Amyloidogenesis: What Do We Know So Far?

The study of protein aggregation, and amyloidosis in particular, has gained considerable interest in recent times. Several neurodegenerative diseases, such as Alzheimer's (AD) and Parkinson's (PD) show a characteristic buildup of proteinaceous aggregates in several organs, especially the brain. Despite the enormous upsurge in research articles in this arena, it would not be incorrect to say that we still lack a crystal-clear idea surrounding these notorious aggregates. In this review, we attempt to present a holistic picture on protein aggregation and amyloids in particular. Using a chronological order of discoveries, we present the case of amyloids right from the onset of their discovery, various biophysical techniques, including analysis of the structure, the mechanisms and kinetics of the formation of amyloids. We have discussed important questions on whether aggregation and amyloidosis are restricted to a subset of specific proteins or more broadly influenced by the biophysiochemical and cellular environment. The therapeutic strategies and the significant failure rate of drugs in clinical trials pertaining to these neurodegenerative diseases have been also discussed at length. At a time when the COVID-19 pandemic has hit the globe hard, the review also discusses the plausibility of the far-reaching consequences posed by the virus, such as triggering early onset of amyloidosis. Finally, the application(s) of amyloids as useful biomaterials has also been discussed briefly in this review.

SARS-CoV-2 spike protein aggregation is triggered by bacterial lipopolysaccharide.

SARS-CoV-2 spike (S) protein is crucial for virus invasion in COVID-19. Here, we showed that lipopolysaccharide (LPS) can trigger S protein aggregation at high doses of LPS and S protein. We demonstrated the formation of S protein aggregates by microscopy analyses, aggregation and gel shift assays. LPS at high levels boosts the formation of S protein aggregates as detected by amytracker and thioflavin T dyes that specifically bind to aggregating proteins. We validated the role of LPS by blocking the formation of aggregates by the endotoxin-scavenging thrombin-derived peptide TCP-25. Aggregation-prone sequences in S protein are predicted to be nearby LPS binding sites, while molecular simulations showed stable formation of S protein-LPS higher-order oligomers. Collectively, our results provide evidence of LPS-induced S protein aggregation.

COVID-19 and Parkinson's Disease: Possible Links in Pathology and Therapeutics.

The outbreak of SARs-CoV-2 with emerging new variants is leading to global health crisis and has brought a major concern for patients with comorbidities. Parkinson's disease (PD) is a motor neurodegenerative disease involving various metabolic and psychological ailments along with the common occurrence of hyposmia as observed in COVID-19 patients. In addition, the observed surplus inflammatory responses in both diseases are also alarming. Alongside, angiotensin-converting enzyme 2 (ACE2) receptor, essentially required by SARS-CoV-2 to enter the cell and dopamine decarboxylase (DDC), required for dopamine synthesis is known to co-regulate in the non-neuronal cells. Taken together, these conditions suggested the probable reciprocal pathological relation between COVID-19 and PD and also suggested that during comorbidities, the disease diagnosis and therapeutics are critical and may engender severe health complications. In this review, we discuss various events and mechanisms which may have implications for the exacerbation of PD conditions and must be taken into account during the treatment of patients.

Interactions between SARS-CoV-2 N-Protein and α-Synuclein Accelerate Amyloid Formation.

First cases that point at a correlation between SARS-CoV-2 infections and the development of Parkinson's disease (PD) have been reported. Currently, it is unclear if there is also a direct causal link between these diseases. To obtain first insights into a possible molecular relation between viral infections and the aggregation of α-synuclein protein into amyloid fibrils characteristic for PD, we investigated the effect of the presence of SARS-CoV-2 proteins on α-synuclein aggregation. We show, in test tube experiments, that SARS-CoV-2 spike protein (S-protein) has no effect on α-synuclein aggregation, while SARS-CoV-2 nucleocapsid protein (N-protein) considerably speeds up the aggregation process. We observe the formation of multiprotein complexes and eventually amyloid fibrils. Microinjection of N-protein in SH-SY5Y cells disturbed the α-synuclein proteostasis and increased cell death. Our results point toward direct interactions between the N-protein of SARS-CoV-2 and α-synuclein as molecular basis for the observed correlation between SARS-CoV-2 infections and Parkinsonism.

In silico analysis of the aggregation propensity of the SARS-CoV-2 proteome: Insight into possible cellular pathologies.

The SARS-CoV-2 virus causes the coronavirus disease 19 emerged in 2020. The pandemic triggered a turmoil in public health and is having a tremendous social and economic impact around the globe. Upon entry into host cells, the SARS-CoV-2 virus hijacks cellular machineries to produce and maintain its own proteins, spreading the infection. Although the disease is known for prominent respiratory symptoms, accumulating evidence is also demonstrating the involvement of the central nervous system, with possible mid- and long-term neurological consequences. In this study, we conducted a detailed bioinformatic analysis of the SARS-CoV-2 proteome aggregation propensity by using several complementary computational tools. Our study identified 10 aggregation prone proteins in the reference SARS-CoV-2 strain: the non-structural proteins Nsp4, Nsp6 and Nsp7 as well as ORF3a, ORF6, ORF7a, ORF7b, ORF10, CovE and CovM. By searching for the available mutants of each protein, we have found that most proteins are conserved, while ORF3a and ORF7b are variable and characterized by the occurrence of a large number of mutants with increased aggregation propensity. The geographical distribution of the mutants revealed interesting differences in the localization of aggregation-prone mutants of each protein. Aggregation-prone mutants of ORF7b were found in 7 European countries, whereas those of ORF3a in only 2. Aggregation-prone sequences of ORF7b, but not of ORF3a, were identified in Australia, India, Nepal, China, and Thailand. Our results are important for future analysis of a possible correlation between higher transmissibility and infection, as well as the presence of neurological symptoms with aggregation propensity of SARS-CoV-2 proteins.

Karyopherin abnormalities in neurodegenerative proteinopathies.

Neurodegenerative proteinopathies are characterized by progressive cell loss that is preceded by the mislocalization and aberrant accumulation of proteins prone to aggregation. Despite their different physiological functions, disease-related proteins like tau, α-synuclein, TAR DNA binding protein-43, fused in sarcoma and mutant huntingtin, all share low complexity regions that can mediate their liquid-liquid phase transitions. The proteins' phase transitions can range from native monomers to soluble oligomers, liquid droplets and further to irreversible, often-mislocalized aggregates that characterize the stages and severity of neurodegenerative diseases. Recent advances into the underlying pathogenic mechanisms have associated mislocalization and aberrant accumulation of disease-related proteins with defective nucleocytoplasmic transport and its mediators called karyopherins. These studies identify karyopherin abnormalities in amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, and synucleinopathies including Parkinson's disease and dementia with Lewy bodies, that range from altered expression levels to the subcellular mislocalization and aggregation of karyopherin α and ß proteins. The reported findings reveal that in addition to their classical function in nuclear import and export, karyopherins can also act as chaperones by shielding aggregation-prone proteins against misfolding, accumulation and irreversible phase-transition into insoluble aggregates. Karyopherin abnormalities can, therefore, be both the cause and consequence of protein mislocalization and aggregate formation in degenerative proteinopathies. The resulting vicious feedback cycle of karyopherin pathology and proteinopathy identifies karyopherin abnormalities as a common denominator of onset and progression of neurodegenerative disease. Pharmacological targeting of karyopherins, already in clinical trials as therapeutic intervention targeting cancers such as glioblastoma and viral infections like COVID-19, may therefore represent a promising new avenue for disease-modifying treatments in neurodegenerative proteinopathies.

Adenosine triphosphate energy-independently controls protein homeostasis with unique structure and diverse mechanisms.

Proteins function in the crowded cellular environments with high salt concentrations, thus facing tremendous challenges of misfolding/aggregation which represents a pathological hallmark of aging and an increasing spectrum of human diseases. Recently, intrinsically disordered regions (IDRs) were recognized to drive liquid-liquid phase separation (LLPS), a common principle for organizing cellular membraneless organelles (MLOs). ATP, the universal energy currency for all living cells, mysteriously has concentrations of 2-12 mM, much higher than required for its previously-known functions. Only recently, ATP was decoded to behave as a biological hydrotrope to inhibit protein LLPS and aggregation at mM. We further revealed that ATP also acts as a bivalent binder, which not only biphasically modulates LLPS driven by IDRs of human and viral proteins, but also bind to the conserved nucleic-acid-binding surfaces of the folded proteins. Most unexpectedly, ATP appears to act as a hydration mediator to antagonize the crowding-induced destabilization as well as to enhance folding of proteins without significant binding. Here, this review focuses on summarizing the results of these biophysical studies and discussing their implications in an evolutionary context. By linking triphosphate with unique hydration property to adenosine, ATP appears to couple the ability for establishing hydrophobic, π-π, π-cation and electrostatic interactions to the capacity in mediating hydration of proteins, which is at the heart of folding, dynamics, stability, phase separation and aggregation. Consequently, ATP acquired a category of functions at ~mM to energy-independently control protein homeostasis with diverse mechanisms, thus implying a link between cellular ATP concentrations and protein-aggregation diseases.

SARS-CoV-2 spike protein interactions with amyloidogenic proteins: Potential clues to neurodegeneration.

The post-infection of COVID-19 includes a myriad of neurologic symptoms including neurodegeneration. Protein aggregation in brain can be considered as one of the important reasons behind the neurodegeneration. SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) binds to heparin and heparin binding proteins. Moreover, heparin binding accelerates the aggregation of the pathological amyloid proteins present in the brain. In this paper, we have shown that the SARS-CoV-2 S1 RBD binds to a number of aggregation-prone, heparin binding proteins including Aß, α-synuclein, tau, prion, and TDP-43 RRM. These interactions suggests that the heparin-binding site on the S1 protein might assist the binding of amyloid proteins to the viral surface and thus could initiate aggregation of these proteins and finally leads to neurodegeneration in brain. The results will help us to prevent future outcomes of neurodegeneration by targeting this binding and aggregation process.

Invertebrate Models Untangle the Mechanism of Neurodegeneration in Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease, afflicting ~10 million people worldwide. Although several genes linked to PD are currently identified, PD remains primarily an idiopathic disorder. Neuronal protein α-synuclein is a major player in disease progression of both genetic and idiopathic forms of PD. However, it cannot alone explain underlying pathological processes. Recent studies demonstrate that many other risk factors can accelerate or further worsen brain dysfunction in PD patients. Several PD models, including non-mammalian eukaryotic organisms, have been developed to identify and characterize these factors. This review discusses recent findings in three PD model organisms, i.e., yeast, Drosophila, and Caenorhabditis elegans, that opened new mechanisms and identified novel contributors to this disorder. These non-mammalian models share many conserved molecular pathways and cellular processes with humans. New players affecting PD pathogenesis include previously unknown genes/proteins, novel signaling pathways, and low molecular weight substances. These findings might respond to the urgent need to discover novel drug targets for PD treatment and new biomarkers for early diagnostics of this disease. Since the study of neurodegeneration using simple eukaryotic organisms brought a huge amount of information, we include only the most recent or the most important relevant data.

Seeding Brain Protein Aggregation by SARS-CoV-2 as a Possible Long-Term Complication of COVID-19 Infection.

Postinfection complications of coronavirus disease 2019 (COVID-19) are still unknown, and one of the long-term concerns in infected people are brain pathologies. The question is that severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection may be an environmental factor in accelerating the sporadic neurodegeneration in the infected population. In this regard, induction of protein aggregation in the brain by SARS-CoV-2 intact structure or a peptide derived from spike protein subunits needs to be considered in futures studies. In this paper, we discuss these possibilities using pieces of evidence from other viruses.