Emerging roles of O-glycosylation in regulating protein aggregation,phase separation,and functions

Protein O-glycosylation is widely identified in various proteins involved in diverse biological processes. Recent studies have demonstrated that O-glycosylation plays crucial and multifaceted roles in modulating protein amyloid aggregation and liquid–liquid phase separation (LLPS) under physiological conditions. Dysregulation of these processes is closely associated with human diseases such as neurodegenerative diseases (NDs) and cancers. In this review, we first summarize the distinct roles of O-glycosylation in regulating pathological aggregation of different amyloid proteins related to NDs and elaborate the underlying mechanisms of how O-glycosylation modulates protein aggregation kinetics, induces new aggregated structures, and mediates the pathogenesis of amyloid aggregates under diseased conditions. Furthermore, we introduce recent discoveries on O-GlcNAc-mediated regulation of synaptic LLPS and phase separation potency of low-complexity domain-enriched proteins. Finally, we identify challenges in future research and highlight the potential for developing new therapeutic strategies of NDs by targeting protein O-glycosylation.     

High pressure modulates amyloid formation

A common mechanism of conformational changes and pathological aggregation of proteins associated with amyloid diseases remains to be proven. High pressure is emerging as a new strategy for studying aspects of amyloid formation. Pressure provides a convenient means to populate and characterize partially folded states, which are thought to have a key role in assembly processes of proteins into amyloid fibrils. High pressure can also be used to dissociate aggregates and amyloid fibrils or on the opposite to generate such species.     

Apolipoproteins and amyloid fibril formation in atherosclerosis

Amyloid fibrils arise from the aggregation of misfolded proteins into highly-ordered structures. The accumulation of these fibrils along with some non-fibrillar constituents within amyloid plaques is associated with the pathogenesis of several human degenerative diseases. A number of plasma apolipoproteins, including apolipoprotein (apo) A-I, apoA-II, apoC-II and apoE are implicated in amyloid formation or influence amyloid formation by other proteins. We review present knowledge of amyloid formation by apolipoproteins in disease, with particular focus on atherosclerosis. Further insights into the molecular mechanisms underlying their amyloidogenic propensity are obtained from in vitro studies which describe factors affecting apolipoprotein amyloid fibril formation and interactions. Additionally, we outline the evidence that amyloid fibril formation by apolipoproteins might play a role in the development and progression of atherosclerosis, and highlight possible molecular mechanisms that could contribute to the pathogenesis of this disease.     

Amyloids, prions and the inherent infectious nature of misfolded protein aggregates

Misfolded aggregates present in amyloid fibrils are associated with various diseases known as "protein misfolding" disorders. Among them, prion diseases are unique in that the pathology can be transmitted by an infectious process involving an unprecedented agent known as a "prion". Prions are infectious proteins that can transmit biological information by propagating protein misfolding and aggregation. The molecular mechanism of prion conversion has a striking resemblance to the process of amyloid formation, suggesting that misfolded aggregates have an inherent ability to be transmissible. Intriguing recent data suggest that other protein misfolding disorders might also be transmitted by a prion-like infectious process.     

Implications of protein structure instability: from physiological to pathological secondary structure

Proteins are folded during their synthesis; this process may be spontaneous or assisted. Both phenomena are carefully regulated by the "housekeeping" mechanism and molecular chaperones to avoid the appearance of misfolded proteins. Unfolding process generally occurs during physiological degradation of protein, but in some specific cases it results from genetic or environmental changes and does not correspond to metabolic needs. The main outcome of these phenomena is the appearance of nonfunctional pathologically unfolded proteins with a strong tendency to aggregation. Moreover, for some of these unfolded proteins, the agglomeration that follows initial proteins association may give rise to highly structured soluble aggregates. These aggregates have been identified as the main cause of the so-called amyloidosis or amyloid diseases, such as Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases, and type II diabetes mellitus. Although some common mechanisms of amyloid protein aggregation have been identified, the roles of the environmental conditions inducing amyloidosis remain to be clarified. In this review, we will summarize recent studies identifying the origin of amyloid nucleation and will try to predict the therapeutic prospects that may be opened by elucidation of the amyloidosis mechanisms.     

Protein folding and diseases

For most of proteins to be active, they need well-defined three-dimensional structures alone or in complex. Folding is a process through which newly synthesized proteins get to the native state. Protein folding inside cells is assisted by various chaperones and folding factors, and misfolded proteins are eliminated by the ubiquitin-proteasome degradation system to ensure high fidelity of protein expression. Under certain circumstances, misfolded proteins escape the degradation process, yielding to deposit of protein aggregates such as loop-sheet polymer and amyloid fibril. Diseases characterized by insoluble deposits of proteins have been recognized for long time and are grouped as conformational diseases. Study of protein folding mechanism is required for better understanding of the molecular pathway of such conformational diseases.     

Potential roles of abundant extracellular chaperones in the control of amyloid formation and toxicity

The in vivo formation of fibrillar proteinaceous deposits called amyloid is associated with more than 40 serious human diseases, collectively referred to as protein deposition diseases. In many cases the amyloid deposits are extracellular and are found associated with newly identified abundant extracellular chaperones (ECs). Evidence is presented suggesting an important regulatory role for ECs in amyloid formation and disposal in the body. A model is presented which proposes that, under normal conditions, ECs stabilize extracellular misfolded proteins by binding to them, and then guide them to specific cell receptors for uptake and subsequent degradation. Thus ECs and their receptors may be critical parts of a quality control system to protect the body against dangerously hydrophobic proteins/peptides. However, it also appears possible that in the presence of a high molar excess of misfolded protein, such as might occur during disease, the limited amounts of ECs available may actually exacerbate pathology. Further advances in understanding of the mechanisms that control extracellular protein folding are likely to identify new strategies for effective disease therapies.     

Misfolded protein aggregates: mechanisms, structures and potential for disease transmission

Some of the most prevalent human degenerative diseases appear as a result of the misfolding and aggregation of proteins. Compelling evidence suggest that misfolded protein aggregates play an important role in cell dysfunction and tissue damage, leading to the disease. Prion protein (Prion diseases), amyloid-beta (Alzheimer's disease), alpha-synuclein (Parkinson's disease), Huntingtin (Huntington's disease), serum amyloid A (AA amyloidosis) and islet amyloid polypeptide (type 2 diabetes) are some of the proteins that trigger disease when they get misfolded. The recent understanding of the crucial role of misfolded proteins as well as the structural requirements and mechanism of protein misfolding have raised the possibility that these diseases may be transmissible by self-propagation of the protein misfolding process in a similar way as the infamous prions transmit prion diseases. Future research in this field should aim to clarify this possibility and translate the knowledge of the basic disease mechanisms into development of novel strategies for early diagnosis and efficient treatment.     

Amyloidosis: a model of misfolded protein disorder

Amyloidosis bears many characteristics of orphan diseases. Its diagnosis is difficult and often delayed. The main reasons thereof are its quite various clinical presentation: amyloidosis behaves as a new great masquerader, and the need to get a tissue sample to submit to specific dyes. Although we have been able for a long time to recognize amyloid, its intimate nature has remained quite completely enigmatic until recently. In fact, major advances in this way have appeared only in the last decade and it is now possible to consider the mechanisms of amyloidosis as a multistep phenomenon. Amyloidosis is no more thought only as a < storage disease > of the extracellular space. This archaic viewpoint has shifted to the emerging paradigm of misfolded protein disorders. Amyloid proteins thus appear as a subgroup of misfolded proteins, where misfolding leads to subsequent aggregation. This aggregation may be a generic property of polypeptide chains possibly linked to their common peptide backbone that does not depend on specific amino acid sequences. And, in fact, many proteins can in vitro form amyloid-like aggregates, while in vivo, only 20 amyloid proteins have been so far identified. Although misfolding and aggregation are quite well studied in vitro, the last step of amyloid deposition, i.e. anchorage to the extracellular matrix, can not be so easily approached. Proteoglycans and serum amyloid P component have nevertheless been identified as key elements involved in extracellular deposition of amyloid proteins. These advances have opened new avenues in the therapeutic of amyloid disorders. Current treatment consists of support or replacement of impaired organ function and measures to reduce the production of amyloidogenic precursor proteins. Potential novel therapeutic strategies include stabilisation of the native fold of precursor proteins with targeted small molecules, reversion of misfolded proteins to their native state with < beta-sheet breakers >, inhibition of amyloid fibril propagation and enhancement of amyloid clearance either through immunotherapy or by reducing the stability of deposits through depletion of serum amyloid P component, and breaking the anchorage to the extracellular matrix with glycosaminoglycan analogs.     

Dynamical Phase Transitions Reveal Amyloid-like States on Protein Folding Landscapes

Developing an understanding of protein misfolding processes presents a crucial challenge for unlocking the mysteries of human disease. In this article, we present our observations of β-sheet-rich misfolded states on a number of protein dynamical landscapes investigated through molecular dynamics simulation and Markov state models. We employ a nonequilibrium statistical mechanical theory to identify the glassy states in a protein’s dynamics, and we discuss the nonnative, β-sheet-rich states that play a distinct role in the slowest dynamics within seven protein folding systems. We highlight the fundamental similarity between these states and the amyloid structures responsible for many neurodegenerative diseases, and we discuss potential consequences for mechanisms of protein aggregation and intermolecular amyloid formation.     

Monitoring the process of HypF fibrillization and liposome permeabilization by protofibrils

Much information has appeared in the last few years on the low resolution structure of amyloid fibrils and on their non-fibrillar precursors formed by a number of proteins and peptides associated with amyloid diseases. The fine structure and the dynamics of the process leading misfolded molecules to aggregate into amyloid assemblies are far from being fully understood. Evidence has been provided in the last five years that protein aggregation and aggregate toxicity are rather generic processes, possibly affecting all polypeptide chains under suitable experimental conditions. This evidence extends the number of model proteins one can investigate to assess the molecular bases and general features of protein aggregation and aggregate toxicity. We have used tapping mode atomic force microscopy to investigate the morphological features of the pre-fibrillar aggregates and of the mature fibrils produced by the aggregation of the hydrogenase maturation factor HypF N-terminal domain (HypF-N), a protein not associated to any amyloid disease. We have also studied the aggregate-induced permeabilization of liposomes by fluorescence techniques. Our results show that HypF-N aggregation follows a hierarchical path whereby initial globules assemble into crescents; these generate large rings, which evolve into ribbons, further organizing into differently supercoiled fibrils. The early pre-fibrillar aggregates were shown to be able to permeabilize synthetic phospholipid membranes, thus showing that this disease-unrelated protein displays the same amyloidogenic behaviour found for the aggregates of most pathological proteins and peptides. These data complement previously reported findings, and support the idea that protein aggregation, aggregate structure and toxicity are generic properties of polypeptide chains.     

Inclusion bodies: specificity in their aggregation process and amyloid-like structure

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, Abeta42 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic Abeta42 intracellular aggregates to act as effective seeds in the formation of Abeta42 amyloid fibrils. Overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.     

胰岛淀粉样多肽的研究进展

由蛋白错误折叠后聚集所产生的淀粉样蛋白沉积是导致老年痴呆症、疯牛病、2型糖尿病等多种疾病的重要因素。由胰岛淀粉样多肽(islet amyloid polypeptide,IAPP)所形成的淀粉样蛋白沉积,具有破坏胰岛β细胞膜结构、诱导β细胞凋亡和损伤β细胞功能的作用,被认为是2型糖尿病的重要致病原因之一。对IAPP的聚集性、聚集体的结构,以及其对β细胞的毒性作用研究,不但有助于明确2型糖尿病的发病机制,而且最新研究也表明抑制IAPP的聚集可有效减少β细胞的凋亡,提高胰岛移植的成功率。因此,IAPP已成为2型糖尿病治疗中一个具有良好前景的靶点。该文对IAPP研究的最新进展进行了简要介绍。     

Molecular chaperones antagonize proteotoxicity by differentially modulating protein aggregation pathways

The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates.Key words: chaperone, heat shock protein, protein aggregation, amyloid, Hsp70, Hsp40, prion     

Sedimentation velocity analysis of amyloid oligomers and fibrils using fluorescence detection

The assembly of proteins into large fibrillar aggregates, known as amyloid fibrils, is associated with a number of common and debilitating diseases. In some cases, proteins deposit extracellularly, while in others the aggregation is intracellular. A common feature of these diseases is the presence of aggregates of different sizes, including mature fibrils, small oligomeric precursors, and other less well understood structural forms such as amorphous aggregates. These various species possess distinct biochemical, biophysical, and pathological properties. Here, we detail a number of techniques that can be employed to examine amyloid fibrils and oligomers using a fluorescence-detection system (FDS) coupled with the analytical ultracentrifuge. Sedimentation velocity analysis using fluorescence detection is a particularly useful method for resolving the complex heterogeneity present in amyloid systems and can be used to characterize aggregation in exceptional detail. Furthermore, the fluorescence detection module provides a number of particularly attractive features for the analysis of aggregating proteins. It expands the practical range of concentrations of aggregating proteins under study, which is useful for greater insight into the aggregation process. It also enables the assessment of aggregation behavior in complex biological solutions, such as cell lysates, and the assessment of processes that regulate in-cell or extracellular aggregation kinetics. Four methods of fluorescent detection that are compatible with the current generation of FDS instrumentation are described: (1) Detection of soluble amyloid fibrils using a covalently bound fluorophore. (2) Detection of amyloid fibrils using an extrinsic dye that emits fluorescence when bound to fibrils. (3) Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates. (4) Detection of green fluorescence protein (GFP) constructs and their interactions within mammalian cell lysates.     

An Intrinsically Disordered Region of the Acetyltransferase p300 with Similarity to Prion-Like Domains Plays a Role in Aggregation

Several human diseases including neurodegenerative disorders and cancer are associated with abnormal accumulation and aggregation of misfolded proteins. Proteins with high tendency to aggregate include the p53 gene product, TAU and alpha synuclein. The potential toxicity of aberrantly folded proteins is limited via their transport into intracellular sub-compartments, the aggresomes, where misfolded proteins are stored or cleared via autophagy. We have identified a region of the acetyltransferase p300 that is highly disordered and displays similarities with prion-like domains. We show that this region is encoded as an alternative spliced variant independently of the acetyltransferase domain, and provides an interaction interface for various misfolded proteins, promoting their aggregation. p300 enhances aggregation of TAU and of p53 and is a component of cellular aggregates in both tissue culture cells and in alpha-synuclein positive Lewy bodies of patients affected by Parkinson disease. Down-regulation of p300 impairs aggresome formation and enhances cytotoxicity induced by misfolded protein stress. These data unravel a novel activity of p300, offer new insights into the function of disordered domains and implicate p300 in pathological aggregation that occurs in neurodegeneration and cancer.     

The amyloid state of proteins in human diseases

Amyloid fibers and oligomers are associated with a great variety of human diseases including Alzheimer's disease and the prion conditions. Here we attempt to connect recent discoveries on the molecular properties of proteins in the amyloid state with observations about pathological tissues and disease states. We summarize studies of structure and nucleation of amyloid and relate these to observations on amyloid polymorphism, prion strains, coaggregation of pathogenic proteins in tissues, and mechanisms of toxicity and transmissibility. Molecular studies have also led to numerous strategies for biological and chemical interventions against amyloid diseases.     

Dynamical Phase Transitions Reveal Amyloid-like States on Protein Folding Landscapes

Developing an understanding of protein misfolding processes presents a crucial challenge for unlocking the mysteries of human disease. In this article, we present our observations of β-sheet-rich misfolded states on a number of protein dynamical landscapes investigated through molecular dynamics simulation and Markov state models. We employ a nonequilibrium statistical mechanical theory to identify the glassy states in a protein’s dynamics, and we discuss the nonnative, β-sheet-rich states that play a distinct role in the slowest dynamics within seven protein folding systems. We highlight the fundamental similarity between these states and the amyloid structures responsible for many neurodegenerative diseases, and we discuss potential consequences for mechanisms of protein aggregation and intermolecular amyloid formation.     

Membranes as modulators of amyloid protein misfolding and target of toxicity

Abnormal protein aggregation is a hallmark of various human diseases. α-Synuclein, a protein implicated in Parkinson's disease, is found in aggregated form within Lewy bodies that are characteristically observed in the brains of PD patients. Similarly, deposits of aggregated human islet amyloid polypeptide (IAPP) are found in the pancreatic islets in individuals with type 2 diabetes mellitus. Significant number of studies have focused on how monomeric, disaggregated proteins transition into various amyloid structures leading to identification of a vast number of aggregation promoting molecules and processes over the years. Inasmuch as these factors likely enhance the formation of toxic, misfolded species, they might act as risk factors in disease. Cellular membranes, and particularly certain lipids, are considered to be among the major players for aggregation of α-synuclein and IAPP, and membranes might also be the target of toxicity. Past studies have utilized an array of biophysical tools, both in vitro and in vivo, to expound the membrane-mediated aggregation. Here, we focus on membrane interaction of α-synuclein and IAPP, and how various kinds of membranes catalyze or modulate the aggregation of these proteins and how, in turn, these proteins disrupt membrane integrity, both in vitro and in vivo. The membrane interaction and subsequent aggregation has been briefly contrasted to aggregation of α-synuclein and IAPP in solution. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

Structure-activity relationship of amyloid fibrils

Protein aggregation is a process in which proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as either amorphous or highly ordered, the most common form of the latter being amyloid fibrils. Amyloid fibrils composed of cross-β-sheet structure are the pathological hallmarks of several diseases including Alzheimer’s disease, but are also associated with functional states such as the fungal HET-s prion. This review aims to summarize the recent high-resolution structural studies of amyloid fibrils in light of their (potential) activities. We propose that the repetitive nature of the cross-β-sheet structure of amyloids is key for their multiple properties: the repeating motifs can translate a rather non-specific interaction into a specific one through cooperativity.