Annular Protofibrils Are a Structurally and Functionally Distinct Type of Amyloid Oligomer

Amyloid oligomers are believed to play causal roles in several types of amyloid-related neurodegenerative diseases. Several different types of amyloid oligomers have been reported that differ in morphology, size, or toxicity, raising the question of the pathological significance and structural relationships between different amyloid oligomers. Annular protofibrils (APFs) have been described in oligomer preparations of many different amyloidogenic proteins and peptides as ring-shaped or pore-like structures. They are interesting because their pore-like morphology is consistent with numerous reports of membrane-permeabilizing activity of amyloid oligomers. Here we report the preparation of relatively homogeneous preparations of APFs and an antiserum selective for APFs (αAPF) compared with prefibrillar oligomers (PFOs) and fibrils. PFOs appear to be precursors for APF formation, which form in high yield after exposure to a hydrophobic-hydrophilic interface. Surprisingly, preformed APFs do not permeabilize lipid bilayers, unlike the precursor PFOs. APFs display a conformation-dependent, generic epitope that is distinct from that of PFOs and amyloid fibrils. Incubation of PFOs with phospholipids vesicles results in a loss of PFO immunoreactivity with a corresponding increase in αAPF immunoreactivity, suggesting that lipid vesicles catalyze the conversion of PFOs into APFs. The annular anti-protofibril antibody also recognizes heptameric α-hemolysin pores, but not monomers, suggesting that the antibody recognizes an epitope that is specific for a β barrel structural motif.Many age-related neurodegenerative diseases are characterized by the accumulation of amyloid deposits derived from a variety of misfolded proteins (1). These diseases typically have both sporadic and inherited forms, and in many cases the mutations associated with the familial forms are in the gene encoding the protein that accumulates or in genes directly related to its production, processing, or accumulation (2). The genetic linkage between the mutant allele and disease is evidence of the causal relationship of amyloid accumulation to pathogenesis, and many of the mutations either destabilize the natively folded state, produce more amyloidogenic protein, or they increase its propensity to aggregate (3). Although fibrillar amyloid deposits are among the most obvious pathognomonic features of disease, their role in pathogenesis is not clear. The extent of fibrillar amyloid plaque deposition does not correlate well with Alzheimer''s disease pathogenesis, and there are a significant number of non-demented individuals that have equivalent amounts of amyloid plaques as disease patients (4). Pathological changes are observed in transgenic animals before the onset of amyloid plaque accumulation (5, 6), and it has been reported that soluble Aβ oligomers correlate better with dementia than insoluble, fibrillar deposits (7, 8), suggesting that oligomeric forms of Aβ may represent the primary toxic species. Soluble oligomers have been implicated as the primary toxic species in many degenerative diseases where the accumulation of large fibrillar deposits may be either inert, protective, or pathological by a different mechanism (for review, see Refs. 9 and 10).Aβ aggregates have been described ranging in size from dimers up to particles of one million daltons or larger (11–16). In the atomic force microscope prefibrillar oligomers (PFOs)³ appear as spherical particles of ∼3–10 nm. PFOs appear at early times of incubation and disappear as mature fibrils appear (16–18). At longer times of incubation PFOs appear to coalesce to form curvilinear beaded strings that have been called protofibrils and ring-shaped, pore-like structures referred to as annular protofibrils (APFs) (17). APFs appear to be formed from the circularization of PFO subunits. A similar spectrum of PFOs and APFs has been observed for many types of amyloids, such as α-synuclein (19), islet amyloid (20), and non-disease associated “neoamyloids” (21). Although PFOs, APFs, and fibrils have been observed for many different types of amyloidogenic proteins and peptides (22), their structures, interrelationships, and contributions to disease pathogenesis are not entirely clear.Insoluble fibrils and small soluble pieces of fibrils known as fibrillar oligomers appear to have a distinct and mutually exclusive underlying structure than PFOs because they display generic epitopes that are recognized by distinct conformation-dependent monoclonal antibodies (23, 24) and antisera (25, 26). It is not yet known whether APFs represent a unique conformation or whether they are structurally related to PFOs or fibrils. So far APFs have only been defined morphologically as pore-like structures and have been observed in preparations of PFOs and in fibril-containing preparations (27–29). Familial mutations associated with inherited forms of Parkinson and Alzheimer diseases increase the formation of APFs, suggesting that their formation is related to pathogenic activity (17, 30). Based on the close resemblance between APFs and bacterial pore-forming toxins, it has been proposed that APFs permeabilize membranes (22). Because membrane permeabilization is a common pathogenic activity of prefibrillar amyloid oligomers (31) and PFOs are a precursor to annular protofibril formation, the formation of APFs is an attractive explanation for the membrane permeabilization of oligomers because annular protofibril formation is also a common assembly state and they resemble pores morphologically.Investigating the pathological properties of Aβ APFs has been impeded by a lack of homogeneous preparations of annular structures and the lack of a facile means of distinguishing them from other aggregations states in vivo. Here we report the preparation of relatively homogeneous populations of APFs that have the same pore-like morphology previously described. We have used these preparations to examine their aggregation potential and membrane-permeabilizing properties and as an immunogen for the preparation of an antiserum that selectively recognizes APFs, compared with monomers, PFOs, and fibrils. APFs are stable and do not convert into fibrils or PFOs within months of incubation. APFs also exhibit much lower membrane-permeabilizing activity compared with the prefibrillar oligomer precursors to APF formation. Interaction with a hydrophobic-hydrophilic interface accelerates the conversion of PFOs into APFs. Incubation of PFOs with lipid vesicles results in a rapid loss of the prefibrillar oligomer specific epitope and the coordinate appearance of an annular protofibril-specific epitope. APFs display a unique conformation-dependent epitope that is distinct from PFOs and fibrils. Anti-annular protofibril antibody recognizes mature heptameric pores from α-hemolysin, suggesting that APFs may form β-barrel pore structures.     

Distinct Annular Oligomers Captured along the Assembly and Disassembly Pathways of Transthyretin Amyloid Protofibrils

Background

Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated.

Methodology/Principal Findings

We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway.

Conclusions/Significance

Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders.     

Carnosine's Effect on Amyloid Fibril Formation and Induced Cytotoxicity of Lysozyme

Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptide''s anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosine''s mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosine''s mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosine''s amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.     

The Non-Core Regions of Human Lysozyme Amyloid Fibrils Influence Cytotoxicity

Identifying the cause of the cytotoxicity of species populated during amyloid formation is crucial to understand the molecular basis of protein deposition diseases. We have examined different types of aggregates formed by lysozyme, a protein found as fibrillar deposits in patients with familial systemic amyloidosis, by infrared spectroscopy, transmission electron microscopy, and depolymerization experiments, and analyzed how they affect cell viability. We have characterized two types of human lysozyme amyloid structures formed in vitro that differ in morphology, molecular structure, stability, and size of the cross-β core. Of particular interest is that the fibrils with a smaller core generate a significant cytotoxic effect. These findings indicate that protein aggregation can give rise to species with different degree of cytotoxicity due to intrinsic differences in their physicochemical properties.     

Analysis of Core Region from Egg White Lysozyme Forming Amyloid Fibrils

Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.     

Characterization of the Heterogeneity and Specificity of Interpolypeptide Interactions in Amyloid Protofibrils by Measurement of Site-Specific Fluorescence Anisotropy Decay Kinetics

The aggregation of proteins often results in highly ordered fibrillar structures. While significant insights have been obtained on structural aspects of amyloid fibrils, little is known about the structures of protofibrils, which are presumed to be the precursors of fibrils. An understanding of the molecular mechanism of the formation of protofibrils and fibrils requires information on the landscape of interpeptide interactions. This work addresses this question by using, as a model protein, barstar, which forms protofibrils and fibrils at low (< 3) pH. Use was made of the heterogeneity of aggregate populations encountered during fibril formation. Population heterogeneity was scored through rotational dynamics monitored by time-resolved fluorescence anisotropy of an environment-sensitive fluorophore, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS), attached to specific locations in the protein. Firstly, it was observed that barstar, when labeled at certain locations with 1,5-IAEDANS, did not form mixed protofibrils with the corresponding unlabeled protein. Labeled and unlabeled proteins formed protofibrils as separate populations. A two-population model of fluorescence anisotropy decay kinetics exhibiting a ‘dip-and-rise’ behavior was the main readout in arriving at this conclusion. Additional support for this conclusion came from the fluorescence lifetime of the probe 1,5-IAEDANS. Subsequently, the location of the fluorophore was moved along the length of the protein in nine mutant proteins, and the capability to form mixed fibrils was assessed. The results revealed that about two-thirds of the protein sequence at the C-terminal end of the protein was intimately involved in the formation of ordered protofibrils, probably forming the core, while the remaining one-third of the protein (i.e., the N-terminal region) remained largely noninteractive and flexible. This methodology can be used as a general strategy to identify regions of a given protein sequence involved in interprotein interactions in amyloid protofibrils.     

N-Terminal T4 Lysozyme Fusion Facilitates Crystallization of a G Protein Coupled Receptor

A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β₂ adrenergic receptor (β₂AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.     

原生质体融合获得柑桔种间体细胞杂种

粗柠檬(Citrus jambhiri Lush)叶肉原生质体与哈姆林甜橙(C.sinensis L.Osbeck)胚性悬浮细胞系原生质体经PEG诱导融合,培养7天时原生质体恢复分裂。再生的胚状体在含有GA_3的培养基中萌发出茎芽。茎芽经生根诱导成为完整植株。对首批再生的5棵植株进行染色体检查,结果表明,全为四倍体,2n=4x=36。淀粉胶电泳分析过氧化物酶同工酶,结果显示这5棵植株为体细咆杂种。粗柠檬和哈姆林甜橙在该位点上均为同质结合,基因型分别是MM和FF。体细胞杂种含有双亲的酶带,基因型为MMFF。杂种植株生长旺盛,根系发达,叶片及植株形态介于双亲之间。本文对其作为砧木品种的可能性等问题作了讨论。     

抗菌肽Cecropin B-人溶菌酶融合蛋白表达载体的构建

目的是构建抗菌肽B(Cecropin B)和人溶菌酶(hLyso)的融合蛋白表达载体。从pUC118～hLyso上卸下人溶菌酶基因后,通过重叠区扩增法人工合成抗菌肽B基因,并将其融合到人溶菌酶基因的5’端。将抗菌肽B基因和人溶菌酶基因按正确的阅读框架定向克隆至大肠杆菌高效表达载体pET32a,终止子位于人溶菌酶基因的3’端。PCR鉴定及序列分析表明,所转化的BL21(DE3)菌落中含有插入Cecropin B-hLyso基因的重组质粒pET32a-CB-hLyso。     

Regeneration of Hybrid Plantlets Via Pollen-Hypocotyl Protoplast Fusion in Brassica spp.

It has been reported that "gameto-somatic hybridization" was induced by fusion of microspore tetrad protoplasts with somatic protoplasts in Nicotiana and Petunia. However, since the success of isolation of pollen protoplasts in recent years, the use of protoplasts at pollen stage as one of the fusion partners in such hybridization is a novel experimentation. Young pollen protoplasts were isolated from the pollen grains of Brassica chinensis at mid-late unicellular to early bicellular stage the pollens for 1.5--2.5 h at 25℃ in a CPW solution containing 0.8 % of eellulase, 0.5 % pectinase, 0.1% pectolyase, 1 3 % mannitol, 1 0 % glucose, 0. 3% potassium dextran sulphate and 3 mmol/L MES. The purified pollen protoplasts were then fused with the hypocotyl protoplasts of B. napus by PEG method. Heterokaryons were identified by means of visualization of the fluorescence from FITC-prela-beled pollen protoplasts. In order to increase heterokaryons and reduce hypocotyls homokaryons, the denstity of hypocotyl protoplasts were lowered and the ratio of the number of hypocotyl vs. pollen protoplasts were adjusted from 1 : 3 to 1 : 6. The fusion products were cultured in a liquid KM8p medium supplemented with 0.4 mol/L glucose, 0.8 mg/L 2, 4-D, 0.25 mg/L NAA. 0. 5 mg/L BA, 500 mg/L glutamine and 3 mmol/L MES where cell division and callus formation took place. The calli, after being transferred to a MS medium supplemented with 2.0 mg/L BA, 3 % sucrose and 0.4 % agarose, differentiated into a few shoots. The shoots were transferred onto a half-strength MS medium supplemented with 2% sucrose, 0.1--0. 2 mg/L NAA, 0.5 mg/L IBA and 20% potato juice for root formation. Finally, three plantlets were regenerated. Chromosome counts by roottip squash method revealed that one plantlet was 2n= 48, corresponding to an allotriploid resulted from a fusion between one pollen protoplast of B. chinensis (2n = 20) and one hypocotyl protoplast of B. napus (2n = 38), and the other two plantlets were 2n = 58, which might be an allotetraploid originated from a fusion between two pollen protoplasts and one hypocotyl protoplast. The isozyme patterns of leaf esterases showed that all the three plantlets had bands characteristic of both parents. This is the first case of success in "gameto-somatic hybridization" by using pollen protoplasts rather than tetrad protoplasts as the haploid partner.     

朊粒蛋白PrP~(Sc)寡聚体的形成与跨膜毒性

朊粒蛋白(prionprotein,PrP)传染致病机制一直是朊粒(prion)研究领域的焦点.由正常型朊粒蛋白(PrPC)向致病型朊粒蛋白(PrPSc)的转变是致病的关键步骤.本文综述了近年来PrPC向PrPSc转变的结构变化特征、PrPSc由单体形成寡聚体的组装机制、以及PrPSc寡聚体的跨膜机制与细胞毒性间的关系等方面的研究进展.     

Design and Unique Expression of a Novel Antibacterial Fusion Protein Cecropin B-Human Lysozyme to Be Toxic to Prokaryotic Host Cells

Probiotics and Antimicrobial Proteins - A novel antibacterial fusion protein, cecropin B-human lysozyme (CB-hLyso), was designed and expressed in a prokaryotic system. The full-length CB gene was...     

Genetic Dissection of the Amyloid Precursor Protein in Developmental Function and Amyloid Pathogenesis

Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, β-amyloid (Aβ) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aβ sequence was replaced by the human Aβ. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aβ pathogenesis.     

Internal Dynamics and Overall Motion of Lysozyme Studied by Fluorescence Depolarization of the Eosin Lysozyme Complex

Abstract

Time-resolved fluorescence depolarization on the nanosecond and sub-nanosecond time scales is a powerful technique for the study of rapid motions in the condensed phase. We apply this technique to measure the motions of proteins using both extrinsic and intrinsic probes. Eosin, which absorbs and fluoresces in the visible, forms a one-to-one complex with lysozyme binding in the hydrophobic box region and is used as an extrinsic probe of lysozyme motion. The long-time anisotropy of bound eosin is used to measure the overall rotation time of lysozyme for which refined values are presented. In addition, our measurements show a rapid restricted motion of the eosin molecule on the time scale of ～ 100 ps. The order parameter, a model independent measure of the extent of the restriction of the rapid motions, decreases with increasing temperature, indicating that the motion of the eosin is less hindered as temperature increases. We compare our results with the crystallographic measurements of least square displacements for the hydrophobic box region. Our measurements provide direct time resolved confirmation that the displacements observed in this region correspond to rapid motion.     

The Rcs Two-Component System Regulates Expression of Lysozyme Inhibitors and Is Induced by Exposure to Lysozyme

The Escherichia coli Rcs regulon is triggered by antibiotic-mediated peptidoglycan stress and encodes two lysozyme inhibitors, Ivy and MliC. We report activation of this pathway by lysozyme and increased lysozyme sensitivity when Rcs induction is genetically blocked. This lysozyme sensitivity could be alleviated by complementation with Ivy and MliC.In gram-negative bacteria, the cell envelope represents an important functional compartment that extends from the cytoplasmic membrane to the outer membrane and supports a number of essential processes, such as solute transport, protein translocation, and respiratory energy generation (15). In addition, the cell envelope accommodates the bacterial peptidoglycan layer, a distinct and structurally vital element of the cell. Most recently, Laubacher and Ades (10) have demonstrated that the Rcs phosphorelay system of Escherichia coli, originally described as regulator of capsule synthesis, is activated by β-lactam antibiotics that inhibit penicillin-binding proteins and consequently interfere with peptidoglycan synthesis. Moreover, mutational activation of the Rcs pathway provided significant protection against these antibiotics, indicating that members of this regulon can prevent or repair the peptidoglycan damage caused by β-lactam antibiotics (10).Interestingly, ivy and ydhA, two genes encoding specific lysozyme inhibitors, were found to reside under this Rcs regulon (8, 10). Ivy (inhibitor of vertebrate lysozyme, formerly known as YkfE) was discovered in 2001 as the first bacterial lysozyme inhibitor (1, 14), while the inhibitory activity of YdhA was only recently revealed by our research group (3). Although Ivy and YdhA are both able to inhibit c-type lysozymes, such as human lysozyme and hen egg white lysozyme (HEWL), they are structurally unrelated (1, 16). Interestingly, YdhA belongs to a group of proteins with a common conserved COG3895 domain that are widely spread among the Proteobacteria (3, 16). Unlike Ivy, which resides in the periplasm, YdhA is a lipoprotein and was therefore renamed MliC (membrane-bound lysozyme inhibitor of c-type lysozyme) (3).Given the elementary observation that the two currently known lysozyme inhibitors of E. coli are both part of the Rcs regulon that can in turn be induced by antibiotic-mediated peptidoglycan stress, we wondered whether Rcs induction could also result from exposure to lysozyme itself. To test this, we introduced a tolA knockout from MG1655 tolA (3) into strain DH300 that is equipped with a genomic rprA-lacZ fusion able to report Rcs activation (12), in order to increase outer membrane permeability for HEWL (Table ​(Table11 lists all strains). A stationary-phase culture of the resulting strain, designated LC100, was diluted 1/100 in 4 ml fresh LB medium with different final concentrations of HEWL (0, 5, 10, 25, and 50 μg/ml), and after 2.5 h of further growth at 37°C, β-galactosidase activity was measured (13). Interestingly, rprA-lacZ was significantly induced at HEWL concentrations of >10 μg/ml, up to 4.4-fold at 50 μg/ml (Fig. ​(Fig.1A).1A). This induction could be completely abolished upon the additional introduction of a knockout of rcsB (strain LC102), the response regulator required to activate gene expression in the Rcs pathway. Moreover, knocking out rcsF (strain LC101), the outer membrane lipoprotein sensor that triggers the Rcs pathway upon antibiotic-mediated peptidoglycan stress (10), also resulted in a loss of lysozyme induction. As a comparison, rprA-lacZ induction in DH300 treated with amdinocillin (Sigma-Aldrich, Bornem, Belgium), as previously described (10), resulted in a 16-fold increase in β-galactosidase activity (Fig. ​(Fig.1B).1B). Please note that the difference in basal β-galactosidase levels between LC100 and DH300 (Fig. 1A and B) is probably due to the tolA mutation in LC100, which is known to result in a higher basal expression of the Rcs pathway (5). These data clearly demonstrate that the Rcs phosphorelay can indeed be activated by exposure to lysozyme and that this induction is mediated by the outer membrane sensor rcsF. This also implies that the Rcs pathway responds to different types of peptidoglycan stress, as β-lactam antibiotics block the formation of peptide side-chain cross-links by binding irreversibly to the transpeptidases, while lysozyme hydrolyzes the heteropolysaccharide backbone.Open in a separate windowFIG. 1.Induction of the Rcs pathway in LC100 (tolA::Kn Rcs⁺) with different HEWL concentrations (0 to 50 μg/ml) (A) and in DH300 (Rcs⁺) with (+) or without (−) amdinocillin treatment (B). Rcs induction is measured as β-galactosidase activity originating from a genomic rprA-lacZ reporter fusion and expressed in Miller units (13). Error bars indicate standard deviations of results from three replicate experiments. The corresponding RcsB⁻ strain (LC102) and the RcsF⁻ strain (LC101) showed rprA-lacZ inductions of <10 Miller units when subjected to lysozyme treatments and are therefore not shown.

TABLE 1.

Bacterial strains and plasmids used in the study

Stability and Inter-domain Interactions Modulate Amyloid Binding Activity of a General Amyloid Interaction Motif

The M13 tip protein, g3p, binds the C-terminal domain of the bacterial membrane protein TolA via β-sheet augmentation, facilitating viral entry into Escherichia coli. G3p binding leads to rearrangement of the β strands and partial unfolding of TolA. G3p also binds multiple amyloid assemblies with high affinity, and it can remodel them into amorphous aggregates. We previously showed that amyloid binding activity is defined by the two g3p N-terminal domains, which we call the general amyloid interaction motif (GAIM). GAIM–hIgG1Fc fusions, which add immune effector function to amyloid targeting of GAIM, mediate reduction of two CNS amyloid deposits, Aβ plaques and tau tangles, in transgenic animal models of neurodegenerative disease. We carried out site-directed mutagenesis of GAIM to identify variants with altered amyloid binding and remodeling activity. A small set of residues along the inner strands of the two domains regulates both activities. The specificity of amyloid binding is governed by individual domain stability and inter-domain interactions. Our studies reveal several lines of similarity between GAIM binding to amyloids and g3p binding to its E. coli membrane target, TolA. Based on these studies, we designed new GAIM fusions that show enhanced binding potency towards multiple amyloid aggregates.     

Differential Regulation of Amyloid Precursor Protein/Presenilin 1 Interaction during Ab40/42 Production Detected Using Fusion Constructs

Beta amyloid peptides (Aβ) play a key role in the pathogenesis of Alzheimer disease (AD). Presenilins (PS) function as the catalytic subunits of γ-secretase, the enzyme that releases Aβ from ectodomain cleaved amyloid precursor protein (APP) by intramembrane proteolysis. Familial Alzheimer disease (FAD)-linked PSEN mutations alter APP processing in a manner that increases the relative abundance of longer Aβ42 peptides to that of Aβ40 peptides. The mechanisms by which Aβ40 and Aβ42 peptides are produced in a ratio of ten to one by wild type presenilin (PS) and by which Aβ42 is overproduced by FAD-linked PS variants are not completely understood. We generated chimeras of the amyloid precursor protein C-terminal fragment (C99) and PS to address this issue. We found a chimeric protein where C99 is fused to the PS1 N-terminus undergoes in cis processing to produce Aβ and that a fusion protein harboring FAD-linked PS1 mutations overproduced Aβ42. To change the molecular interactions within the C99-PS1 fusion protein, we made sequential deletions of the junction between C99 and PS1. We found differential effects of deletion in C99-PS1 on Aβ40 and 42 production. Deletion of the junction between APP CTF and PS1 in the fusion protein decreased Aβ40, while it did not decrease Aβ42 production in the presence or absence of FAD-linked PS1 mutation. These results are consistent with the idea that the APP/PS interaction is differentially regulated during Aβ40 and 42 production.     

Inactivation of Gram-Negative Bacteria by Lysozyme, Denatured Lysozyme, and Lysozyme-Derived Peptides under High Hydrostatic Pressure

We have studied the inactivation of six gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens, Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei, and Shigella flexneri) by high hydrostatic pressure treatment in the presence of hen egg-white lysozyme, partially or completely denatured lysozyme, or a synthetic cationic peptide derived from either hen egg white or coliphage T4 lysozyme. None of these compounds had a bactericidal or bacteriostatic effect on any of the tested bacteria at atmospheric pressure. Under high pressure, all bacteria except both Salmonella species showed higher inactivation in the presence of 100 μg of lysozyme/ml than without this additive, indicating that pressure sensitized the bacteria to lysozyme. This extra inactivation by lysozyme was accompanied by the formation of spheroplasts. Complete knockout of the muramidase enzymatic activity of lysozyme by heat treatment fully eliminated its bactericidal effect under pressure, but partially denatured lysozyme was still active against some bacteria. Contrary to some recent reports, these results indicate that enzymatic activity is indispensable for the antimicrobial activity of lysozyme. However, partial heat denaturation extended the activity spectrum of lysozyme under pressure to serovar Typhimurium, suggesting enhanced uptake of partially denatured lysozyme through the serovar Typhimurium outer membrane. All test bacteria were sensitized by high pressure to a peptide corresponding to amino acid residues 96 to 116 of hen egg white, and all except E. coli and P. fluorescens were sensitized by high pressure to a peptide corresponding to amino acid residues 143 to 155 of T4 lysozyme. Since they are not enzymatically active, these peptides probably have a different mechanism of action than all lysozyme polypeptides.