities by fermentation has been shown to improve protein yields by 10–100-fold. Unfortunately, the cost of the isotopically enriched fermentation media components, particularly 15NH4OH, is prohibitively high. We report fermentation methods that allow for both 15N- labeling from (15NH4)2SO4 and 13C-labeling from 13C-glucose or 13C-glycerol of proteins produced in Pichia pastoris. Expression of an 83 amino acid fragment of thrombomodulin with two N-linked glycosylation sites shows that fermentation is more cost effective than shake flask growth for isotopic enrichment.     

Thrombin-binding affinities of different disulfide-bonded isomers of the fifth EGF-like domain of thrombomodulin.

The fifth EGF-like domain of thrombomodulin (TM), both with and without the amino acids that connect the fifth domain to the sixth domain, has been synthesized and refolded to form several different disulfide-bonded isomers. The domain without the connecting region formed three disulfide-bonded isomers upon refolding under redox conditions. Of these three isomers, the (1-2,3-4,5-6) bonded isomer was the best inhibitor of fibrinogen clotting and also of the thrombin-TM interaction that results in protein C activation, but all the isomers were inhibitors in both assays. The isomer containing an EGF-like disulfide-bonding pattern (1-3,2-4,5-6) was not found among the oxidation products. The domain with the connecting region amino acids (DIDE) at the C-terminus formed two isolable products upon refolding in redox buffer. These products had the same two disulfide-bonding patterns as the earliest and latest eluting isomers of the domain without the DIDE. In order to compare the thrombin-binding affinities of these isomers to the isomer with the EGF-like disulfide bonds, acetamidomethyl protection of the second and fourth cysteines was used to force the disulfide bonds into the EGF-like pattern. Thrombin-binding affinity, measured as inhibition of fibrinogen clotting and as inhibition of protein C activation correlated inversely with the number of crossed disulfide bonds. As was found for the domain without the connecting region, the isomer that was the best inhibitor of fibrinogen clotting and of protein C activation was the isomer with no crossing disulfide bonds (1-2,3-4,5-6).(ABSTRACT TRUNCATED AT 250 WORDS)     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Inhibition of interferon secretion by vinblastine

The plant alkaloids vinblastine and colchicine are known to arrest cells in mitosis by virtue of their binding to spindle protein. These drugs are also capable of binding to microtubule protein and causing these structures to disaggregate into nonfunctional subunits (1, 2). Microtubular structures are thought to be involved in the secretory process of a number of proteins including insulin (7), collagen (4), and thyroid hormone (12). In this report we present our findings on the effects of these two drugs on the synthesis and secretion of interferon in a high producing human foreskin fibroblast strain (FS-4) (11).