The Aggregation of Huntingtin and α-Synuclein

Huntington’s and Parkinson’s diseases are neurodegenerative disorders associated with unusual protein interactions. Although the origin and evolution of these diseases are completely different, characteristic deposits of protein aggregates (huntingtin and α-synuclein resp.), are a common feature in both diseases. After these observations, many studies are performed with both proteins. Some of them try to understand the nature and driving forces of the aggregation process; others try to find a correlation between the genetic and failure in protein function. Finally with the combination of both approaches, it was proposed that possible strategies deal with pathologic aggregation. Unfortunately, if protein aggregation is a cause or a consequence of the neurodegeneration observed in these pathologies, it is still debatable. This paper describes the process of aggregation of two proteins: huntingtin and α synuclein. The characteristics of the aggregation reaction of these proteins have been followed with novel methods both in vivo and in vitro; these studies include both the combination with other proteins and the presence of various chemical compounds. The ultimate goal of this study was to summarize recent findings on protein aggregation and its possible role as a therapeutic target in neurodegenerative diseases and their role in biomaterial science.

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Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/156.1.21 ◽

2000 ◽

Vol 156 (1) ◽

pp. 21-29 ◽

Cited By ~ 1

Author(s):

David R H Evans ◽

Brian A Hemmings

Keyword(s):

Protein Interactions ◽

Active Site ◽

Protein Function ◽

Mutational Analysis ◽

Yeast Cells ◽

Temperature Sensitive ◽

Active Site Residues ◽

Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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SET7/9-dependent methylation of ARTD1 at K508 stimulates poly-ADP-ribose formation after oxidative stress

Open Biology ◽

10.1098/rsob.120173 ◽

2013 ◽

Vol 3 (10) ◽

pp. 120173 ◽

Cited By ~ 23

Author(s):

Ingrid Kassner ◽

Anneli Andersson ◽

Monika Fey ◽

Martin Tomas ◽

Elisa Ferrando-May ◽

...

Keyword(s):

Oxidative Stress ◽

Protein Interactions ◽

Protein Function ◽

Dependent Manner ◽

Protein Protein Interactions ◽

Post Translational Modification ◽

Target Proteins ◽

Protein Methyltransferase

ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) is localized in the nucleus, where it ADP-ribosylates specific target proteins. The post-translational modification (PTM) with a single ADP-ribose unit or with polymeric ADP-ribose (PAR) chains regulates protein function as well as protein–protein interactions and is implicated in many biological processes and diseases. SET7/9 (Setd7, KMT7) is a protein methyltransferase that catalyses lysine monomethylation of histones, but also methylates many non-histone target proteins such as p53 or DNMT1. Here, we identify ARTD1 as a new SET7/9 target protein that is methylated at K508 in vitro and in vivo . ARTD1 auto-modification inhibits its methylation by SET7/9, while auto-poly-ADP-ribosylation is not impaired by prior methylation of ARTD1. Moreover, ARTD1 methylation by SET7/9 enhances the synthesis of PAR upon oxidative stress in vivo . Furthermore, laser irradiation-induced PAR formation and ARTD1 recruitment to sites of DNA damage in a SET7/9-dependent manner. Together, these results reveal a novel mechanism for the regulation of cellular ARTD1 activity by SET7/9 to assure efficient PAR formation upon cellular stress.

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The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504822112 ◽

2015 ◽

Vol 112 (23) ◽

pp. 7189-7194 ◽

Cited By ~ 484

Author(s):

Shana Elbaum-Garfinkle ◽

Younghoon Kim ◽

Krzysztof Szczepaniak ◽

Carlos Chih-Hsiung Chen ◽

Christian R. Eckmann ◽

...

Keyword(s):

Phase Separation ◽

Phase Boundary ◽

Single Molecule ◽

Protein Interactions ◽

Driving Forces ◽

Early Embryo ◽

P Granules ◽

Intrinsically Disordered

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA–protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA–protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.

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Design and Prototyping of Genetically Encoded Arsenic Biosensors Based on Transcriptional Regulator AfArsR

Biomolecules ◽

10.3390/biom11091276 ◽

2021 ◽

Vol 11 (9) ◽

pp. 1276

Author(s):

Salma Saeed Khan ◽

Yi Shen ◽

Muhammad Qaiser Fatmi ◽

Robert E. Campbell ◽

Habib Bokhari

Keyword(s):

Protein Interactions ◽

Protein Function ◽

Fluorescent Proteins ◽

Transcriptional Factor ◽

Thiol Groups ◽

Protein Protein Interactions ◽

Protein Thiol ◽

New Family

Genetically encoded biosensors based on engineered fluorescent proteins (FPs) are essential tools for monitoring the dynamics of specific ions and molecules in biological systems. Arsenic ion in the +3 oxidation state (As3+) is highly toxic to cells due to its ability to bind to protein thiol groups, leading to inhibition of protein function, disruption of protein–protein interactions, and eventually to cell death. A genetically encoded biosensor for the detection of As3+ could potentially facilitate the investigation of such toxicity both in vitro and in vivo. Here, we designed and developed two prototype genetically encoded arsenic biosensors (GEARs), based on a bacterial As3+ responsive transcriptional factor AfArsR from Acidithiobacillus ferrooxidans. We constructed FRET-based GEAR biosensors by insertion of AfArsR between FP acceptor/donor FRET pairs. We further designed and engineered single FP-based GEAR biosensors by insertion of AfArsR into GFP. These constructs represent prototypes for a new family of biosensors based on the ArsR transcriptional factor scaffold. Further improvements of the GEAR biosensor family could lead to variants with suitable performance for detection of As3+ in various biological and environmental systems.

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DNA-Encircled Lipid Bilayers

10.1101/285957 ◽

2018 ◽

Author(s):

Katarina Iric ◽

Madhumalar Subramanian ◽

Jana Oertel ◽

Nayan P. Agarwal ◽

Michael Matthies ◽

...

Keyword(s):

Membrane Proteins ◽

Lipid Bilayer ◽

Lipid Bilayers ◽

Protein Interactions ◽

Protein Function ◽

Dna Nanotechnology ◽

Lipid Vesicles ◽

Associated Proteins

ABSTRACTLipid bilayers and lipid-associated proteins play a crucial role in biology. As in vivo studies and manipulation are inherently difficult, several membrane-mimetic systems have been developed to enable investigation of lipidic phases, lipid-protein interactions, membrane protein function and membrane structure in vitro. Controlling the size and shape, or site-specific functionalization is, however, difficult to achieve with established membrane mimetics based on membrane scaffolding proteins, polymers or peptides. In this work, we describe a route to leverage the unique programmability of DNA nanotechnology and create DNA-encircled bilayers (DEBs), which are made of multiple copies of an alkylated oligonucleotide hybridized to a single-stranded minicircle. To stabilize the hydrophobic rim of the lipid bilayer, and to prevent formation of lipid vesicles, we introduced up to 2 alkyl chains per helical that point to the inside of the toroidal DNA ring and interact with the hydrophobic side chains of the encapsulated lipid bilayer. The DEB approach described herein provides unprecedented control of size, and allows the orthogonal functionalizations and arrangement of engineered membrane nanoparticles and will become a valuable tool for biophysical investigation of lipid phases and lipid-associated proteins and complexes including structure determination of membrane proteins and pharmacological screenings of membrane proteins.

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In-vivo crystals reveal protein interactions

10.1101/778779 ◽

2019 ◽

Author(s):

Eleanor R. Martin ◽

Alessandro Barbieri ◽

Robert C. Ford ◽

Robert C. Robinson

Keyword(s):

Protein Interactions ◽

Protein Function ◽

Pdz Domain ◽

Cell Types ◽

Binding Motif ◽

Protein Protein Interactions ◽

Reporter System ◽

X Ray Crystallography

AbstractCrystallisation of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under non-physiological extremes of protein, salt, and H+ concentrations. Here, we describe the development of the robust iBox-PAK4cat system that spontaneously crystallises in several mammalian cell types. The developments described here allow the quantitation of in-vivo protein-protein interactions using a novel GFP-linked reporter system. Here, we have combined this assay with in-vitro X-ray crystallography and molecular dynamics studies characterise the molecular determinants of the interaction between NHERF1 PDZ2 and CFTR, a protein complex pertinent to the genetic disease cystic fibrosis. These studies have revealed the crystal structure of the extended PDZ domain of NHERF1, and indicated, contrary to previous reports, that residue selection at −1 and −3 PDZ-binding motif positions influence the affinity and specificity of the interaction. The results presented here demonstrate that the iBox-PAK4cat assay could easily be utilised to screen other protein-protein interactions.

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Bacterial production and direct functional screening of expanded molecular libraries for discovering inhibitors of protein aggregation

Science Advances ◽

10.1126/sciadv.aax5108 ◽

2019 ◽

Vol 5 (10) ◽

pp. eaax5108 ◽

Cited By ~ 5

Author(s):

Dafni C. Delivoria ◽

Sean Chia ◽

Johnny Habchi ◽

Michele Perni ◽

Ilias Matis ◽

...

Keyword(s):

Protein Aggregation ◽

Early Stage ◽

Amyloid Β ◽

Amyloid Β Peptide ◽

Functional Screening ◽

Parkinson’S Diseases ◽

Aggregation Inhibitors ◽

Molecular Libraries

Protein misfolding and aggregation are associated with a many human disorders, including Alzheimer’s and Parkinson’s diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid-β peptide (Aβ42), linked to Alzheimer’s disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce Aβ42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.

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Inhaled Insulin Forms Toxic Pulmonary Amyloid Aggregates

Endocrinology ◽

10.1210/en.2010-0457 ◽

2010 ◽

Vol 151 (10) ◽

pp. 4717-4724 ◽

Cited By ~ 34

Author(s):

Cristian A. Lasagna-Reeves ◽

Audra L. Clos ◽

Terumi Midoro-Hiriuti ◽

Randall M. Goldblum ◽

George R. Jackson ◽

...

Keyword(s):

Protein Aggregation ◽

Protein Misfolding ◽

Diabetic Patients ◽

Amyloid Formation ◽

Pulmonary Tissue ◽

Inhaled Insulin ◽

Parkinson’S Diseases ◽

Pulmonary Air Flow

It is well known that interfaces, such as polar-nonpolar or liquid-air, play a key role in triggering protein aggregation in vitro, in particular the aggregation of peptides and proteins with the predisposition of misfolding and aggregation. Here we show that the interface present in the lungs predisposes the lungs to form aggregation of inhaled insulin. Insulin inhalers were introduced, and a large number of diabetic patients have used them. Although inhalers were safe and effective, decreases in pulmonary capacity have been reported in response to inhaled insulin. We hypothesize that the lung air-tissue interface provides a template for the aggregation of inhaled insulin. Our studies were designed to investigate the harmful potential that inhaled insulin has in pulmonary tissue in vivo, through an amyloid formation mechanism. Our data demonstrate that inhaled insulin rapidly forms amyloid in the lungs causing a significant reduction in pulmonary air flow. Our studies exemplify the importance that interfaces play in protein aggregation in vivo, illustrating the potential aggregation of inhaled proteins and the formation of amyloid deposits in the lungs. These insulin deposits resemble the amyloid structures implicated in protein misfolding disorders, such as Alzheimer’s and Parkinson’s diseases, and could as well be deleterious in nature.

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An emerging cyberinfrastructure for biodefense pathogen and pathogen–host data

Nucleic Acids Research ◽

10.1093/nar/gkm903 ◽

2007 ◽

Vol 36 (Supplement_1) ◽

pp. D884-D891 ◽

Cited By ~ 14

Author(s):

C. Zhang ◽

O. Crasta ◽

S. Cammer ◽

R. Will ◽

R. Kenyon ◽

...

Keyword(s):

Protein Interactions ◽

Protein Function ◽

Expression Profiles ◽

Protein Structures ◽

Gene Expression Profiles ◽

Structural Data ◽

Data Types ◽

Individual Gene

Abstract The NIAID-funded Biodefense Proteomics Resource Center (RC) provides storage, dissemination, visualization and analysis capabilities for the experimental data deposited by seven Proteomics Research Centers (PRCs). The data and its publication is to support researchers working to discover candidates for the next generation of vaccines, therapeutics and diagnostics against NIAID's Category A, B and C priority pathogens. The data includes transcriptional profiles, protein profiles, protein structural data and host–pathogen protein interactions, in the context of the pathogen life cycle in vivo and in vitro. The database has stored and supported host or pathogen data derived from Bacillus, Brucella, Cryptosporidium, Salmonella, SARS, Toxoplasma, Vibrio and Yersinia, human tissue libraries, and mouse macrophages. These publicly available data cover diverse data types such as mass spectrometry, yeast two-hybrid (Y2H), gene expression profiles, X-ray and NMR determined protein structures and protein expression clones. The growing database covers over 23 000 unique genes/proteins from different experiments and organisms. All of the genes/proteins are annotated and integrated across experiments using UniProt Knowledgebase (UniProtKB) accession numbers. The web-interface for the database enables searching, querying and downloading at the level of experiment, group and individual gene(s)/protein(s) via UniProtKB accession numbers or protein function keywords. The system is accessible at http://www.proteomicsresource.org/.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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