Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM

A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.

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SAT0012 ANTIBODY REACTIVITY AGAINST NATIVE PROTEINS IN RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.6124 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 936.2-936

Author(s):

T. B. G. Poulsen ◽

D. Damgaard ◽

M. M. Jørgensen ◽

L. Senolt ◽

J. Blackburn ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Histone Deacetylases ◽

Protein Microarray ◽

Treatment Modalities ◽

Igg Antibodies ◽

Calcium Calmodulin Dependent ◽

Native Proteins ◽

Healthy Donors ◽

Human Proteins ◽

Dependent Protein

Background:The majority of patients with rheumatoid arthritis (RA) produce autoantibodies against proteins that have undergone post-translational modfication, e.g. citrullination or carbamylation. There is growing evidence of their relevance and their potential utility to improve diagnosis, patient stratification, and prognosis for precision medicine. Investigating new autoantibody patterns may allow further stratification of patients and identifying subsets of patients that benefit from different treatment modalities. Following the discovery of high autoantibody reactivity against multiple modified proteins the interest in native targets decreased. Even though antibodies reacting with native proteins may also have a role in RA pathogenesis, their reactivity patterns are much less studied.Objectives:To identify novel native autoantigens in RA patients and elucidate patterns within autoantibody reactivity against native autoantigens.Methods:We investigated the reactivity of autoantibodies in plasma pools from 15 anti-CCP positive and 10 anti-CCP negative RA patients and 10 healthy donors against more than 1600 human proteins in native configuration using the Immunome high-density protein microarray.Results:We identified 86 native proteins that were recognized by IgG antibodies from anti-CCP positive RA patients and 76 native proteins recognized by IgG antibodies from anti-CCP negative RA patients, but not by antibodies from healthy donors. Examples of proteins recognized by both patient subgroups are calcium/calmodulin-dependent protein kinase type II subunits, histone deacetylases, keratin, and vimentin. Reactivity against the ribonucleic protein SSB was observed in anti-CCP negative RA patients only.Conclusion:Several human proteins in their native conformation are recognized by autoantibodies from anti-CCP positive as well as anti-CCP negative RA patients. In general, anti-CCP positive patients had higher autoantibody activity than anti-CCP negative patients and healthy donors.References:[1] Konig, M.F., Giles, J.T., Nigrovic, P.A., Andrade, F., 2016. Antibodies to native and citrullinated RA33 (hnRNP A2/B1) challenge citrullination as the inciting principle underlying loss of tolerance in rheumatoid arthritis. Ann. Rheum. Dis. 75, 2022–2028.[2] Zheng, Z., Mergaert, A.M., Fahmy, L.M., Bawadekar, M., Holmes, C.L., Ong, I.M., Bridges, A.J., Newton, M.A., Shelef, M.A., 2019. Disordered Antigens and Epitope Overlap Between Anti-Citrullinated Protein Antibodies and Rheumatoid Factor in Rheumatoid Arthritis. Arthritis Rheumatol. art.41074.[3] Sirotti, S., Generali, E., Ceribelli, A., Isailovic, N., De Santis, M., Selmi, C., 2017. Personalized medicine in rheumatology: the paradigm of serum autoantibodies. Autoimmun. Highlights 8.Acknowledgments :The Department of Clinical Immunology at Rigshospitalet Copenhagen is acknowledged for providing the healthy donor blood. The study is part of the PROCIT study financed by the Danish Council for Independent Research (grant no. DFF - 7016-00233). Moreover, the Obelske Family Foundation, the Svend Andersen Foundation, the Spar Nord Foundation and the Danish National Mass Spectrometry Platform for Functional Proteomics (PRO-MS; grant no. 5072-00007B) are acknowledged for grants to the analytical platform are acknowledged for the funding to enabling parts of this study.Disclosure of Interests:Thomas B.G. Poulsen: None declared, Dres Damgaard: None declared, Malene Møller Jørgensen: None declared, Ladislav Senolt: None declared, Jonathan Blackburn Shareholder of: Sengenics Corporation, Consultant of: Director of Sengenics Corporation, Employee of: Director of Sengenics Corporation, Claus Henrik Nielsen: None declared, Allan Stensballe: None declared

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C1-Inhibitor: Structure, Functional Diversity and Therapeutic Development

Current Medicinal Chemistry ◽

10.2174/0929867328666210804085636 ◽

2021 ◽

Vol 28 ◽

Author(s):

Elena Karnaukhova

Keyword(s):

Biochemical Characterization ◽

Biological Activities ◽

Serine Proteinase ◽

Endotoxin Shock ◽

C1 Inhibitor ◽

Recent Advances ◽

Therapeutic Development ◽

Inhibitory Activities ◽

Endogenous Proteins ◽

C1inh Deficiency

: Human C1-Inhibitor (C1INH), also known as C1-esterase inhibitor, is an important multifunctional plasma glycoprotein that is uniquely involved in a regulatory network of complement, contact, coagulation, and fibrinolytic systems. C1INH belongs to a superfamily of serine proteinase inhibitor (serpins) and exhibits its inhibitory activities towards several target proteases of plasmatic cascades, operating as a major anti-inflammatory protein in the circulation. In addition to its inhibitory activities, C1INH is also involved in non-inhibitory interactions with some endogenous proteins, polyanions, cells and infectious agents. While C1INH is essential for multiple physiological processes, it is better known for its deficiency with regards to Hereditary Angioedema (HAE), a rare autosomal dominant disease clinically manifested by recurrent acute attacks of increased vascular permeability and edema. Since the link was first established between functional C1INH deficiency in plasma and HAE in the 1960s, tremendous progress has been made in the biochemical characterization of C1INH and its therapeutic development for replacement therapies in patients with C1INH-dependent HAE. Various C1INH biological activities, recent advances in the HAE-targeted therapies, and availability of C1INH commercial products have prompted intensive investigation of the C1INH potential for treatment of clinical conditions other than HAE. This article provides an updated overview of the structure and biological activities of C1INH, its role in HAE pathogenesis, and recent advances in the research and therapeutic development of C1INH; it also considers some trends for using C1INH therapeutic preparations for applications other than angioedema, from sepsis and endotoxin shock to severe thrombotic complications in COVID-19 patients.

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A Mega-High-Throughput Screening Platform for the Discovery of Biologically Relevant Sequence-Defined Non-Natural Polymers

10.26434/chemrxiv.13106069 ◽

2020 ◽

Author(s):

Michal Avital-Shmilovici ◽

Xiaohe Liu ◽

Thomas Shaler ◽

Andrew Lowenthal ◽

Pauline Bourbon ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Binding Affinities ◽

Natural Polymers ◽

Protein Targets ◽

Biologically Relevant ◽

Novel Technology ◽

Major Bottleneck ◽

Screening Platform ◽

Optic Array

Combinatorial methods enable the synthesis of chemical libraries on scales of millions to billions of compounds, but the ability to efficiently screen and sequence such large libraries has remained a major bottleneck for molecular discovery. We developed a novel technology for screening and sequencing libraries of synthetic molecules of up to a billion compounds in size. This method utilizes Fiber-optic Array Scanning Technology (FAST) to screen bead-based libraries of synthetic compounds at a rate of 5 million compounds per minute (~83,000 Hz). This ultra-high-throughput screening platform has been used to screen libraries of synthetic “self-readable” non-natural polymers that can be sequenced at femtomole scale by chemical fragmentation and highresolution mass spectrometry. The versatility and throughput of the platform was demonstrated by screening two libraries of non-natural polyamide polymers with sizes of 1.77M and 1B compounds against the protein targets K-Ras, asialoglycoprotein receptor (ASGPR), IL-6, IL-6 receptor and TNFα. Hits with nanomolar binding affinities were found against all targets, including competitive inhibitors of K-Ras binding to Raf and functionally active uptake ligands for ASGPR facilitating intracellular delivery.

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The Curious Case of Earthworms and COVID-19

Biology ◽

10.3390/biology10101043 ◽

2021 ◽

Vol 10 (10) ◽

pp. 1043

Author(s):

Janeck J. Scott-Fordsmand ◽

Monica J. B. Amorim

Keyword(s):

Eisenia Fetida ◽

Standard Test ◽

Careful Consideration ◽

Test Species ◽

Native Proteins ◽

Therapeutic Development ◽

Pore Forming Proteins ◽

Corona Viruses ◽

Therapeutic Model

Earthworms have been used for centuries in traditional medicine and are used globally as an ecotoxicological standard test species. Studies of the earthworm Eisenia fetida have shown that exposure to nanomaterials activates a primary corona-response, which is covering the nanomaterial with native proteins, the same response as to biological invaders such as a virus. We outline that the earthworm Eisenia fetida is possibly immune to COVID-19 (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2), and we describe the likely mechanisms of highly receptor-specific pore-forming proteins (PFPs). A non-toxic version of this protein is available, and we hypothesize that it is possible to use the earthworm’s PFPs based anti-viral mechanism as a therapeutic model for human SARS-CoV-2 and other corona viruses. The proteins can be used as a drug, for example, delivered with a nanoparticle in a similar way to the current COVID-19 vaccines. Obviously, careful consideration should be given to the potential risk of toxicity elicited by lysenin for in vivo usage. We aim to share this view to activate its exploration by the wider scientific community while promoting a potential therapeutic development.

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Localizing frustration in native proteins and protein assemblies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0709915104 ◽

2007 ◽

Vol 104 (50) ◽

pp. 19819-19824 ◽

Cited By ~ 181

Author(s):

D. U. Ferreiro ◽

J. A. Hegler ◽

E. A. Komives ◽

P. G. Wolynes

Keyword(s):

Protein Assemblies ◽

Native Proteins

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Identification of Novel Native Autoantigens in Rheumatoid Arthritis

Biomedicines ◽

10.3390/biomedicines8060141 ◽

2020 ◽

Vol 8 (6) ◽

pp. 141 ◽

Cited By ~ 1

Author(s):

Thomas B. G. Poulsen ◽

Dres Damgaard ◽

Malene Møller Jørgensen ◽

Ladislav Senolt ◽

Jonathan M. Blackburn ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

High Density ◽

Protein Array ◽

Post Translational Modification ◽

Native Proteins ◽

Healthy Donors ◽

Array Technology ◽

Human Proteins ◽

Patient Subgroups ◽

Citrullinated Peptide

The majority of patients diagnosed with rheumatoid arthritis (RA) have developed autoantibodies against neoepitopes in proteins that have undergone post-translational modification, e.g., citrullination or carbamylation. There is growing evidence of their molecular relevance and their potential utility to improve diagnosis, patient stratification, and prognosis for precision medicine. Autoantibodies reacting to native proteins may also have a role in RA pathogenesis, however, their reactivity patterns remain much less studied. We hypothesized that a high-density protein array technology could shed light onto the normal and disease-related autoantibodies produced in healthy and RA patient subgroups. In an exploratory study, we investigated the global reactivity of autoantibodies in plasma pools from 15 anti-cyclic citrullinated peptide (CCP)-positive and 10 anti-CCP-negative RA patients and 10 healthy donors against more than 1600 native and unmodified human proteins using a high-density protein array. A total of 102 proteins recognized by IgG autoantibodies were identified, hereof 86 were recognized by antibodies from CCP-positive RA patients and 76 from anti-CCP-negative RA patients, but not by antibodies from healthy donors. Twenty-four of the identified autoantigens have previously been identified in synovial fluid. Multiple human proteins in their native conformation are recognized by autoantibodies from anti-CCP-positive as well as anti-CCP-negative RA patients.

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A less invasive method for orthotopic injection of breast cancer cells into the mouse mammary gland

Laboratory Animals ◽

10.1177/0023677216640706 ◽

2016 ◽

Vol 51 (1) ◽

pp. 85-88 ◽

Cited By ~ 5

Author(s):

Luz E Tavera-Mendoza ◽

Myles Brown

Keyword(s):

Breast Cancer ◽

Mammary Gland ◽

Cancer Cells ◽

Cancer Biology ◽

Mouse Mammary Gland ◽

Breast Cancers ◽

Biologically Relevant ◽

Therapeutic Development ◽

Invasive Method ◽

Animal Handling

Breast cancer is the most common type of cancer diagnosed in women, and the second most common cause of cancer-related deaths in women in North America. The use of laboratory mice in research is an essential tool for the study of breast cancer biology and for pre-clinical therapeutic development. While subcutaneous flank injections of cancer cells are widely used for studying breast cancer biology and for exploring novel therapies, orthotopic xenografting of tumors into the mouse mammary gland allow for the study of breast cancers in a biologically relevant microenvironment. In this study we report a modification of the method of orthotopic injections of cancer cells into the mouse mammary gland which greatly reduces the effects of surgery in mice including decreased wound size, procedure time and anesthesia. It also removes the risk of accidentally puncturing the peritoneal cavity. Consequently post-operative animal handling and stress are significantly reduced. All of these advantages are present without compromising procedure success rate. Therefore, this modification makes orthotopic mammary gland injection a more efficient procedure and greatly improves animal welfare.

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A Mega-High-Throughput Screening Platform for the Discovery of Biologically Relevant Sequence-Defined Non-Natural Polymers

10.26434/chemrxiv.13106069.v1 ◽

2020 ◽

Author(s):

Michal Avital-Shmilovici ◽

Xiaohe Liu ◽

Thomas Shaler ◽

Andrew Lowenthal ◽

Pauline Bourbon ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Binding Affinities ◽

Natural Polymers ◽

Protein Targets ◽

Biologically Relevant ◽

Novel Technology ◽

Major Bottleneck ◽

Screening Platform ◽

Optic Array

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Automated evaluation of quaternary structures from protein crystals

10.1101/224717 ◽

2017 ◽

Cited By ~ 1

Author(s):

Spencer Bliven ◽

Aleix Lafita ◽

Althea Parker ◽

Guido Capitani ◽

Jose M Duarte

Keyword(s):

Protein Data Bank ◽

Quaternary Structure ◽

Protein Structures ◽

Data Bank ◽

New Method ◽

Protein Assemblies ◽

Native Solution ◽

Biologically Relevant ◽

Crystal Contacts ◽

Structure Of Proteins

AbstractA correct assessment of the quaternary structure of proteins is a fundamental prerequisite to understanding their function, physico-chemical properties and mode of interaction with other proteins. Currently about 90% of structures in the Protein Data Bank are crystal structures, in which the correct quaternary structure is embedded in the crystal lattice among a number of crystal contacts. Computational methods are required to 1) classify all protein-protein contacts in crystal lattices as biologically relevant or crystal contacts and 2) provide an assessment of how the biologically relevant interfaces combine into a biological assembly In our previous work we addressed the first problem with our EPPIC (Evolutionary Protein Protein Interface Classifier) method. Here, we present our solution to the second problem with a new method that combines the interface classification results with symmetry and topology considerations. The new algorithm enumerates all possible valid assemblies within the crystal using a graph representation of the lattice and predicts the most probable biological unit based on the pairwise interface scoring. Our method achieves 85% precision on a new dataset of 1,481 biological assemblies with consensus of PDB annotations. Although almost the same precision is achieved by PISA, currently the most popular quaternary structure assignment method, we show that, due to the fundamentally different approach to the problem, the two methods are complementary and could be combined to improve biological assembly assignments. The software for the automatic assessment of protein assemblies (EPPIC version 3) has been made available through a web server at http://www.eppic-web.org.Author summaryX-ray diffraction experiments are the main experimental technique to reveal the detailed atomic 3-dimensional structure of proteins. In these experiments, proteins are packed into crystals, an environment that is far away from their native solution environment. Determining which parts of the structure reflect the protein’s state in the cell rather than being artifacts of the crystal environment can be a difficult task. How the different protein subunits assemble together in solution is known as the quaternary structure. Finding the correct quaternary structure is important both to understand protein oligomerization and for the understanding of protein-protein interactions at large. Here we present a new method to automatically determine the quaternary structure of proteins given their crystal structure. We provide a theoretical basis for properties that correct protein assemblies should possess, and provide a systematic evaluation of all possible assemblies according to these properties. The method provides a guidance to the experimental structural biologist as well as to structural bioinformaticians analyzing protein structures in bulk. Assemblies are provided for all proteins in the Protein Data Bank through a public website and database that is updated weekly as new structures are released.

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