Dissertations / Theses on the topic 'Proteolysis'

La maladie de Huntington (MH) est une maladie neurodégénérative héréditaire autosomique dominante. Elle est due à l’expansion anormale de polyglutamine dans la partie N-terminal de la protéine huntingtine (HTT). Une des étapes clés de la pathologie est le clivage de la HTT pleine longueur en fragments N-terminaux plus petits, contenant l’expansion de polyglutamine, et qui sont toxiques pour les neurones. En effet, les clivages de la HTT mutée génère des fragments N-terminaux (N-ter) de tailles comprises entre les acides aminés 1-105 et 1-586 observés dans des extraits de cerveaux de patients MH post-mortem et dont l’implication dans la mort neuronal est bien caractérisée. Mes travaux de thèse ont visé à modéliser le clivage de la HTT et à évaluer les conséquences sur la survie neuronale.Au cours de ma thèse, j’ai développé un outil permettant de contrôler le clivage de la HTT dans le temps et à des sites spécifiques. J’ai étudié le clivage de la HTT à deux sites stratégiques : les positions clivées par la caspase-6 et par la bléomycine hydrolase/cathepsine Z. A l’aide de cet outil, j’ai montré que le clivage de la HTT confère une toxicité cellulaire qui dépend du profil du clivage. Plus précisément, J’ai décrit une interaction intramoléculaire au sein des domaines de la HTT. Mes résultats indiquent que cette interaction protège les cellules de la toxicité induite par le clivage de la HTT mutée. En effet, les clivages successifs de la HTT annulent cette interaction, ce qui induit la libération des fragments N-ter mutants et provoque la mort cellulaire à l’issue de leur translocation nucléaire. Pour conclure, au cours de ma thèse, j’ai montré que la protéolyse successive de la HTT induit des processus cytotoxiques différents
Huntington’s disease (HD) is an autosomal dominant inherited neurodegenerative disorder caused by an abnormal polyglutamine (polyQ) expansion in the N-terminus of the protein huntingtin (HTT). A crucial step in HD pathogenesis is the cleavage of full-length HTT into smaller N-terminal (N-ter) fragments that contain the polyQ stretch and that are toxic to neurons. HTT cleavage generates short N-ter fragments whose amino-acid positions range from 1-105 to 1-586. These fragments are observed in HD post mortem brain samples and their participation in neuronal death in HD is well characterized. During my PhD research, I investigated the consequences of full-length mutant HTT proteolysis by developing a time and site-specific controlled system for HTT proteolysis. I have assessed HTT cleavage on two sites caspase-6 and cathepsin Z. My results show that HTT cleavage induces neurotoxicity in vitro as well as in vivo, toxicity which depends on HTT proteolysis pattern. Briefly, we described an intramolecular interaction within the HTT domains which is impaired upon successive proteolysis of HTT. We found that HTT intramolecular interaction buffer mutant N-ter HTT-induced toxicity. Moreover, specific cleavages of the mutant HTT generated toxic N-ter fragments as they translocate into the nucleus. To conclude, my PhD work has shown that additional cleavage of mutant HTT induces cytotoxicity by different mechanisms

The destruction of mitotic cyclins and other key regulators uses ubiquitin mediated proteolysis controlled via the activation of the ubiquitin ligase the Anaphase Promoting Complex/Cyclosome (APC/C), and its adaptor proteins Cdc20 and Cdh1. The spindle assembly checkpoint coordinates the APC/C with microtubule attachment and sets the timing from NEBD to anaphase. Cdc20 is inactivated by the spindle assembly checkpoint to prevent premature anaphase onset. Once the spindle assembly checkpoint is satisfied, Cdc20 can be released and activate the APC/C. However, cyclin A is degraded independently of the spindle assembly checkpoint before cyclin B1 and the anaphase inhibitor Securin. How Cdc20 can target different substrates for degradation at different times during mitosis is not yet clear. Using live-cell imaging and RNA interference and immunofluorescence techniques, I have studied the degradation of endogenous and ectopicly expressed APC/C substrates in cells with reduced Cdc20 levels. In this dissertation, I show that cyclin A degradation strongly depends on Cdc20, whereas cyclin B1 and Securin degradation do not. I identified the region of Cdc20 required for cyclin A binding and by mutating this site, I found that Cdc20 was no longer properly localised. I also show that Cdc20 proteolysis in human somatic cells does not require the KEN motif and gone on to find the motif required for Cdc20 degradation. I also show that the function of Cdc20 in the spindle assembly checkpoint can be influenced by the serine/threonine kinase Aurora A. Co-expression of a fluorescently tagged Cdc20 and Aurora A in human somatic cells causes an accelerated progression through mitosis and premature substrate degradation.

Proteolysis is a component of protein turnover, controlled by multiple proteolytic systems. Alterations in system components within skeletal muscle has been associated with hypertrophy, remodelling, atrophy, apoptosis and metabolic dysregulation. Key components may have novel regulatory roles, e. g. calpain-3 and cathepsin-L. Experiments described within this thesis investigated the hypothesis that the gene expression of specific proteolytic system components within skeletal muscle may be co-ordinately regulated and altered during nutritional and pharmacological states known to modify protein turnover and induce muscle growth. Gene expression for multiple components of the calpain system was analysed in calf LD (Longissmus dorsi) by Quantitative Real-Time PCR in a plane of nutrition trial. There were three groups: low (LOW), high (HIGH) plane of nutrition and LOW to HIGH (REFED). Half of each group were slaughtered 48 hrs after refeeding, whilst the remainder were slaughtered 13 days later. Total RNA yield/g LD increased (P < 0.05) across all groups between slaughter dates. Calpain-3 expression increased in LOW and REFED and calpastatin in all groups between slaughter dates, with a trend towards significance (P = 0.073, P=0.085, respectively). In the 1St slaughter, calpain-3 expression had a trend to be lower in the LOW group and values for REFED were similar to HIGH value level. cDNA probes for unique and novel proteolytic system components were generated by RT-PCR and used to investigate the effects of acute and chronic Q-adrenergic stimulation, on the gene expression of those specific components in pig LD, by northern blotting. The ß2-adrenergic agonist clenbuterol (5 ppm) decreased glycogen levels (mg/g LD) (P < 0.001), increased cathepsin-L expression (P < 0.001) and increased E2G 1 values numerically within 24 hrs of treatment. Cathepsin-L was unchanged by adrenaline administration. Calpain-3 was unchanged with either clenbuterol or adrenaline treatment. The significance and implications of the data are discussed.

Ubiquitination provides a means of rapidly and irreversibly eliminating an unwanted protein from the cell, and is therefore a potentially effective tool for regulating cellular behaviour. Ubiquitin-mediated proteolysis is involved in such diverse physiological functions as growth control, cell signalling, differentiation and the immune response. The aim of this research has been to investigate its role in Drosophila embryogenesis. Protein ubiquitination is a stepwise process carried out by three classes of enzyme known as E1s, E2s and E3s. The E1 (ubiquitin-activating enzyme), generates a thiolester linkage with a ubiquitin cysteine residue. The activated ubiquitin is then transferred to an E2 (ubiquitin-conjugating enzyme) which, with the help of an E3 (ubiquitin-protein ligase), recruits the substrate protein which is to be degraded. I examined the embryonic expression patterns of several known and novel genes encoding each type of ubiquitinating enzyme. The E2 UbcD4 is transcribed during early to mid-embryogenesis in a variety of tissues, with specific germcell expression in stage 10 embryos. This suggested a possible role for UbcD4 in germ cell migration towards the somatic gonadal precursors. UbcD4 mRNA was also abundant in git and nervous system during germband retraction and dorsal closure. I screened for UbcD4 - interacting proteins using the yeast two-hybrid system, and identified several putative substrates for, as well as ancillary factors involved in, ubiquitination by UbcD4. These included a novel E3 of the Hect-domain family. In an attempt to examine the function of UbcD4 directly, I used RNA interference to disrupt UbcD4 function. The results suggest a post-germband retraction requirement for UbcD4.

The effect of cancer cachexia on protein metabolism has been studied in mice transplanted with the MAC16 adenocarcinoma. The progressive cachexia induced by the MAC16 tumour was characterised by a reduction in carcass nitrogen between 16-30% weight loss and a reciprocal increase in tumour nitrogen content. Carcass nitrogen loss was accompanied by a concomitant decrease in gastrocnemius muscle weight and nitrogen content and also by a decrease in liver nitrogen content. The loss of gastrocnemius muscle throughout the progression of cachexia was attributable to a 60% decrease in the rate of protein synthesis and a 240% increase in the rate of protein degradation. The loss of skeletal muscle protein that may be mediated by an increased rate of protein degradation has been correlated with a circulatory catabolic factor present only in cachectic tumour-bearing animals, that degrades host muscle in vitro. The proteolysis-inducing factor was found to be heat stable, not a serine protease and was inhibited by indomethacin and eicosapentaenoic acid (EPA) in a dose-related manner. The proteolytic factor induced prostaglandin E2 formation in the gastrocnemius muscle of non tumour-bearing animals and this effect was inhibited by indomethacin and EPA. In vivo studies show EPA (2.0g/kg-1 by gavage) to effectively reverse the decrease in body weight in animals bearing the MAC16 tumour with a concomitant reduction in tumour growth. Muscle from animals treated with EPA showed a decrease (60%) in protein degradation without an effect on protein synthesis. The action of the factor was largely mimicked by triarachidonin and trilinoleia. The increased serum levels of arachidonic acid in cachectic tumour-bearing animals may thus be responsible for increased protein degradation through prostanoid metabolism.

Fujinami sarcoma virus encodes a 140/000 m.w. polypeptide (P140) which has been correlated as the agent of transformation in host chicken fibroblasts and mammalian fibroblasts. To conclusively identify the role of P140 in the transformation process it will be necessary to obtain intact/ purified P140. The availability of an antibody monoclonally specific to the N-terminal gag encoded portion of P140 suggested a one-step immunoaffinity purification of P140. After purification of the antibody out of mouse ascites fluid, by 50% ammonium sulfate fractionation and ion exchange chromatography, antibody was linked to a Sepharose 4-B matrix activated with cyanogen bromide. The anti-pl9 affinity matrix bound intact P140 as a doublet relative to a polyclonal anti-pl9. Chaotropic agents, high pH and low pH treatments all failed to elute the bound P140 from the affinity matrix. Failing the purification of intact P140 a method of partial proteolysis was used to produce varying sized fragments of P140/ with the goal of purifying these fragments for further work on the role of P140. Trypsin alone in a limited proteolysis produced small, unstable peptides too close in size distribution to be effectively purified. Chymotrypsin alone produced a broad range of more stable peptides, with a predominance of a 45,000 m.w. peptide. Chymotrypsin-trypsin consecutive proteolysis produced a very stable 35,000 m.w. peptide. Gel filtration of the chymotryptic peptides was ineffective as the peptides coraplexed and were not fractionated. Ion exchange chromatography fractionated the complexing chymotryptic peptides, making possible the purification of these peptides. The stable 45,000 m.w. peptide retained some kinase activity, as it phosphorylated the substrate enolase, similar to but less intense than intact P140. A 30,000 m.w. peptide only phosphorylating after ion exchange did not phosphorylate enolase.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

Chopped alfalfa and chopped whole-plant corn were ensiled and the proteolytic changes which occurred during ensiling were investigated. Proteolysis was measured in terms of end-products of protein degradation and by protein isolation followed by electrophoresis. The effects of formic acid and ammonia, applied at the time of ensiling, on proteolytic changes were investigated.
Alfalfa treated with formic acid contained significantly reduced levels of NH$ sb3$-N and NPN compared to control silage; ammonia (NH$ sb3$)-treated alfalfa silage had significantly less NPN (P $<$ 0.05). After 90d of storage, formic acid-treated and NH$ sb3$-treated alfalfa silage contained lower levels of both branched and non-branched amino acids, sulfur containing, and basic amino acids compared to control silage; formic acid-treated and NH$ sb3$-treated corn silage contained lower levels of branched chain amino acids and sulfur containing amino acids after the same time period. Formic acid and ammonia were most effective in the reduction of proteolysis in alfalfa silage and corn silage, respectively. The protein ribulose 1,5-diphosphate carboxylase (RuDPCase) was depleted completely after 2d of fermentation in control silage. Conditions in NH$ sb3$-treated alfalfa silage stabilized RuDPCase during the first 24h of storage.

Changes in intramuscular connective tissue brought about by conditioning were investigated in bovine muscles of different quality. Perimysial and endomysial collagens were solubilized to a small extent during conditioning and residual insoluble collagens in both connective tissue domains were damaged by proteolytic processing. Yields of soluble perimysial material from unconditioned muscles were significantly lower (p = 0.096) than from conditioned muscles. Solubilized perimysial collagen from unconditioned muscles was significantly lower (p = 0.015) than from conditioned muscles with 1±0.8 % of original collagen solubilized for unconditioned muscles and 3.4 ± 3.3 % for conditioned muscles. 87.5 % of the muscles examined showed an increase in percentage solubilized collagen due to conditioning. The main peptide components observed on analysis of insoluble perimysial fractions after CNBr digestion were derived from types I and III collagen. No changes were observed in the major peptide bands due to conditioning. Yields of soluble endomysial fractions representedo, n average,9 4.5 % of total extracted endomysial material for unconditioned muscles compared with 97.5 % for conditioned muscles. Soluble endomysial fractions contained, on average, 0.13 % collagen from unconditioned muscles and 0.22 % collagen from conditioned muscles. The main peptide components observed on analysis of insoluble endomysial fractions after CNBr-digestion were derived from types I and III collagen. Changes observed on the peptide maps, evident as the appearanceo f a number of new bandsf rom conditioned samples,a ppearedt o be muscle specific. % Type III collagen decreased on conditioning, indicating that endomysial type III collagen was preferentially destroyed during - conditioning. In model systems, insoluble perimysium treated with pepsin over 24 h resulted in little damage to the insoluble collagenous residue remaining. Insoluble perimysium treated with cathepsin resulted in changes to the major peptide bands on one-dimensional SDSpolyacrylamide gel electrophoresisw hich were evident after 24 h treatment. Two-dimensional peptide maps obtained from conditioned insoluble perimysium and from insoluble perimysium treated with cathepsin for 24 h were altered relative to the unconditioned insoluble perimysium, indicating proteolytic damage to high molecular weight fractions. The in vitro case was extreme, but was comparable with conditioned insoluble perimysium. In addition, new peptide material in conditioned perimysium and endomysium in the molecular weight range 40 000 to 50 000 was observed, while perimysial samples revealed loss of peptide material, due to conditioning. Percentage solubilized collagen was higher (p < 0.05) from three muscles of varying quality when pre-injected with 0.1 M lactic acid and conditioned from 1 to 14 days than from untreated muscles. Analysis of the high molecular weight collagen peptides from lactic acid treated muscles by two-dimensional SDS-polyacrylamide gel electrophoresis revealed increased incidence of degradation in this region compared with untreated controls. Sensory profiling using quality descriptive analysis (QDA) was carried out on three muscles of varying quality, pre-injected with 0.1 M lactic acid and results compared with untreated muscles. The results obtained failed to correlate the observed biochemical changes due to lactic acid treatment with perceived textural changes in these muscles. However, variability of the taste panel scores contributed significantly to the results obtained.

L-selectin (CD62L) is a type I transmembrane protein expressed by lymphocytes which directs their migration from the bloodstream into lymph nodes and infected tissues. Stimulation of the T cell receptor (TCR) activates the enzyme A Disintegrin and Metalloproteinase 17 (ADAM 17), which cleaves L-selectin at the ectodomain generating a metalloproteinase product (MP product) comprising of a transmembrane region and a 17-amino acid intracellular domain (ICD). ϒ-secretase is a multi-subunit protease that cleaves up to 90 identified type I transmembrane proteins in the intramembrane region following ectodomain proteolysis by metalloproteinases. Presenilin (PS), the catalytic component of γ-secretase is activated during an intramolecular cleavage called endoproteolysis separating the carboxy (C) and amino (N) termini. The catalytically active C-terminal fragment of PS then induces intramembrane proteolysis of substrates. The aim of my thesis was to firstly determine whether the MP product of L-selectin was a substrate for PS. Subsequently, I analysed whether stimulation of the TCR activates PS, inducing intramembrane proteolysis of the MP product releasing the ICD into the intracellular region. My data showed for the first time that in a resting T-cell, L-selectin forms a multi-component complex with both ADAM 17 and PS. TCR-activation induces ADAM 17 dependent proteolysis of L-selectin generating an MP product. Stimulation of the TCR also causes endoproteolysis of PS, where activated PS then cleaves the bound MP product. After PS cleavage, the released ICD was unstable and therefore difficult to detect, however I was able to block its formation using either PS inhibitor treatment or generating I351W mutated L-selectin, which was resistant to intramembrane proteolysis.

Solving the problem of inconsistent meat tenderness is a top priority of the meat industry. This requires a greater understanding of the processes that affect meat tenderness and the adoption of such information by the meat industry. It is essential that we understand the mechanism of meat tenderisation of which, the calpain protease system is believed to play a central role. This thesis focuses on three aspects; characterisation of calpain activity, the effect of porcine μ-calpain on myofibril degradation and the effect of μ-calpain on specific proteins desmin and troponin-T. To study the effect of calpain activity, fluorogenic assays were used to determine: μ-calpain concentration for optimal peptide cleavage; calcium requirements and the effect of chelating substances on the activity of μ-calpain. In addition, the affinity of μ-calpain for substrates CalS-I and CalS-III were assessed. The effect of μ-calpain on myofibril degradation was evaluated through the use of myofibrillar fragmentation index and density marker beads. Myofibrils were digested at three different temperatures for varying time periods. Conflicting results were displayed and it was concluded that these methods are not accurate, thus further research should be conducted to ensure inconsistencies are eliminated. Specific proteins desmin and troponin-T have previously been shown to exhibit degradation in the presence of calcium and μ-calpain. SDS-polyacrylamide electrophoresis, western blotting and densitometry measurements were utilized to investigate this effect. It was concluded that μ-calpain plays a significant role in the post mortem proteolysis of myofibrillar protein. This thesis provides information and strives to give a better understanding of the proteolytic changes that occur within muscle. Understanding how these mechanisms affect meat on a cellular level, can help to control the influence they inflict on meat quality.

String cheese, a Mozzarella cheese, has the unique ability to string in fibrous strands when pulled apart. Graders judge string cheese by its stringy texture; samples with copious amounts of string are awarded high ratings. But just as the texture of natural cheeses softens with time, the stringy texture of string cheese can diminish with age too. Age related softening in cheese is due primarily to an important biochemical event known as proteolysis, which is attributed to inherent milk proteinases, residual coagulant activity, and enzymes from the lysis of starter culture microorganisms. It is hypothesized that a post manufacture heat treatment of string cheese could inactivate these proteolytic enzymes and slow or eliminate proteolysis during storage. Therefore, the main objective of this study was to determine the effects of a post manufacture thermal dip treatment on proteolytic activity in packaged, commercial string cheese. Proteolysis was examined qualitatively by Urea-PAGE electrophoresis, quantitatively by measuring percentage of water-soluble nitrogen (%WSN), and by using a scoring method to analyze stringy texture during refrigerated storage. Fresh, commercial string cheese was sourced on two separate occasions and treated six days after manufacture. Treatment consisted of dipping the packaged cheese sticks in water baths at 55°C, 75°C, and 95°C for 30 and 60 seconds. String cheese that did not undergo treatment served as the control. Treated and control cheeses were stored at 4°C until sampling for Urea-PAGE, WSN extraction, and texture analysis on days 1, 11, 22, 29, 49, 91, and 172 after treatment. The degree of β-CN breakdown was not observed to be different between all treatment levels throughout the storage period. This was not expected since Mozzarella cheese exposed to a higher temperature should have more plasmin activity than that of cheese exposed to a lower temperature. There was a trend of slightly more intact αs1-CN in the most severely treated string cheese (95°C for 60s) when compared to the control at the final time point of the study. This suggests the possibility of successful inactivation of residual coagulant, intracellular enzymes, or other proteolytic enzymes in the string cheese at this treatment. However, only storage time had a significant effect on %WSN (p The research completed in this study provides insight of the proteolytic effects from a thermal treatment process applied post string cheese manufacture. Though relationships between the treatments to the extent of secondary proteolysis and stringy texture were not significant, it was still found that there was more intact αs1-CN due to one of the treatments. These results suggest that it is possible that the use of other heat treatment parameters, longer storage period, or a combination of the two could show a significant relationship between thermal treatment and proteolysis. These results also suggest that further work to improve shelf life of string cheese or other cheese varieties through the concept of a post manufacture heat treatment may be promising.

Mycobacterium tuberculosis uses the ESX-1 type VII secretion system to export proteins to its cell surface, which permeabilize the host macrophage phagosomal membrane, allowing the bacterium to escape and spread to new cells. The structure of the type VII membrane complex and how it mediates this function is unknown, but it is hypothesized that some of the secreted proteins form an extracellular appendage that facilitates membrane lysis or direct secretion of virulence factors into the host cytoplasm. This thesis investigates the structural relationship between one of these secreted proteins, EspB, and a protease that processes it, MycP1. The x-ray crystallographic structures of both proteins are determined and described. EspB is shown to form a multimer with heptameric stoichiometry, and an EM reconstruction of this multimer is generated and used to create a model of the oligomer using symmetric Rosetta docking. The final model is supported by mass spectrometry-based detection of chemically cross-linked peptides from adjacent subunits. We use mass spectrometry to determine how EspB is proteolytically processed during secretion and discuss the effect of this processing event on the EspB ultrastructure. Finally, the structure of one of the membrane apparatus proteins, EccB1 is determined, revealing structural homology to a phage lysin. The combination of x-ray crystallography, EM, modeling, and mass-spectrometry provides an exciting first glimpse at the structure and function of the type VII secretion system - a critical factor in the TB pathogenesis cycle.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

In order to maintain genetic fidelity and to avoid aberrant proliferation, the cell cycle must be carefully regulated. Progression through mitosis is controlled by ubiquitin-mediated proteolysis of cell cycle proteins, allowing each stage in the cell division process to begin only when the previous stage is successfully completed and the various checkpoints are satisfied. As proteolysis contributes to the correct timing of exit from mitosis and cytokinesis, I have investigated the mechanisms responsible for degrading specific proteins at specific times during the cell cycle and how this is regulated. By mislocalising Cdc20 substrates such as Cyclin B1 by targeting them away from the spindle, I have shown that localisation of these substrates to the mitotic spindle is a requirement for the correct timing of their degradation. When substrates are localised away from the spindle, the timing of their degradation shifts from metaphase to anaphase. I have also shown that this localised metaphase-degradation is dependent on specific regions of the mitotic spindle. Localisation to the centromeres allows substrates to be degraded at metaphase, whereas localisation to the microtubules and centrosomes prevents degradation during metaphase and results in anaphase degradation. Using a combination of live cell imaging and biochemistry, I have also investigated the mechanisms that allow global degradation during anaphase.

Neutral proteinases, released by inflammatory leukocytes, have been implicated in the pathogenesis of pneumoconiosis but there has been no systematic study of the proteolytic acitivty of leukocytes from dust-exposed lung. The aim of the present study was, therefore, to assess the bronchoalveolar leukocyte profile and proteolytic activity of the leukocytes in a rat model of pneumoconiosis. An assay, based on the breakdown of [¹²⁵I] fibronectin, that would measure the overall proteolytic activity of the bronchoalveolar leukocytes and indicate their potential to damage the connective tissue of the alveolar septum was developed and validated. Increased proteolytic activity was found in the inflammatory bronchoalveolar leukocyte populations and so the relative role of macrophages and neutrophils was assessed by separation into distinct populations; both inflammatory macrophages and neutrophils had increased proteolytic activity. The important features governing the inflammogenicity of particles were addressed by measuring the inflammation-generating properties of a variety of fibrogenic and non-fibrogenic particles in rats exposed by intratracheal instillation or inhalation. The number of leukocytes recruited to the alveolar region was measured by bronchoalveolar lavage. Only fibrogenic mineral dusts had the ability to produce a sustained alveolitis in which the proteolytic activity of the bronchoalveolar leukocytes remained elevated. The pathogenicity of fibrogenic particles is likely, therefore, to be related to their ability to evoke and sustain an increased lung proteinase burden. The sustained alveolitis with fibrogenic particles was not related to lack of clearance of dust from the lung. Both quartz and coalmine dust elicited persistent alveolitis, but titanium dioxide, which is no more readily cleared than silica or coalmine dust, failed to sustain the inflammation. Properties of the particle surface, at least in the case of quartz, appear to play a part in their inflammogenicity. Altering the surface of quartz particles by coating them with aluminium lactate reduced their ability to recruit inflammatory leukocytes but did not alter the proteolytic activity of the leukocytes. The tissue response to the aluminium-coated quartz particles was also less than that elicited by native quartz with fewer and less-severe lesions. The foregoing serve to substantiate the role of inflammatory leukocytes in the pathogenesis of pneumoconiosis. Only when there is a sustained alveolitis with an overall increased proteinase burden, does pathological change occur in the lung. Reducing the magnitude and/or the duration of the alveolitis markedly suppresses the development of the tissue lesions.

Chromatin is the combination of DNA and proteins in the nucleus that is used to aid in the compaction of DNA. Histones are a group of proteins used to condense DNA by forming a complex (nucleosome) around which DNA wraps around; there are two of each type of histone in a nucleosome: H2A, H2B, H3 and H4. Once the DNA is wrapped around the histones, the genome is further compacted. A shortened, "clipped" version of histone H3 has been found in some organisms including yeasts, flies, mammalian stem cells, and the ciliated protozoan, Tetrahymena thermophila. In each organism, clipping occurs at a different site on the N-terminus, usually before an alanine residue. Clipping is important as it may affect other epigenetic modifications and gene regulation in cell differentiation, but the regulation of this histone proteolysis has remained largely unstudied. In Tetrahymena thermophila, approximately half of the histone H3 molecules are clipped between residues 6 and 7 on histone H3, solely in the transcriptionally silent micronucleus. The histones in the micronuclei are deacetylated, while histones in the macronuclei can be acetylated or deacetylated. It is hypothesized that the post-translational acetylation modification to the histone tails may inhibit histone H3 clipping. Immunoblot analyses were carried out with acetylated and deacetylated micronuclei, demonstrating an increase of clipping when acetylated. Additionally, mutations were created at lysine 9 upstream of the clip site on the histone H3 tails to mimic acetylation and deacetylation to study whether the modification of that site has a regulatory effect.

Proteolysis inducing factor (PIF) is a pro-cachectic glycopeptide purified from the urines of mice inoculated with the MAC16 tumour and from the urines of weight losing patients with pancreatic carcinoma. It arises from the dermcidin gene which produces 2 other peptides, Y-P30, an unglycosylated neuronal survival factor with homology to the PIF peptide core and DCD-1, an antibiotic peptide secreted by eccrine sweat glands which has no homology with PIF. We sought to investigate PIF / dermcidin expression in cell lines and pancreatic carcinoma tissue, the role of proteolysis inducing factor as a growth and survival factor in tumour cells and the influence on these effects of the structural features known to be important to the induction of cachexia by PIF and the functions of Y-P30 and dermcidin. Dermcidin was expressed by pancreatic carcinoma cell lines and primary human hepatocytes but not by the HuH7 cell line. In HuH7 cells induced expression promoted cell growth and improved survival following oxidative stress. The YP-30 / PIF core peptide sequence was sufficient to induce cell growth. Survival promotion did not require glycosylation but was prevented by mutagenesis of the asparagines residues of the PIF core peptide. Analysis of mRNA expression suggested dermidin was expressed in a low number of pancreatic carcinomas. Non-specific antibody binding prompted the development of mass spectrometry for detection of PIF in urine samples of patients with these tumours. This did not reveal PIF but did demonstrate other proteomic changes. We have demonstrated growth and survival functions of PIF / dermcidin which may be relevant in a range of physiological and pathological processes including cachexia and neoplasia. The PIF core peptide sequence appears to be important for these effects but its glycosylation does not appear to be required.

Fbxl13 (F-box and leucine-rich repeat protein 13) is an orphan F-box protein. Fbox proteins are a family of substrate-targeting specificity factors for the SCF superfamily of E3 ubiquitin ligases. Since their discovery, many F-box proteins have been shown to have oncogenic and tumour suppressive roles. The importance of Fbxl13 itself in tumourigenesis is reflected in several genome-wide shRNA screens. Fbxl13 depletion in human cancer cells correlates with increased ionising radiation sensitivity and increased genomic instability. Furthermore, Fbxl13 depletion reduces proliferation in mouse embryonic epidermis. Conversely, Fbxl13 amplification is frequently observed in several cancer patient cohorts. However, the main function of Fbxl13 is unknown and its biochemical mechanism of action remains uncharacterised. The aim of this study was to identify the interactors, substrates, and functions of Fbxl13, in order to elucidate its role in tumourigenesis. In this study, I identify and validate Fbxl13 interactors Centrin-2, Centrin-3, Cep152, and Cep192. I show that Fbxl13 is enriched at the centrosome, and present evidence that Fbxl13 targets Cep192-3 for ubiquitin mediated proteolysis. In line with this, Fbxl13 overexpression downregulated centrosomal Cep192 and γ-tubulin, and disrupted the microtubule nucleation activity at the centrosome. Finally, Fbxl13 amplification in U2OS cells is associated with increased cell motility. Thus, we propose that Fbxl13 is a novel regulator of centrosome microtubule nucleation activity.

Recent studies have identified a novel 600 kDa neutrophil membrane associated protease which degrades fibrinogen, fibrin and C-reactive protein (CRP) during incubation of these ligands with phorbol 12-myristate 13-acetate (PMA, 5-10 ng/ml) stimulated neutrophils. This proteolysis is predominantly an extracellular event which occurs through a ligand dependent release of this protease from the neutrophil. Degradation products arising from this proteolysis not only become neutrophil associated but influence a number of important processes occurring in inflammation and coagulation. The aim of the present 'study was to purify and further characterize this protease and investigate the location of the neutrophil associated fibrinogen and fibrin degradation products. Whilst enzyme purification procedures were unsuccessful, several observations made during these attempts suggested that the neutrophil membrane associated proteolytic activity displayed similar characteristics to proteases of the azurophil granule. The proteolytic activity of the membrane was concluded from inhibitor profiles, zymography, and the apparent molecular mass values and hydrophobicity of the fibrinogen degradation products that it generated, to be the composite action of the azurophil granule proteases, human neutrophil elastase, cathepsin G and possibly proteinase 3. Electron microscopy analysis of PMA stimulated neutrophils incorporated within fibrin clots revealed morphological changes suggestive of neutrophil degranulation, and the proteolytic activity released by these cells was shown to be identical to that of azurophil granule proteases with respect to the apparent molecular mass values of the fibrin products that it generated. Immunoelectron microscopy revealed minimal internalization of fibrin like material during this process suggesting that neutrophil mediated fibrinolysis under these conditions is predominantly an extracellular event. Immunoelectron microscopy was used to localise fibrinogen degradation products previously reported to be associated with the neutrophil following incubation with fibrinogen. This revealed neutrophil associated fibrinogen products to be intracellular. Internalisation appears to be the result of pinocytosis which is stimulated in the presence of PMA. Although internalisation may be enhanced by an initial interaction of fibrinogen with the neutrophil membrane, a large proportion of uptake occurs via the fluid phase. Both intact and degraded forms of fibrinogen can associate with the neutrophil. Internalised material is rapidly degraded intracellularly into low molecular weight products which are partially released into the surrounding medium. This intracellular degradation, however, contributes minimally to the overall degradation of fibrinogen by neutrophils; the major pathway is extracellular. The demonstration in this· study, that the previously identified fibrinogen- fibrin- and CRP-degrading activity of the neutrophil membrane is due to azurophil granule proteases co-incides with numerous recent reports suggesting that membrane bound forms of these proteases, due to their ability to evade naturally occurring protease inhibitors, are the biologically relevant forms of these proteases. The membrane expression of azurophil granule proteases has recently been shown to be under the control of a variety of inflammatory mediators. Thus, neutrophil mediated degradation of fibrinogen, fibrin and CRP in vivo may be tightly controlled by the regulated expression of azurophil granule proteases on the neutrophil membrane.

The pattern and sequence of zein degradation in the endosperm of germinating maize seeds were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. The proteases involved in the degradation of various zein components (α, ß and γ) were extracted with three buffer systems and partially characterized with respect to their ability to degrade various zein components. They were also investigated in vivo by germinating the seeds in the presence of protease inhibitors used singly and in combination. Of the various zein components, γ-zein (27-kD) was the first to be degraded and its degradation was complete by the third day after germination (DAG). Beta-zeins (17- and 18-kD) began to be degraded on the second DAG, degradation being complete by the seventh day for the l7-kD polypeptide, and the fourth day for the 18-kD polypeptide. The degradation of 10-kD- zein began on the fourth DAG and was complete by the eighth day. The α-zein fraction (22-and 24-kD) was degraded beginning on the faith day and continued gradually until after the tenth day. From the results of these studies, the arrangement of various zein fractions within the protein bodies can be deduced and this was consistent with the immunocytochemical data published by others. Gamma-zein is situated in the peripheral region of the protein bodies and could be a structural component of the protein body membrane or it may be directly anchored in the membrane. Beta-zeins are internal to γ-zein with the l0-kD in the interface between the 17-kD and γ-zein. The 10- kD zein is located between the 17-kD and α-zein or interlacing with α-zein in the protein body core. Finally, a-zeins are in the protein body core. Based on these observations the proteolysis of the protein in protein bodies of maize would start from the periphery and proceed towards their core. The proteases involved in degradation of various zein components were synthesized de novo. The mRNAs pre-existing in dry seeds appeared to direct the synthesis of active proteases required for zein degradation at least during the initial stages of germination. Serine protease was responsible for the degradation of a- and ß-zeins while aspartic (acid) protease may play some role in ß-zein degradation. Serine and cysteine (thiol) proteases worked synergistically in γ-zein degradation. Enzymes extractable from the endosperm of germinating seeds with 0.2 M acetate buffer (pH 3.8) were able to degrade the α-, ß-, and γ-zeins in an in vito assay.
Ph. D.

Pulmonary surfactant is a critical surface-active substance consisting of dipalmitoylphosphatidylcholine (DPPtdCho) and key apoproteins that are produced and secreted into the airspace from alveolar type II epithelial cells. Surfactant deficiency leads to severe lung atelectasis, ventilatory impairment, and gas-exchange abnormalities. These are features of the acute lung injury syndrome, characterized by a strong pro-inflammatory component where cytokines or bacteria infections greatly impair surfactant DPPtdCho biosynthesis. The key enzyme needed to produce surfactant DPPtdCho is a rate-limiting enzyme CTP: phosphocholine cytidylyltransferase (CCTalpha). Calmodulin (CaM), rather than disruption of an NH2-terminal PEST sequence, stabilizes CCTalpha from actions of the proteinase, calpain. Mapping and site-directed mutagenesis of CCTalpha uncovered a motif (LQERVDKVK) harboring a vital recognition site, Q243, whereby CaM directly binds to the enzyme. Mutagenesis of CCTalpha Q243 not only resulted in loss of CaM binding, but also led to complete calpain resistance in vitro and in vivo. These data suggest that CaM, by antagonizing calpain, serves as a novel binding partner for CCTalpha that stabilizes the enzyme under pro-inflammatory stress. We further show that CCTalpha does not undergo polyubiquitination and proteasomal degradation. Rather, the enzyme is monoubiquitinated at a molecular site (K57) juxtaposed near its NLS resulting in disruption of its interaction with importin, nuclear exclusion, and subsequent degradation within the lysosome. Importantly, by using CCTalpha-ubiquitin hybrid constructs that vary in the intermolecular distance between ubiquitin and the NLS, we show that CCTalpha monoubiquitination masks its NLS resulting in cytoplasmic retention. These results unravel a unique molecular mechanism whereby monoubiquitination governs the trafficking of a critical regulatory enzyme in vivo. Last, we identify FBXL2 as a novel F-box E3 ubiquitin ligase that targets CCTalpha for degradation. Interestingly, FBXL2 also interacts with CaM, and CaM directly disrupts CCTalpha and FBXL2 interaction. This study demonstrates in the first time that adenoviral gene transfer of CaM attenuates the deleterious effects of P. aeruginosa infection by improving several parameters of pulmonary mechanics in animal models of sepsis-induced acute pulmonary injury. Collectively, these studies reveal a novel regulatory mechanism for phosphatidylcholine synthesis that may provide important clues to understanding the pathobiology of acute lung injury.

ABSTRACT Influence of chelating agents on proteolysis of micellar casein slurries Azucena Becerril Díez The focus of this research was to analyze the effect of Ca chelators on the proteolytic activity of rennet during ripening of a micellar casein slurry. This research was divided in two phases. During the first phase, preliminary studies were performed to understand the behavior of rennet and the milk system in the presence of chelating agents. A solution of reconstituted skim milk at 10% (w/v) was prepared. Individual samples of this solution were divided in two study groups, samples with chelators and rennet and samples with only chelators. Two different chelators were used, sodium hexametaphosphate (SHMP or NaHMP) at concentration levels of 2.5 and 10 mM; and sodium citrate (SC) at concentration levels of 5 and 25 mM. These samples were incubated in a water bath at 32°C to measure coagulation time (CT) and Urea-PAGE electrophoresis was performed to identify presence of caseins and smaller peptides. For the second experiment, micellar casein slurries were prepared with a 25% protein content to simulate protein content in cheese. Samples were classified in two study groups, samples with chelators and rennet and samples with only chelators. Percentage of water soluble nitrogen analysis (%WSN) was performed following Kjeldahl procedures to measure the break down of caseins into free soluble nitrogen and Urea-PAGE electrophoresis was used to measure the appearance of smaller peptides due to protein break down. Bands with a higher density on electrophoretic gels were an indication of a higher concentration of peptides due to possible increased exposure of caseins to proteolysis. For the second phase, samples with the 2 most significant chelator levels were selected to add a protease and measure with the same tests if there was a higher rate of proteolysis. The data showed no particular trend or significant difference between treatments (p>0.05) for the %WSN test while some treatments showed a significant effect in samples treated for Urea-PAGE gels. In the Urea-PAGE analysis a different ANOVA was conducted on the percentage of alpha, beta, and gamma caseins for treatments before and after the incubation period separately and for the difference between both periods. These ANOVAs showed that the percentage difference in each casein before and after incubation was affected by the choice and level of chelator, and that these effects were modified by the presence or absence of rennet (p<0.001).

The inconsistent quality of pork meat is a major worry for the industry, and is mainly blamed on variable tenderness. Meat tenderness depends in part on the degree of alteration of the structural components of muscle and associated proteins post-mortem. Reduced activity of the calpain proteinase system is associated with tough meat. However, it is known that other proteases such caspase and proteasome proteolytic enzyme systems are involved in skeletal muscle development and remodeling, and it is their proteolytic attributes that make them possible contributors to improved meat tenderness. This research work investigated the effects of growth promoters (beta-agonist - Ractopamine ™ and growth hormone - Reporcin TM) on gilts' carcass characteristics, and longissimus muscle meat quality and proteolytic system activities (caspase 3/7, calpains and proteasome). Two experiments were carried out consisting of a short period (7 day study) of treatment aimed at assessing the immediate response of the protease systems to the two growth promoters and a time course study that aimed to assess the growth promoters' effects in a longer treatment period (13 and 27 days), simulating the commercial set up in which feed additives are used in finishing phases that can be up to 60 days. Gilts were fed a standard commercial diet ad-libitum (control) or with the same feed supplemented with beta-agonist at 10mg/kg (7days) or 20mg/kg (13 and 27days), or administered growth hormone (10mg every 2 days) in both experiments. Carcass characteristics were assessed at slaughter and longissimus muscle samples were assessed for tenderness (shear force), caspase 3/7 and proteasome (chymotrypsin-like, trypsin-like and caspase-like) activities and calpastatin protein level in the 7 day experiment, with calpains activities, MuRF, MAFbx protein levels and lipid and fatty acid content in the long treatment study of 13 and 27 days. Growth hormone increased liver weights (P < 0.001) in both experiments, but had no effect on muscle weights in both short and long treatment studies. Beta-agonist increased muscle weights over the 13 and 27 day (P < 0.05) and 7 day (P = 0.062) treatments and tended to increase shear force values (P = 0.09 and 0.107, 7day and 27day respectively) after 8 days of ageing . Caspase 3/7 and total proteasome activities decreased significantly (P < 0.001) with time during the 7 day treatment period. GH significantly increased activity of trypsin-like proteasome subunit at day 13 (P = 0.048). Beta-agonist Significantly increased activity of chymotrypsin-like proteasome subunit (P = 0.041) at 27 days, whereas GH decreased caspase-like proteasome activity (P = 0.025) at day 27. Beta-agonist decreased glycogen levels at 7 day (P = 0.015). Both growth promoters significantly decreased lipid content after 27 days of treatment (P < 0.001). v All correlations between shear force and proteases were negative for the growth hormone treated gilts and significant except for the Caspase 3/7 (P = 0.081) in the 7 day treatment period. MuRF protein level negatively and significantly correlated with shear force at slaughter day 27 (P = 0.053) for data pooled across treatments, and for gilts treated with the growth hormone (P = 0.035). These were the only negative correlations with shear force and in the expected direction, where high protease activities should translate to lower shear force to indicate a role in meat quality of improved tenderness. But the correlations between the protease systems studied and shear force were very inconsistent across this research work, indicating no role played by the systems in meat quality. No differences were observed in weights across treatments (live and carcass) in all the experiments conducted in this research work. This work indicated that caspase 3/7 and proteasome activities are not associated with meat quality (tenderness) attributes of growth promoter treated gilts, and that the slight increase observed in shear force in beta-agonist treated pigs might have been due to larger (hypertrophied) fibres that also had reduced lubrication during shearing due to low intra-muscular fat levels. vi

Iron is one of the essential elements involved in various fundamental biological activities. However, excess iron may bypass the negative feedback regulatory systems, leading to the formation of iron overload. The increase of iron deposition generates cellular toxicity and subsequently damages vital organs. Primary and secondary iron overload are affecting patients worldwide. Iron overload cardiomyopathy is the primary cause of cardiac dysfunction and cardiovascular mortality in β-thalassaemia major patients. Current effective therapy includes chelation treatment with conventional and new iron chelators, while potential new therapies are currently under development. The pathophysiology of iron overload cardiomyopathy remains unclear. Controversial findings on the mechanism of excessive iron entry into cardiomyocytes exist. Using novel real-time approach to trace iron entry into HL-1 cardiomyocytes, the only beating cardiac cell line with mature cardiac phenotype available currently, we visualized the patterns of iron entry following ferric iron incubation with and without ascorbate. Iron entry could be partly blocked by pretreatment with L-type calcium channel blockers but not T-type calcium channel blocker. Such blockage effect by L-type calcium channel blockers occurred in ferric iron overload. This finding suggested a role of L-type calcium channels for ferric iron uptake into cardiomyocytes under iron overload condition. For the pathophysiology of iron cardiac toxicity, we assessed the iron overload induced apoptosis using both in vitro and in vivo approaches. The results demonstrated that iron-overloaded mouse HL-1 atrial cardiomyocytes and human embryonic stem cell derived ventricular cardiomyocytes underwent apoptosis via the mitochondria-mediated caspase-3 dependent pathway. Supportive data was found in iron-overloaded mouse myocardium by an increase in DNA fragmentation. However, despite the blockage of iron entry, L-type calcium channel blockers did not significantly prevent iron induced apoptosis in vitro. The mechanism of cardiac contractile dysfunction caused by iron overload on cardiomyopathy has not yet been fully characterized. Given the central role of titin, the giant myofilament protein, as the main determinant in myocardial passive tension, stiffness, diastolic and systolic cardiac function, as well as myocardial twisting and untwisting motion, we investigated its expression in iron-overloaded cardiomyocytes in vitro and in vivo. Our results indicated that significant degradation of cardiomyocytes titin was induced by iron overload. This was associated with the cleavage at the elastic domain. Its potential upstream protease, calpain, was further identified to be activated under iron overload. The specific role of titin proteolysis in iron-overloaded cardiomyocytes merited further investigation. The findings in this project provided new insights to the pathophysiology of iron overload cardiomyopathy, in terms of the route for iron entry, iron induced cardiac apoptosis, and titin proteolysis. Novel therapeutic approaches for prevention and treatment of iron overload cardiomyopathy can focus on inhibiting excessive iron uptake, as well as by targeting pathways involved in cardiac apoptosis and titin proteolysis.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Neuregulin-1 (NRG1) type III is a growth factor on the surface of neurons in the peripheral nervous system (PNS). It is required for initial myelination of nerves by Schwann cells after birth and for remyelination after injury. Neuregulin-1 type III is activated by cleavage (shedding) in its extracellular juxtamembrane region generating a membrane-bound N-terminal fragment (NTF) that contains a bioactive epidermal growth factor (EGF)-like domain. This domain signals to neighboring Schwann cells in a contact-dependent manner prompting the cells to initiate myelination. The β-site APP cleaving enzyme 1 (BACE1) was identified as the enzyme that cleaves NRG1 type III and promotes myelination. Consequently, loss of BACE1 cleavage results in dramatically reduced myelin sheaths around nerves in the PNS of BACE1 knockout mice. Besides its role in myelination, BACE1, better known as β-secretase, is also involved in the generation of the neurotoxic amyloid β-peptide (Aβ) which is the main component of amyloid plaques in the brain of patients suffering from Alzheimer’s disease (AD). The Aβ peptide is derived through sequential cleavage of the amyloid precursor protein APP, first by BACE1 in the extracellular domain and subsequently by the γ-secretase in the transmembrane domain (TMD). Inhibition of BACE1 and γ-secretase is therefore considered a promising therapeutic strategy for AD. However, this approach harbors the risk of mechanism-based side effects due to impaired processing of substrates beside APP such as NRG1 type III which is not only a substrate for BACE1 but like APP is also cleaved in its TMD by the γ-secretase. Adding another layer of complexity, ADAM10 and ADAM17, the so-called α-secretases of AD, also cleave NRG1 type III. In the first part of this study, the proteolytic processing of NRG1 type III in its ectodomain was investigated in detail. The precise juxtamembrane shedding sites of BACE1, ADAM10 and ADAM17 were determined by mass spectrometry and two novel cleavage sites of BACE1 and ADAM17 N-terminal of the EGF-like domain were discovered. Cleavage at these novel sites by ADAM17 and BACE1 results in the secretion of the EGF-like domain from NRG1 type III as α-sEGF and β-sEGF, respectively. Using novel monoclonal antibodies generated against the identified cleavage sites the processing of NRG1 type III could also be confirmed in primary neurons. The soluble EGF-like domains were found to be functionally active and induced signaling pathways required for myelination in cultured Schwann cells. Furthermore, β-sEGF rescued the myelination deficit in the PNS of a zebrafish model lacking BACE1, thereby demonstrating its activity in vivo. Using cell culture and the zebrafish model the effects of BACE1- and ADAM17-mediated shedding on the activity of the soluble EGF-like domains were carefully dissected. In contrast to published evidence, however, both the BACE1- as well as the ADAM17-shed sEGF were found to be equally active and to promote myelination in vivo. Together this suggests that NRG1 type III dependent myelination is not only controlled by membrane-retained NRG1 type III but also in a contact-independent manner via proteolytic liberation of the EGF-like domain. The second part of this study investigates the processing of the C-terminal fragment (CTF) which remains after shedding of NRG1 type III. Intramembranous cleavage of the CTF by the γ-secretase was previously shown to release the NRG1 intracellular domain, which acts as transcriptional regulator of proteins involved in neuronal maturation and brain plasticity. Interestingly, a mutation within the TMD of NRG1 type III is associated with an increased risk of schizophrenia linking γ-secretase processing of NRG1 type III to this neurological disorder. Using a novel antibody against the N-terminus of the NRG1 CTF it was possible to detect a NRG1 β-peptide that is secreted during γ-secretase cleavage and could potentially serve as marker for this processing. Moreover, by means of mass spectrometry, the precise cleavage sites within the TMD of NRG1 could be identified. Strikingly, the ɛ-like cleavage site was found to be located exactly at the position of the schizophrenia-associated mutation providing a possible mechanism for the reported interference of this mutation with γ-secretase cleavage. The evidence presented unambiguously establishes NRG1 type III as a γ-secretase substrate and provides a basis for further investigation of the mechanisms which link its processing to the development of schizophrenia. In summary and with regard to BACE1 and γ-secretase being prime targets for a potential AD therapy, the results of this work call for further careful investigation of the consequences of altered NRG1 type III signaling due to chronic treatment with inhibitors.
Als Wachstumsfaktor auf der Oberfläche von Neuronen des peripheren Nervensystems (PNS) ist Neuregulin-1 (NRG1) Typ III nach der Geburt essentiell für die Ausbildung der die Nerven umgebenden Myelinscheiden durch Schwann-Zellen sowie zur Re-myelinisierung nach einer Verletzung. Hierfür wird NRG1 Typ III durch proteolytische Spaltung seiner extrazellulären Domäne (so genanntes Shedding) durch die Protease BACE1 (engl. β-site APP cleaving enzyme 1) aktiviert. Dabei entsteht ein membranständiges N-terminales Fragment (NTF), das in kontaktabhängiger Weise durch seine bioaktive, dem Epidermalen Wachstumsfaktor ähnliche (engl. epidermal growth factor, EGF) Domäne die Myelinisierung durch benachbarte Schwann-Zellen einleitet. Folglich führt der Verlust der BACE1-vermittelten Spaltung von NRG1 Type III in BACE1 Knockout-Mäusen zu stark reduzierten Myelinscheiden der peripheren Nerven. Neben seiner Rolle bei der Myelinisierung ist BACE1, besser bekannt als β-Sekretase, auch an der Bildung des neurotoxischen Amyloid β Peptides (Aβ), Haupt¬bestandteil der Amyloidplaques im Gehirn von Alzheimer-Patienten, beteiligt. Das Aβ Peptid entsteht durch die aufeinanderfolgende Spaltung des Amyloid-Vorläufer-Proteins APP (engl. amyloid precursor protein) erst durch BACE1 innerhalb der extra¬zellulären Domäne und anschließend durch die γ-Sekretase in der Transmembrandomäne (TMD). Die Hemmung von BACE1 und der γ-Sekretase gilt deshalb als vielversprechender Ansatz für die Therapie von Alzheimer. Allerdings könnte dies zu starken Nebenwirkungen führen, weil beispiels¬weise NRG1 Typ III wie APP auch von BACE1, der γ-Sekretase sowie von ADAM10 und ADAM17, den α-Sekretasen der Alzheimer Krankheit, prozessiert wird. Im ersten Teil dieser Studie wurde die proteolytische Prozessierung der Ektodomäne von NRG1 genauer untersucht. Mithilfe massenspektrometrischer Untersuchungen wurden die genauen Schnittstellen von BACE1, ADAM10 und ADAM17 in der extrazellulären membrannahen Region von NRG1 Typ III bestimmt und zusätzlich zwei neue Schnittstellen auf der N-terminalen Seite der EGF-ähnlichen Domäne identifiziert. Die proteolytische Spaltung von NRG1 Typ III durch ADAM17 und BACE1 an diesen zuvor unbekannten Stellen setzt die EGF-ähnliche Domäne von NRG1 Typ III als α-sEGF und β-sEGF frei. Mit neuen gegen die Schnittstellen gerichteter monoklonaler Antikörpern gelang es zudem, die Prozessierung von NRG1 Typ III auch in primären Nervenzellen nachzuweisen. Es wurde gezeigt, dass die löslichen EGF-ähnlichen Domänen funktional sind und die zur Ausbildung von Myelinscheiden notwendigen Signalkaskaden in Schwann-Zellen auslösen. Außerdem war β-sEGF in der Lage, den Myelinisierungsdefekt im PNS eines BACE1-defizienten Zebrafish-Modells zu beheben, was die Aktivität von β-sEGF in vivo bestätigt. Der Einfluss, den das Shedding durch BACE1 und ADAM17 auf die Aktivität der löslichen EGF-ähnlichen Domäne hat, wurde in Zellkultur und im Zebrafish-Modell ausführlich untersucht. Im Widerspruch zu bisher veröffentlichten Daten wurde festgestellt, dass sowohl das von BACE1 als auch das von ADAM17 geschnittene sEGF gleichermaßen aktiv ist und die Ausbildung von Myelinscheiden fördert. Zusammengenommen deutet dies darauf hin, dass die von NRG1 Typ III abhängige Myelinisierung nicht nur von membrangebundenem NRG1 Typ III gesteuert wird, sondern auch auf eine kontaktunabhängige Weise von der durch Proteolyse freigesetzten löslichen EGF-ähnlichen Domäne. Der zweite Teil dieser Studie befasst sich mit der Prozessierung des durch das Shedding von NRG1 Typ III entstandenen C-terminalen Fragments (CTF). Wie bereits früher gezeigt wurde, führt die Intramembranproteolyse des CTFs durch die γ-Sekretase zur Freisetzung der intrazellulären Domäne von NRG1, die an der Regulierung der neuronalen Reifung und der Plastizität des Gehirns beteiligt ist. Interessanterweise ist eine Mutation innerhalb der TMD von NRG1 Typ III mit einem erhöhten Risiko an Schizophrenie zu erkranken verbunden und stellt damit einen Zusammenhang zwischen der Prozessierung von NRG1 Typ III durch die γ-Sekretase und dieser neurologischen Erkrankung dar. Die Verwendung eines neuen Antikörpers gegen den N-Terminus des NRG1 CTFs ermöglichte es, ein NRG1 β Peptid zu detektieren, das während der Spaltung durch die γ-Sekretase freigesetzt wird und möglicherweise als Biomarker für diese Prozessierung dienen könnte. Des Weiteren konnten massen¬spektrometrisch die genauen Schnittstellen innerhalb der TMD von NRG1 identifiziert werden. Bemerkenswerterweise liegt die ɛ-ähnliche Schnittstelle genau an der Position der mit Schizophrenie assoziierten Mutation, was möglicherweise die von dieser Mutation ausgehende Beeinträchtigung der γ-Sekretase-bedingten Spaltung, über die früher schon berichtet wurde, erklären könnte. Die hier vorgelegten Daten zeigen eindeutig, dass NRG1 Typ III ein Substrat der γ-Sekretase ist und bereiten die Grundlage für weiterführende Untersuchungen des Zusammenhangs zwischen der Prozessierung von NRG1 Typ III und der Entwicklung von Schizophrenie. Vor dem Hintergrund, dass die Hemmung bzw. Modulation von BACE1 und der γ-Sekretase als vielversprechende Strategie zur Behandlung der Alzheimer Krankheit gilt, machen die Ergebnisse dieser Arbeit deutlich, dass es weiterer Untersuchungen der Auswirkungen bedarf, die eine veränderte Signalübertragung von NRG1 Typ III aufgrund der Hemmung dieser beiden Enzyme zur Folge hätte.

The production and aggregation of the amyloid β-peptide (Aβ) is thought to play a central role in Alzheimer’s disease (AD) pathogenesis. The presenilin (PS)-containing γ-secretase complex cleaves the amyloid β-protein precursor C-terminal fragment (APP CTFβ) to generate Aβs of 38-49 residues. Evidence suggests that these Aβs are the result of successive γ-secretase cleavages, which are thought to start at the ε sites to generate Aβ48 or Aβ49, followed by C-terminal trimming mostly every three residues to produce secreted Aβs. Specifically, two product lines have been proposed: the Aβ49-46-43-40 line and the Aβ48-45-42-38 line. An increased proportion of aggregation-prone Aβ42 compared to Aβ40 is believed to be important in AD pathogenesis. Despite the apparent relevance of the production of the Aβ C-terminus in AD, questions surround the mechanisms by which γ-secretase generates the Aβ spectrum and how familial AD-causing (FAD) mutations alter Aβ production. This dissertation first examined the C-terminal trimming function of γ-secretase and how PS FAD mutations alter this activity. We found that synthetic Aβ49, Aβ48, Aβ46, and Aβ45 are trimmed to Aβ40 and Aβ42 by γ-secretase in vitro. Moreover, our results were consistent with the two-pathway model in which Aβ49 is primarily converted to Aβ40 and Aβ48 to Aβ42, but also demonstrated a small degree of crossover between the pathways. Most importantly, we found that PS1 FAD mutations dramatically reduce the efficiency of trimming of ε-cleaved Aβs, particularly the trimming of Aβ49 to Aβ40. We also investigated substrate determinants for ε site endoproteolysis and C-terminal trimming of APP CTFβ by γ-secretase. The deletion of residues around the ε sites indicated that upstream sequences, and not depth within the transmembrane domain, are the determinants of ε site specificity. We also show that instability of the APP CTFβ transmembrane helix near the ε site increases endoproteolysis, and that instability near the carboxypeptidase cleavage sites facilitates C-terminal trimming by γ-secretase. Last, the potential role of Aβ45-49 in AD pathogenesis was considered. We did not detect these Aβ species in AD brains by immunoprecipitation and western blot. However, we developed cellular systems to investigate their toxicity and obtained preliminary data suggesting that these Aβs may be neurotoxic.
Medical Sciences