Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease

The structural proteins of Sindbis virus are translated as a polyprotein precursor that is cleaved upon translation. The capsid protein is postulated to be a serine protease that releases itself from the N terminus of the nascent polyprotein by autoproteolysis. We have tested the importance in autoproteolysis of His-141, Asp-147, and Ser-215, previously postulated to form the catalytic triad of the protease, and of Asp-163. Several site-specific mutations were constructed at each of these positions, and the release of the capsid protein during translation in a cell-free system was examined. Because proteolysis occurs in cis during translation, the kinetics of release cannot be determined in this system, but the extent of proteolysis can be ascertained. Ser-215 appears to be the catalytic serine of the proteinase. Cys or Thr could substitute inefficiently for Ser-215, but substitution with Ala or Ile led to complete loss of activity. His-141 was also important for proteolysis. Substitution with Ala or Pro led to total loss of activity. Surprisingly, substitution with Arg resulted in complete proteolysis in vitro. Changes at the two Asp residues resulted in complete proteolysis of the substrate in vitro. All mutations that resulted in at least partial cleavage in vitro were incorporated into a full-length clone of Sindbis virus and an attempt was made to recover mutant virus. All changes tested were lethal for the virus except Asp-163 to Asn. Thus, production of infectious virus is either a more sensitive measure of the catalytic rate than the extent of in vitro cleavage, or these residues have necessary functions in addition to their possible role in proteolysis

The model of proteolysis

This document presents the original approach for estimating parameters of proteolysis process. Data used to fit the model are taken from mass
spectrometric experiments. For parameters estimation the Levenberg-Marquadt algorithm is used. The motivation for model is a hypothesis
that discrimination between cancer patients and healthy donors can be based on activity of peptide cleaving enzymes (i.e. peptidases)

IKKα and IKKβ Regulation of DNA Damage-Induced Cleavage of Huntingtin

Background: Proteolysis of huntingtin (Htt) plays a key role in the pathogenesis of Huntington's disease (HD). However, the environmental cues and signaling pathways that regulate Htt proteolysis are poorly understood. One stimulus may be the DNA damage that accumulates in neurons over time, and the subsequent activation of signaling pathways such as those regulated by IκB kinase (IKK), which can influence neurodegeneration in HD. Methodology/Principal Findings: We asked whether DNA damage induces the proteolysis of Htt and if activation of IKK plays a role. We report that treatment of neurons with the DNA damaging agent etoposide or γ-irradiation promotes cleavage of wild type (WT) and mutant Htt, generating N-terminal fragments of 80–90 kDa. This event requires IKKβ and is suppressed by IKKα. Elevated levels of IKKα, or inhibition of IKKβ expression by a specific small hairpin RNA (shRNA) or its activity by sodium salicylate, prevents Htt proteolysis and increases neuronal resistance to DNA damage. Moreover, IKKβ phosphorylates the anti-apoptotic protein Bcl-xL, a modification known to reduce Bcl-xL levels, and activates caspases that can cleave Htt. When IKKβ expression is blocked, etoposide treatment does not decrease Bcl-xL and activation of caspases is diminished. Similar to silencing of IKKβ, increasing the level of Bcl-xL in neurons prevents etoposide-induced caspase activation and Htt proteolysis. Conclusions/Significance: These results indicate that DNA damage triggers cleavage of Htt and identify IKKβ as a prominent regulator. Moreover, IKKβ-dependent reduction of Bcl-xL is important in this process. Thus, inhibition of IKKβ may promote neuronal survival in HD as well as other DNA damage-induced neurodegenerative disorders

Modelling primary proteolysis in cheddar cheese in commercial cool stores : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Bioprocess Engineering at Massey University

One issue identified as a possible problem during the manufacture of cheddar cheese is the possibility of producing a non-uniform product. It was proposed that a pallet of cheese experiencing different time-temperature histories, depending on the position within the pallet, could cause the heterogeneity. This work involved the investigation of that issue. The level of primary proteolysis observed in cheese was measured over time in cheeses of different compositions, stored at different temperatures. The remaining intact α s ₁casein was measured using reverse phase high performance liquid chromatography. Several trends were observed during maturation. High temperatures caused a faster rate of disappearance of α s ₁casein. The temperature relationship followed Arrhenius law. High moisture content caused a faster rate of the disappearance of α s ₁casein. The level of rennet added to the milk during production had a directly proportional effect on the rate of the disappearance of α s ₁casein. Salt had no observable effect in the range investigated here. From the data a kinetic model was developed that described the rate of disappearance of α s ₁casein in terms of the temperature, the moisture content, and the level of rennet in the cheese. The heat transfer occurring in the commercial pallet of cheese was mathematically modelled and solved numerically. The heat transfer model was then applied to produce data describing the time-temperature profile throughout a pallet of cheese for a variety of possible industrial storage conditions. The kinetic model developed was then used to predict the extent of proteolysis in each case. It was found that there would be significantly different levels of proteolysis within a pallet of cheese that had undergone chilling. A 10% difference in the level of proteolysis between the surface and the centre was observed after chilling for 40 days. During freezing the difference in the level of proteolysis after freezing was complete ranged from 10-25%. It was found that the heterogeneity was reduced during the thawing process and that the greatest reduction in non-uniformity was observed when thawed at lower temperatures

Charting the protein complexome in yeast by mass spectrometry

It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks

Aspects of proteolysis in cheese : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Food Technology at Massey University

The purpose of the present study was to elaborate methods for the detailed examination of proteolysis pathways in cheese (reviewed in Chapter 1) and to demonstrate their usefulness. Many techniques, including solvent fractionation, chromatographic separation and electrophoresis have been used previously and were revisited in this study. Gel electrophoresis can be a powerful technique and was examined in detail. The methods investigated were: 1) a slab gel system using the apparatus of the E-C Apparatus Corporation and a polyacrylamide gel in a Tris-EDTA-borate buffer at alkaline pH and containing urea; 2) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide stacking and resolving gel with a discontinuous (Tris-chloride/Tris-EDTA-borate) buffer system that contained urea; 3) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide stacking and resolving gel and acetic acid-ammonium acetate buffers at acidic pH that contained urea; 4) a mini-slab gel system using the Bio-Rad mini-Protean II apparatus, a polyacrylamide gel with a stacking and resolving gel in Tris-HCl buffers containing sodium dodecyl sulphate (SDS) and a Tris-chloride-glycine electrode buffer. The mini-slab alkaline urea polyacrylamide gel electrophoresis (PAGE) method was considered to be the most suitable for monitoring the loss of intact casein during cheese ripening. However, SDS-PAGE gave good resolution of para-κ-casein, β-lactoglobulin and α-lactalbumin and it could therefore be used for the analysis of cheese in which whey proteins have been incorporated or for monitoring the breakdown of para-κ-casein (Chapter 4) in cheese. Two-dimensional PAGE revealed the presence of more bands than were visible using any single method of electrophoresis. Traces of protein were found to lie beneath the α31-casein band and this explained why, even after considerable proteolysis, some α31-casein appeared to remain. Storing cheese samples in such a way that there is a minimum of further change was examined using several different storage methods and temperatures, including storage as: freeze-dried powder at 4°C in the dark, frozen at -9, -16, -35,-75 and -100°C, and dissolved in 6 M urea solution and stored at 4 and -16°C. The trial ran for 6 months and involved the multiple sampling and detailed analysis of three Cheddar cheeses by reversed phase fast protein liquid chromatography (RP-FPLC) for the water-soluble fraction (WSF) and alkaline urea-PAGE for the protein fraction. None of the methods used to store the cheese samples was completely satisfactory. Cheese stored at temperatures of -9 and -16°C was unstable, with proteolysis discernible after 66 days. Storage of cheese samples at these temperatures is, therefore, not recommended. Cheese stored at temperatures of -35, -75 and -100°C was unstable after 94 days, although the samples stored at -100°C were more stable. This lack of stability probably arose during thawing as well as during storage of the frozen cheese samples. Storage of freeze-dried samples at4°C in the dark was equivalent to storing the frozen cheese at -100°C. Storage of samples in alkaline urea sample buffer was better at -16°C than at 4° but should be for no longer than 1 month. An indication of the differences to be expected within the normal range of Cheddar cheese was determined using three very similar Cheddar cheeses ripened at5 and 13°C (Chapter 3, part II). Cheeses ripened at 5°C for 6 months were similar to those ripened at 13°C for 2 months and the proteolytic pathways appeared to the same at both temperatures. The proteolytic pathways in Cheddar and Mozzarella cheeses, manufactured according to standard protocols, ripened at 13°C and sampled at regular intervals over a six month period were examined using a variety of techniques: total nitrogen (TN), non- protein nitrogen (NPN), water-soluble nitrogen (WSN), alkaline urea-PAGE, low molecular weight (LMW) SDS-PAGE, RP-FPLC and size exclusion high performance liquid chromatography (SE-HPLC). The TN and NPN analyses were done at the time of sampling whereas the other assays were done on samples that had been stored at <-75°C so that they could be analysed simultaneously. The increase in WSN and NPN was greater in Cheddar cheese than in Mozzarella cheese and reflected the greater microbial enzyme activity in this cheese type. Alkaline urea-PAGE revealed that there was more α31-casein hydrolysis (with the formation of α31-casein-I) in Cheddar cheese than in Mozzarella cheese, indicating that rennet activity was greater in Cheddar cheese. The presence of peptides believed to be β-I- (β-casein fl-189/192) and β-II-casein (β-casein fl-165) indicated that rennet may have hydrolysed β-casein. The amount of β-casein hydrolysis (and γ-casein formation) was greater in Mozzarella cheese, reflecting the greater plasmin activity in this cheese type. Both LMW SDS-PAGE and SE-HPLC of the whole cheese provided little additional information. Examination of the WSF of each cheese by PAGE analysis showed that many of the larger peptides may have been present in both cheese types. The different concentrations of these peptides in each cheese type were consistent with different rennet and plasmin activities and suggested that they may have been products of these enzymes. RP-FPLC and SE-HPLC analysis of the WSF of Cheddar cheese revealed that, although the larger peptides continued to accumulate during ripening, there was also a large increase in the amount of small peptides and amino acids in the cheese. In the Mozzarella cheese, the larger peptides accumulated and there was little evidence of their further hydrolysis to small peptides and amino acids. The present studies indicate that SE-HPLC using a Toyo-Soda SW 2000 column and a 36% acetonitrile/0.1 % trifluoroacetic acid solvent system is a promising new technique that may be useful in determining cheese type and maturity and in relating changes in the molecular weight distribution of the peptides to changes in the textural, functional and flavour characteristics of cheese. It was concluded that the results are consistent with the concept that differences in the manufacture of Cheddar and Mozzarella cheeses result in the formation of two cheeses, each with different amounts of similar enzymes (rennet, plasmin, and the enzymes of the starter and non-starter lactic acid bacteria), and that these differences in enzyme concentration, combined with the modifying effect of pH, temperature, moisture content and S/M, result in different enzyme activities and patterns of proteolysis in the two types of cheese and these,in turn, result in cheeses with different functional properties

The regions of the sequence most exposed to the solvent within the amyloidogenic state of a protein initiate the aggregation process.

Formation of misfolded aggregates is an essential part of what proteins can do. The process of protein aggregation is central to many human diseases and any aggregating event needs to be prevented within a cell and in protein design. In order to aggregate, a protein needs to unfold its native state, at least partially. The conformational state that is prone to aggregate is difficult to study, due to its aggregating potential and heterogeneous nature. Here, we use a systematic approach of limited proteolysis, in combination with electrospray ionisation mass spectrometry, to investigate the regions that are most flexible and solvent-exposed within the native, ligand-bound and amyloidogenic states of muscle acylphosphatase (AcP), a protein previously shown to form amyloid fibrils in the presence of trifluoroethanol. Seven proteases with different degrees of specificity have been used for this purpose. Following exposure to the aggregating conditions, a number of sites along the sequence of AcP become susceptible to proteolytic digestion. The pattern of proteolytic cleavages obtained under these conditions is considerably different from that of the native and ligand-bound conformations and includes a portion within the N-terminal tail of the protein (residues 6-7), the region of the sequence 18-23 and the position 94 near the C terminus. There is a significant overlap between the regions of the sequence found to be solvent-exposed from the present study and those previously identified to be critical in the rate-determining steps of aggregation from protein engineering approaches. This indicates that a considerable degree of solvent exposure is a feature of the portions of a protein that initiate the process of aggregation

Prion protein in the cerebrospinal fluid of healthy and naturally scrapie-affected sheep

The aim of this study was to characterize the cerebrospinal fluid (CSF) prion protein (PrP) of healthy and naturally scrapie-affected sheep. The soluble form of CSF PrPC immunoblotted with an anti-octarepeat and an anti-C terminus mAb showed two isoforms of approximately 33 and 26 kDa, corresponding to the biglycosylated and unglycosylated isoforms of brain PrPC. Neither the mean concentration nor the electrophoretic profile of CSF PrP differed between healthy and scrapieaffected sheep, whereas a slightly increased resistance of CSF PrP to mild proteolysis by proteinase K was evident in the CSF of scrapie-affected sheep. No difference in susceptibility to proteolysis was observed between the two ARR and VRQ genetic variants of the purified prokaryote recombinant PrP. It was concluded that the physicochemical properties of PrPC in the CSF could be altered during scrapie and that these changes might reflect the physiopathological process of prion disease