Structural mapping of oligomeric intermediates in an amyloid assembly pathway.

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human ß2-microglobulin (ß2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-ß structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.

A new era for understanding amyloid structures and disease.

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-ß structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention.

Why are Functional Amyloids Non-Toxic in Humans?

Amyloids were first identified in association with amyloidoses, human diseases in which proteins and peptides misfold into amyloid fibrils. Subsequent studies have identified an array of functional amyloid fibrils that perform physiological roles in humans. Given the potential for the production of toxic species in amyloid assembly reactions, it is remarkable that cells can produce these functional amyloids without suffering any obvious ill effect. Although the precise mechanisms are unclear, there are a number of ways in which amyloid toxicity may be prevented. These include regulating the level of the amyloidogenic peptides and proteins, minimising the production of prefibrillar oligomers in amyloid assembly reactions, sequestrating amyloids within membrane bound organelles, controlling amyloid assembly by other molecules, and disassembling the fibrils under physiological conditions. Crucially, a better understanding of how toxicity is avoided in the production of functional amyloids may provide insights into the prevention of amyloid toxicity in amyloidoses.

pH-induced molecular shedding drives the formation of amyloid fibril-derived oligomers.

Amyloid disorders cause debilitating illnesses through the formation of toxic protein aggregates. The mechanisms of amyloid toxicity and the nature of species responsible for mediating cellular dysfunction remain unclear. Here, using ß2-microglobulin (ß2m) as a model system, we show that the disruption of membranes by amyloid fibrils is caused by the molecular shedding of membrane-active oligomers in a process that is dependent on pH. Using thioflavin T (ThT) fluorescence, NMR, EM and fluorescence correlation spectroscopy (FCS), we show that fibril disassembly at pH 6.4 results in the formation of nonnative spherical oligomers that disrupt synthetic membranes. By contrast, fibril dissociation at pH 7.4 results in the formation of nontoxic, native monomers. Chemical cross-linking or interaction with hsp70 increases the kinetic stability of fibrils and decreases their capacity to cause membrane disruption and cellular dysfunction. The results demonstrate how pH can modulate the deleterious effects of preformed amyloid aggregates and suggest why endocytic trafficking through acidic compartments may be a key factor in amyloid disease.

ß2-microglobulin amyloid fibrils are nanoparticles that disrupt lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes.

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of ß2-microglobulin (ß2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented ß2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented ß2m fibrils did not, however, cause cell death. Instead, fragmented ß2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.

ß2-Microglobulin amyloid fibril-induced membrane disruption is enhanced by endosomal lipids and acidic pH.

Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of ß2-microglobulin (ß2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which ß2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of ß2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that ß2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between ß2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of ß2m amyloid-associated osteoarticular tissue destruction in DRA.

Analysis of familial hemophagocytic lymphohistiocytosis type 4 (FHL-4) mutant proteins reveals that S-acylation is required for the function of syntaxin 11 in natural killer cells.

Natural killer (NK) cell secretory lysosome exocytosis and cytotoxicity are impaired in familial hemophagocytic lymphohistiocytosis type 4 (FHL-4), a disorder caused by mutations in the gene encoding the SNARE protein syntaxin 11. We show that syntaxin 11 binds to SNAP23 in NK cells and that this interaction is reduced by FHL-4 truncation and frameshift mutation proteins that delete all or part of the SNARE domain of syntaxin 11. In contrast the FHL-4 mutant proteins bound to the Sec-1/Munc18-like (SM) protein Munc18-2. We demonstrate that the C-terminal cysteine rich region of syntaxin 11, which is deleted in the FHL-4 mutants, is S-acylated. This posttranslational modification is required for the membrane association of syntaxin 11 and for its polarization to the immunological synapse in NK cells conjugated to target cells. Moreover, we show that Munc18-2 is recruited by syntaxin 11 to intracellular membranes in resting NK cells and to the immunological synapse in activated NK cells. This recruitment of Munc18-2 is abolished by deletion of the C-terminal cysteine rich region of syntaxin 11. These results suggest a pivotal role for S-acylation in the function of syntaxin 11 in NK cells.

Aggregation modulators interfere with membrane interactions of ß2-microglobulin fibrils.

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer's disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of ß2-microglobulin (ß2m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of ß2m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.

Characterization of the response of primary cells relevant to dialysis-related amyloidosis to ß2-microglobulin monomer and fibrils.

The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of ß(2)-microglobulin (ß(2)m) in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood. In this study we performed a systematic analysis of the interaction of monomeric and fibrillar ß(2)m with primary human cells of the types present in the synovial joints of subjects with DRA. Building upon observations that macrophages infiltrate ß(2)m amyloid deposits in vivo we demonstrate that monocytes, the precursors of macrophages, cannot degrade ß(2)m fibrils, and that both monomeric ß(2)m and fibrillar ß(2)m are cytotoxic to these cells. ß(2)m fibrils also impair the formation of bone resorbing osteoclasts from monocytes and reduce the viability of osteoblasts, the cell type that produces bone. As a consequence, we predict that ß(2)m amyloid will disrupt the remodelling of the bone, which is critical for the maintenance of this tissue. Moreover, we show that ß(2)m fibrils reduce the viability of chondrocytes, rationalizing the loss of cartilage in DRA. Together, our observations demonstrate that ß(2)m cytotoxicity has multiple cellular targets in the osteoarticular tissues and is likely to be a key factor in the bone and joint destruction characteristic of DRA.

Fibril fragmentation in amyloid assembly and cytotoxicity: when size matters.

Amyloid assemblies are associated with several debilitating human disorders. Understanding the intra- and extracellular assembly of normally soluble proteins and peptides into amyloid aggregates and how they disrupt normal cellular functions is therefore of paramount importance. In a recent report, we demonstrated a striking relationship between reduced fibril length caused by fibril fragmentation and enhanced ability of fibril samples to disrupt membranes and to reduce cell viability. These findings have important implications for our understanding of amyloid disease in that changes in the physical dimensions of fibrils, without parallel changes in their composition or molecular architecture, could be sufficient to alter the biological responses to their presence. These conclusions provide a new hypothesis that the physical dimensions and surface interactions of fibrils play key roles in amyloid disease. Controlling fibril length and stability toward fracturing, and thereby the biological availability of fibril material, may provide a new target for future therapeutic strategies towards combating amyloid disease.

Fibril fragmentation enhances amyloid cytotoxicity.

Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.

Natural killer cell cytotoxicity: how do they pull the trigger?

Natural killer (NK) cells target and kill aberrant cells, such as virally infected and tumorigenic cells. Killing is mediated by cytotoxic molecules which are stored within secretory lysosomes, a specialized exocytic organelle found in NK cells. Target cell recognition induces the formation of a lytic immunological synapse between the NK cell and its target. The polarized exocytosis of secretory lysosomes is then activated and these organelles release their cytotoxic contents at the lytic synapse, specifically killing the target cell. The essential role that secretory lysosome exocytosis plays in the cytotoxic function of NK cells is highlighted by immune disorders that are caused by the mutation of critical components of the exocytic machinery. This review will discuss recent studies on the molecular basis for NK cell secretory lysosome exocytosis and the immunological consequences of defects in the exocytic machinery.

Kaposi's sarcoma-associated herpesvirus RTA promotes degradation of the Hey1 repressor protein through the ubiquitin proteasome pathway.

The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation.

Structural and Functional Dissection of the Human Cytomegalovirus Immune Evasion Protein US6.

The human cytomegalovirus (HCMV) protein US6 inhibits the transporter associated with antigen processing (TAP). Since TAP transports antigenic peptides into the endoplasmic reticulum for binding to major histocompatibility class I molecules, inhibition of the transporter by HCMV US6 impairs the presentation of viral antigens to cytotoxic T lymphocytes. HCMV US6 inhibits ATP binding by TAP, hence depriving TAP of the energy source it requires for peptide translocation, yet the molecular basis for the interaction between US6 and TAP is poorly understood. In this study we demonstrate that residues 89 to 108 of the HCMV US6 luminal domain are required for TAP inhibition, whereas sequences that flank this region stabilize the binding of the viral protein to TAP. In parallel, we demonstrate that chimpanzee cytomegalovirus (CCMV) US6 binds, but does not inhibit, human TAP. The sequence of CCMV US6 differs from that of HCMV US6 in the region corresponding to residues 89 to 108 of the HCMV protein. The substitution of this region of CCMV US6 with the corresponding residues from HCMV US6 generates a chimeric protein that inhibits human TAP and provides further evidence for the pivotal role of residues 89 to 108 of HCMV US6 in the inhibition of TAP. On the basis of these observations, we propose that there is a hierarchy of interactions between HCMV US6 and TAP, in which residues 89 to 108 of HCMV US6 interact with and inhibit TAP, whereas other parts of the viral protein also bind to TAP and stabilize this inhibitory interaction.

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils.

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.

Organelle proteomics: identification of the exocytic machinery associated with the natural killer cell secretory lysosome.

Natural killer (NK) cells and cytotoxic T lymphocytes eliminate virally infected and transformed cells. Target cell killing is mediated by the regulated exocytosis of secretory lysosomes, which deliver perforin and proapoptotic granzymes to the infected or transformed cell. Yet despite the central role that secretory lysosome exocytosis plays in the immune response to viruses and tumors, little is known about the molecular machinery that regulates the docking and fusion of this organelle with the plasma membrane. To identify potential components of this exocytic machinery we used proteomics to define the protein composition of the NK cell secretory lysosome membrane. Secretory lysosomes were isolated from the NK cell line YTS by subcellular fractionation, integral membrane proteins and membrane-associated proteins were enriched using Triton X-114 and separated by SDS-PAGE, and tryptic peptides were identified by LC ESI-MS/MS. In total 221 proteins were identified unambiguously in the secretory lysosome membrane fraction of which 61% were predicted to be either integral membrane proteins or membrane-associated proteins. A significant proportion of the proteins identified play a role in vesicular trafficking, including members of both the Rab GTPase and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and protein families. These proteins include Rab27a and the SNARE vesicle-associated membrane protein-7, both of which were enriched in the secretory lysosome fraction and represent potential components of the machinery that regulates the exocytosis of this organelle in NK cells.

Virus subversion of protective immunity.

The major histocompatibility (MHC) class I antigen presentation pathway plays a pivotal role in immunity to viruses. MHC class I molecules are expressed on the cell surface of all nucleated cells and present peptides derived from intracellular proteins to cytotoxic T lymphocytes (CTLs), which then eliminate virally infected cells. However, many viruses have evolved proteins to inhibit the MHC class I pathway, thus enabling virally infected cells to escape CTL lysis. In this review, we summarize recent findings about viral inhibition of the MHC class I pathway.

The ABC-transporter signature motif is required for peptide translocation but not peptide binding by TAP.

The transporter associated with antigen processing (TAP) translocates peptides from their site of generation in the cytosol to the lumen of the endoplasmic reticulum for binding to MHC class I molecules. TAP is a member of the ATP-binding cassette (ABC) transporter family whose members utilize energy from ATP hydrolysis to translocate substrates across membranes. The highly conserved nucleotide-binding domains of ABC transporters couple ATP hydrolysis to substrate translocation by the membrane domains. The conserved 'signature motif' can be identified in the nucleotide-binding domains of all ABC transporters, and may play a role in ATP hydrolysis. Here we show that introduction of mutations into the signature motifs of either TAP1 or TAP2 inhibits the translocation of peptide without affecting binding of either peptide or ATP by TAP. We therefore conclude that the signature motifs in both TAP1 and TAP2 are required after peptide binding to facilitate peptide translocation by TAP.

Powering the peptide pump: TAP crosstalk with energetic nucleotides.

ATP-binding cassette (ABC) transporters represent a large family of membrane-spanning proteins that have a shared structural organization and conserved nucleotide-binding domains (NBDs). They transport a large variety of solutes, and defects in these transporters are an important cause of human disease. TAP (tmacr;ransporter associated with antigen pmacr;rocessing) is a heterodimeric ABC transporter that uses nucleotides to drive peptide transport from the cytoplasm into the endoplasmic reticulum lumen, where the peptides then bind major histocompatibility complex (MHC) class I molecules. TAP plays an essential role in the MHC class I antigen presentation pathway. Recent studies show that the two NBDs of TAP fulfil distinct functions in the catalytic cycle of this transporter. In this opinion article, a model of alternating ATP binding and hydrolysis is proposed, in which nucleotide interaction with TAP2 primarily controls substrate binding and release, whereas interaction with TAP1 controls structural rearrangements of the transmembrane pathway. Viral proteins that inhibit TAP function cause arrests at distinct points of this catalytic cycle.

Synaptotagmin I-DeltaC2B. A novel synaptotagmin isoform with a single C2 domain in the bovine adrenal medulla.

Synaptotagmin I is a 65 kDa type 1 membrane glycoprotein found in secretory organelles that plays a key role in regulated exocytosis. We have characterised two forms (long and short) of synaptotagmin I that are present in the bovine adrenal medulla. The long form is a type I integral membrane protein which has two cytoplasmic C2 domains and corresponds to the previously characterised full-length synaptotagmin I isoform. The short-form synaptotagmin I-DeltaC2B has the same structure in the lumenal and transmembrane sequences, but synaptotagmin I-DeltaC2B is truncated such that it only has a single cytoplasmic C2 domain. Analysis of synaptotagmin I-DeltaC2B expression indicates that synaptotagmin I-DeltaC2B is preferentially expressed in the bovine adrenal medulla. However, it is absent from the dense core chromaffin granules. Furthermore, when expressed in the rat pheochromocytoma cell line PC12 bovine synaptotagmin I-DeltaC2B is largely absent from dense core granules and synaptic-like microvesicles. Instead, indirect immunofluorescence microscopy reveals the intracellular location of synaptotagmin I-DeltaC2B to be the plasma membrane.