Transglutaminase-mediated Intramolecular Cross-linking of Membrane-bound α-Synuclein Promotes Amyloid Formation in Lewy Bodies

Band 3 Memphis variant II is a mutant anion-exchange protein associated with the Diego a+ blood group antigen. There are two mutations in this transporter: Lys-56 → Glu within the cytoplasmic domain, and Pro-854 → Leu within the membrane-bound domain. The Pro-854 mutation, which is thought to give rise to the antigenicity, is located within the C-terminal subdomain of the membrane-bound domain. Yet, there is an apparent enhancement in the rate of covalent binding of H2DIDS (4,4´-di-isothiocyanatodihydro-2,2´-stilbenedisulphonate) to ‘lysine A’ (Lys-539) in the N-terminal subdomain, suggesting widespread conformational changes. In this report, we have used various kinetic assays which differentiate between conformational changes in the two subdomains, to characterize the stilbenedisulphonate site on band 3 Memphis variant II. We have found a significantly higher H2DIDS (a C-terminal-sensitive inhibitor) affinity for band 3 Memphis variant II, due to a lower H2DIDS ‘off’ rate constant, but no difference was found between mutant and control when DBDS (4,4´-dibenzamido-2,2´-stilbenedisulphonate) (a C-terminal-insensitive inhibitor) ‘off’ rates were measured. Furthermore, there were no differences in the rates of covalent binding to lysine A, for either DIDS (4,4´-di-isothiocyanato-2,2´-stilbenedisulphonate) or H2DIDS. However, the rate of covalent intrasubunit cross-linking of Lys-539 and Lys-851 by H2DIDS was abnormally low for band 3 Memphis variant II. These results suggest that the Pro-854 → Leu mutation causes a localized conformational change in the C-terminal subdomain of band 3.

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Endogenous alpha-synuclein monomers, oligomers and resulting pathology: let’s talk about the lipids in the room

npj Parkinson s Disease ◽

10.1038/s41531-019-0095-3 ◽

2019 ◽

Vol 5 (1) ◽

Cited By ~ 8

Author(s):

Bryan A. Killinger ◽

Ronald Melki ◽

Patrik Brundin ◽

Jeffrey H. Kordower

Keyword(s):

Lewy Bodies ◽

Alpha Synuclein ◽

Biological Origin ◽

Lewy Pathology ◽

Molecular Weights ◽

Intrinsically Disordered ◽

Bona Fide ◽

Membrane Bound ◽

Soluble Species ◽

Alpha Synuclein Aggregation

Abstract Alpha-synuclein is an intrinsically disordered, highly dynamic protein that pathogenically aggregates into inclusion structures called Lewy bodies, in several neurogenerative diseases termed synucleinopathies. Despite its importance for understanding disease, the oligomerization status of alpha-synuclein in healthy cells remains unclear. Alpha-synuclein may exist predominantly as either a monomer or a variety of oligomers of different molecular weights. There is solid evidence to support both theories. Detection of apparent endogenous oligomers are intimately dependent on vesicle and lipid interactions. Here we consider the possibility that apparent endogenous alpha-synuclein oligomers are in fact conformations of membrane-bound alpha-synuclein and not a bona fide stable soluble species. This perspective posits that the formation of any alpha-synuclein oligomers within the cell is likely toxic and interconversion between monomer and oligomer is tightly controlled. This differs from the hypothesis that there is a continuum of endogenous non-toxic oligomers and they convert, through unclear mechanisms, to toxic oligomers. The distinction is important, because it clarifies the biological origin of synucleinopathy. We suggest that a monomer-only, lipid-centric view of endogenous alpha-synuclein aggregation can explain how alpha-synuclein pathology is triggered, and that the interactions between alpha-synuclein and lipids can represent a target for therapeutic intervention. This discussion is well-timed due to recent studies that show lipids are a significant component of Lewy pathology.

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VirB8 homolog TraE from plasmid pKM101 forms a hexameric ring structure and interacts with the VirB6 homolog TraD

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1802501115 ◽

2018 ◽

Vol 115 (23) ◽

pp. 5950-5955 ◽

Cited By ~ 6

Author(s):

Bastien Casu ◽

Charline Mary ◽

Aleksandr Sverzhinsky ◽

Aurélien Fouillen ◽

Antonio Nanci ◽

...

Keyword(s):

Molecular Mass ◽

Secretion System ◽

High Molecular Mass ◽

Full Length ◽

Size Exclusion ◽

Cross Linking ◽

X Ray ◽

Exclusion Chromatography ◽

Plasmid Pkm101 ◽

Membrane Bound

Type IV secretion systems (T4SSs) are multiprotein assemblies that translocate macromolecules across the cell envelope of bacteria. X-ray crystallographic and electron microscopy (EM) analyses have increasingly provided structural information on individual T4SS components and on the entire complex. As of now, relatively little information has been available on the exact localization of the inner membrane-bound T4SS components, notably the mostly periplasmic VirB8 protein and the very hydrophobic VirB6 protein. We show here that the membrane-bound, full-length version of the VirB8 homolog TraE from the plasmid pKM101 secretion system forms a high-molecular-mass complex that is distinct from the previously characterized periplasmic portion of the protein that forms dimers. Full-length TraE was extracted from the membranes with detergents, and analysis by size-exclusion chromatography, cross-linking, and size exclusion chromatography (SEC) multiangle light scattering (MALS) shows that it forms a high-molecular-mass complex. EM and small-angle X-ray scattering (SAXS) analysis demonstrate that full-length TraE forms a hexameric complex with a central pore. We also overproduced and purified the VirB6 homolog TraD and show by cross-linking, SEC, and EM that it binds to TraE. Our results suggest that TraE and TraD interact at the substrate translocation pore of the secretion system.

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Lentil seed aquaporins form a hetero-oligomer which is phosphorylated by a Mg2+-dependent and Ca2+-regulated kinase

Biochemical Journal ◽

10.1042/bj3520183 ◽

2000 ◽

Vol 352 (1) ◽

pp. 183-190 ◽

Cited By ~ 24

Author(s):

Pol HARVENGT ◽

Ariane VLERICK ◽

Bruno FUKS ◽

Ruddy WATTIEZ ◽

Jean-Marie RUYSSCHAERT ◽

...

Keyword(s):

Stress Responses ◽

Lens Culinaris ◽

Kinase Activity ◽

Cross Linking ◽

Protein Storage Vacuoles ◽

Membrane Bound ◽

Flow Through ◽

Growth Development ◽

Chemical Cross Linking ◽

Aquaporin Family

In plants, aquaporins regulate the water flow through membranes during growth, development and stress responses. We have isolated two isoforms of the aquaporin family from the protein-storage vacuoles of lentil (Lens culinaris Med.) seeds. Chemical cross-linking experiments showed that both isoforms belong to the same oligomer in the membrane and are phosphorylated by a membrane-bound protein kinase. We assigned the kinase activity to a 52kDa protein that is magnesium-dependent and calcium-regulated.

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Rat and human neutrophil N-formyl-peptide chemotactic receptors. Species difference in the glycosylation of similar 35-38 kDa polypeptide cores

Biochemical Journal ◽

10.1042/bj2770067 ◽

1991 ◽

Vol 277 (1) ◽

pp. 67-72 ◽

Cited By ~ 7

Author(s):

J J Remes ◽

U E Petäjä-Repo ◽

H J Rajaniemi

Keyword(s):

Ligand Binding ◽

Species Difference ◽

Cross Linking ◽

Complex Type ◽

High Affinity ◽

Affinity Ligand ◽

Pngase F ◽

Formyl Peptide ◽

Membrane Bound ◽

High Affinity Ligand

Rat and human neutrophil N-formyl-peptide chemotactic receptors were subjected to glycosidase and proteinase treatments to determine the extent and species differences of glycosylation and the carbohydrate requirement in the high-affinity ligand binding. N-Formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys was attached to rat and human neutrophils either before or after glycosidase and proteinase treatments, and the labelled receptors were solubilized after glutaraldehyde cross-linking and analysed by SDS/PAGE and autoradiography. Both the rat and human N-formyl-peptide chemotactic receptors contain only N-linked oligosaccharides, as demonstrated by their sensitivity to peptide N-glycosidase F (PNGase F) and resistance to O-glycanase treatment. The N-linked oligosaccharides seem to be of the complex type rather than the high-mannose or hybrid type and lack terminal sialic acid, as demonstrated by their resistance to endoglycosidases D and H and neuraminidase treatments. This sensitivity pattern was similar in both species, and the shift in the molecular size of the receptors to 35-38 kDa after PNGase F treatment occurred through one intermediate product, suggesting that both receptors contain a similar 35-38 kDa polypeptide core with two N-linked complex-type oligosaccharides, the heterogeneity of which is responsible for the species difference in receptor size. Papain treatment alone or followed by PNGase F produced in both species a 33-36 kDa membrane-bound fragment that was still able to bind the ligand, suggesting that the oligosaccharides are located on the approx. 2 kDa papain-cleavable polypeptide fragment of the receptors. The cleavage sites for both papain and PNGase F were hidden in occupied receptors, suggesting a conformational or topographical change in these upon ligand binding. Scatchard analyses and cross-linking experiments demonstrated that carbohydrates are not required for high-affinity ligand binding and that the 33-36 kDa membrane-bound papain fragment of both receptors contains the ligand-binding site.

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Chemical cross-linking of membrane-bound rat liver mitochondrial monoamine oxidase

Pharmacological Research Communications ◽

10.1016/s0031-6989(88)80550-2 ◽

1988 ◽

Vol 20 ◽

pp. 77-78

Author(s):

Anne-Marie O'Carroll ◽

Keith F. Tipton

Keyword(s):

Monoamine Oxidase ◽

Rat Liver ◽

Cross Linking ◽

Mitochondrial Monoamine Oxidase ◽

Membrane Bound ◽

Chemical Cross Linking

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C-terminal α-synuclein truncations are linked to cysteine cathepsin activity in Parkinson's disease

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.008930 ◽

2019 ◽

Vol 294 (25) ◽

pp. 9973-9984 ◽

Cited By ~ 11

Author(s):

Ryan P. McGlinchey ◽

Shannon M. Lacy ◽

Katherine E. Huffer ◽

Nahid Tayebi ◽

Ellen Sidransky ◽

...

Keyword(s):

Parkinson’S Disease ◽

Amino Acids ◽

Parkinson's Disease ◽

Amyloid Fibrils ◽

Lewy Bodies ◽

Amyloid Formation ◽

Pathological Feature ◽

Cysteine Cathepsins ◽

Cathepsin Activity ◽

Cysteine Cathepsin

A pathological feature of Parkinson's disease (PD) is Lewy bodies (LBs) composed of α-synuclein (α-syn) amyloid fibrils. α-Syn is a 140 amino acids–long protein, but truncated α-syn is enriched in LBs. The proteolytic processes that generate these truncations are not well-understood. On the basis of our previous work, we propose that these truncations could originate from lysosomal activity attributable to cysteine cathepsins (Cts). Here, using a transgenic SNCAA53T mouse model, overexpressing the PD-associated α-syn variant A53T, we compared levels of α-syn species in purified brain lysosomes from nonsymptomatic mice with those in age-matched symptomatic mice. In the symptomatic mice, antibody epitope mapping revealed enrichment of C-terminal truncations, resulting from CtsB, CtsL, and asparagine endopeptidase. We did not observe changes in individual cathepsin activities, suggesting that the increased levels of C-terminal α-syn truncations are because of the burden of aggregated α-syn. Using LC-MS and purified α-syn, we identified C-terminal truncations corresponding to amino acids 1–122 and 1–90 from the SNCAA53T lysosomes. Feeding rat dopaminergic N27 cells with exogenous α-syn fibrils confirmed that these fragments originate from incomplete fibril degradation in lysosomes. We mimicked these events in situ by asparagine endopeptidase degradation of α-syn fibrils. Importantly, the resulting C-terminally truncated fibrils acted as superior seeds in stimulating α-syn aggregation compared with that of the full-length fibrils. These results unequivocally show that C-terminal α-syn truncations in LBs are linked to Cts activities, promote amyloid formation, and contribute to PD pathogenesis.

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Interaction Interface of Mason-Pfizer Monkey Virus Matrix and Envelope Proteins

Journal of Virology ◽

10.1128/jvi.01146-20 ◽

2020 ◽

Vol 94 (20) ◽

Author(s):

Jan Prchal ◽

Jakub Sýs ◽

Petra Junková ◽

Jan Lipov ◽

Tomáš Ruml

Keyword(s):

Nuclear Magnetic Resonance ◽

Human Immunodeficiency Virus ◽

Magnetic Resonance ◽

Cytoplasmic Tail ◽

Cross Linking ◽

Terminal Part ◽

Immunodeficiency Virus ◽

The Matrix ◽

Membrane Bound

ABSTRACT Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding. IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

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Potential Treatment of Parkinson's Disease, Using Last-Generation Carbon Nanotubes: A Bimolecular In-Silico Study

10.21203/rs.3.rs-45640/v1 ◽

2020 ◽

Author(s):

Ehsan Alimohammadi ◽

Arash Nikzad ◽

Mohamad Khedri ◽

Milad Rezaeian ◽

Ahmad Miri Jahromi ◽

...

Keyword(s):

Parkinson’S Disease ◽

Molecular Dynamics ◽

Parkinson's Disease ◽

Lewy Bodies ◽

Molecular Study ◽

Amyloid Formation ◽

Simulation Tools ◽

Study Results ◽

Underlying Mechanisms ◽

Synuclein Proteins

Abstract Background: Parkinson's disease (PD) is one of the most common neurodegenerative disorders. One of the underlying mechanisms of the disease is the accumulation of α-synuclein protein aggregates, including amyloids and Lewy bodies in the brain, resulting in the death of dopaminergic cells in the substantia nigra. The current treatments for PD are mainly focused on replacing dopamine. However, if these medications are stopped, the severity of PD will increase. Moreover, the drugs used for the treatment of PD are associated with considerable side effects and dietary restrictions. Therefore, necessary studies to develop more effective medications for PD seem to be indispensable. To prevent the progression of PD, avoiding the development of α-synuclein amyloids could be proposed. Methods: In this study, the effects of three last-generation nanotube-based structures on α-synuclein amyloid formation were investigated for the first time employing Molecular Dynamics (MD) simulation tools. Molecular dynamics provide a deep insight into atomic interactions and can well study α-synuclein amyloid formation at the atomic and molecular scales.Results: The molecular study results indicated that all of the nanotubes studied in this work, had strong energy interactions with α-synuclein. Therefore, nanotubes using phosphorus, nitrogen and boron dopants, have great potential to prevent α-synuclein amyloid formation. Among these nanotubes, phosphorus-doped carbon nanotube (P-CNT) has the most substantial interactions with α-synuclein. The P-CNT caused more hydrogen bonds to be formed between water and α-synuclein molecules. This phenomenon leads to a decrease in the compactness, stability, and contact area of α-synuclein proteins, which results in considerable changes in the secondary structure of α-synuclein.Conclusions: Doping nanotubes especially P-CNT could be very effective for preventing the α-synuclein amyloid formation and hence, halting the progression of PD. This molecular study paves the way for the use of the Doping nanotubes in the treatment of PD. These structures are highly tunable and flexible. Therefore, the results of this work can be developed to computational, experimental and clinical levels.

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Cytosolic Proteins Regulate α-Synuclein Dissociation from Presynaptic Membranes

Journal of Biological Chemistry ◽

10.1074/jbc.m605965200 ◽

2006 ◽

Vol 281 (43) ◽

pp. 32148-32155 ◽

Cited By ~ 39

Author(s):

Sabine Wislet-Gendebien ◽

Cheryl D'Souza ◽

Toshitaka Kawarai ◽

Peter St George-Hyslop ◽

David Westaway ◽

...

Keyword(s):

Lewy Bodies ◽

Physiological Role ◽

Normal Function ◽

Vesicular Trafficking ◽

Metabolic Energy ◽

Cytosolic Proteins ◽

Lewy Body Pathology ◽

Cellular Factors ◽

Membrane Bound ◽

Biochemical Mechanisms

Intracellular accumulation of insoluble α-synuclein in Lewy bodies is a key neuropathological trait of Parkinson disease (PD). Neither the normal function of α-synuclein nor the biochemical mechanisms that cause its deposition are understood, although both are likely influenced by the interaction of α-synuclein with vesicular membranes, either for a physiological role in vesicular trafficking or as a pathological seeding mechanism that exacerbates the propensity of α-synuclein to self-assemble into fibrils. In addition to the α-helical form that is peripherally-attached to vesicles, a substantial portion of α-synuclein is freely diffusible in the cytoplasm. The mechanisms controlling α-synuclein exchange between these compartments are unknown and the possibility that chronic dysregulation of membrane-bound and soluble α-synuclein pools may contribute to Lewy body pathology led us to search for cellular factors that can regulate α-synuclein membrane interactions. Here we reveal that dissociation of membrane-bound α-synuclein is dependent on brain-specific cytosolic proteins and insensitive to calcium or metabolic energy. Two PD-linked mutations (A30P and A53T) significantly increase the cytosol-dependent α-synuclein off-rate but have no effect on cytosol-independent dissociation. These results reveal a novel mechanism by which cytosolic brain proteins modulate α-synuclein interactions with intracellular membranes. Importantly, our finding that α-synuclein dissociation is up-regulated by both familial PD mutations implicates cytosolic cofactors in disease pathogenesis and as molecular targets to influence α-synuclein aggregation.

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