Novel molecular aspects of the CRISPR backbone protein ‘Cas7’ from cyanobacteria

The cyanobacterium Anabaena PCC 7120 shows the presence of Type I-D CRISPR system that can potentially confer adaptive immunity. The Cas7 protein (Alr1562), which forms the backbone of the type I-D surveillance complex, was characterized from Anabaena. Alr1562, showed the presence of the non-canonical RNA recognition motif and two intrinsically disordered regions (IDRs). When overexpressed in E. coli, the Alr1562 protein was soluble and could be purified by affinity chromatography, however, deletion of IDRs rendered Alr1562 completely insoluble. The purified Alr1562 was present in the dimeric or a RNA-associated higher oligomeric form, which appeared as spiral structures under electron microscope. With RNaseA and NaCl treatment, the higher oligomeric form converted to the lower oligomeric form, indicating that oligomerization occurred due to the association of Alr1562 with RNA. The secondary structure of both these forms was largely similar, resembling that of a partially folded protein. The dimeric Alr1562 was more prone to temperature-dependent aggregation than the higher oligomeric form. In vitro, the Alr1562 bound more specifically to a minimal CRISPR unit than to the non-specific RNA. Residues required for binding of Alr1562 to RNA, identified by protein modeling-based approaches, were mutated for functional validation. Interestingly, these mutant proteins, showing reduced ability to bind RNA were predominantly present in dimeric form. Alr1562 was detected with specific antiserum in Anabaena, suggesting that the type I-D system is expressed and may be functional in vivo. This is the first report that describes the characterization of a Cas protein from any photosynthetic organism.

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Molecular and Biochemical Techniques for Deciphering p53-MDM2 Regulatory Mechanisms

Biomolecules ◽

10.3390/biom11010036 ◽

2020 ◽

Vol 11 (1) ◽

pp. 36

Author(s):

Konstantinos Karakostis ◽

Ignacio López ◽

Ana M. Peña-Balderas ◽

Robin Fåhareus ◽

Vanesa Olivares-Illana

Keyword(s):

Protein Complexes ◽

Post Translational Modifications ◽

Intrinsically Disordered ◽

Advantages And Disadvantages ◽

Intrinsically Disordered Regions ◽

Multiple Partners ◽

Allosteric Interactions ◽

Spatio Temporal

The p53 and Mouse double minute 2 (MDM2) proteins are hubs in extensive networks of interactions with multiple partners and functions. Intrinsically disordered regions help to adopt function-specific structural conformations in response to ligand binding and post-translational modifications. Different techniques have been used to dissect interactions of the p53-MDM2 pathway, in vitro, in vivo, and in situ each having its own advantages and disadvantages. This review uses the p53-MDM2 to show how different techniques can be employed, illustrating how a combination of in vitro and in vivo techniques is highly recommended to study the spatio-temporal location and dynamics of interactions, and to address their regulation mechanisms and functions. By using well-established techniques in combination with more recent advances, it is possible to rapidly decipher complex mechanisms, such as the p53 regulatory pathway, and to demonstrate how protein and nucleotide ligands in combination with post-translational modifications, result in inter-allosteric and intra-allosteric interactions that govern the activity of the protein complexes and their specific roles in oncogenesis. This promotes elegant therapeutic strategies that exploit protein dynamics to target specific interactions.

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Unstructured mRNAs form multivalent RNA-RNA interactions to generate TIS granule networks

10.1101/2020.02.14.949503 ◽

2020 ◽

Cited By ~ 4

Author(s):

Weirui Ma ◽

Gang Zhen ◽

Wei Xie ◽

Christine Mayr

Keyword(s):

Endoplasmic Reticulum ◽

Protein Synthesis ◽

Complex Formation ◽

Major Site ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Protein Complex Formation ◽

Unstructured Regions

SummaryThe TIS granule network is a constitutively expressed membraneless organelle that concentrates mRNAs with AU-rich elements and interacts with the major site of protein synthesis, the rough endoplasmic reticulum. Most known biomolecular condensates are sphere-like, but TIS granules have a mesh-like morphology. Through in vivo and in vitro reconstitution experiments we discovered that this shape is generated by extensive intermolecular RNA-RNA interactions. They are mostly accomplished by mRNAs with large unstructured regions in their 3′UTRs that we call intrinsically disordered regions (IDRs). As AU-rich RNA is a potent chaperone that suppresses protein aggregation and is overrepresented in mRNAs with IDRs, our data suggests that TIS granules concentrate mRNAs that assist protein folding. In addition, the proximity of translating mRNAs in TIS granule networks may enable co-translational protein complex formation.

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Molecular determinants of phase separation forDrosophilaDNA replication licensing factors

10.1101/2021.04.12.439485 ◽

2021 ◽

Author(s):

Matthew W. Parker ◽

Jonchee Kao ◽

Alvin Huang ◽

James M. Berger ◽

Michael R. Botchan

Keyword(s):

Phase Separation ◽

Self Assembly ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Partner Proteins

ABSTRACTLiquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separationin vitroand chromosome bindingin vivo, and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. UsingD. melanogaster (Dm)Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificitya priori.

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Cellular labelling favours unfolded proteins

10.1101/274761 ◽

2018 ◽

Cited By ~ 3

Author(s):

David-Paul Minde ◽

Manasa Ramakrishna ◽

Kathryn S. Lilley

Keyword(s):

Large Scale ◽

Protein Structures ◽

Unfolded Proteins ◽

80S Ribosome ◽

Unfolded Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Wide Scale

AbstractFolded enzymes are essential for life, but there is limited in vivo information about how locally unfolded protein regions contribute to biological functions. Intrinsically Disordered Regions (IDRs) are enriched in disease-linked and multiply post-translationally modified proteins. The extent of foldability of predicted IDRs is difficult to measure due to significant technical challenges to survey in vivo protein conformations on a proteome-wide scale. We reasoned that IDRs should be more accessible to targeted in vivo biotinylation than more ordered protein regions, if they retain their flexibility in vivo. Indeed, we observed a positive correlation of predicted IDRs and biotinylation density across four independent large-scale proximity proteomics studies that together report >20 000 biotinylation sites. We show that biotin ‘painting’ is a promising approach to fill gaps in knowledge between static in vitro protein structures, in silico disorder predictions and in vivo condition-dependent subcellular plasticity using the 80S ribosome as an example.

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Molecular determinants of phase separation for Drosophila DNA replication licensing factors

eLife ◽

10.7554/elife.70535 ◽

2021 ◽

Vol 10 ◽

Author(s):

Matthew W Parker ◽

Jonchee A Kao ◽

Alvin Huang ◽

James M Berger ◽

Michael R Botchan

Keyword(s):

Phase Separation ◽

Dna Replication ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Partner Proteins

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.

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Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

10.1101/2021.10.27.466110 ◽

2021 ◽

Author(s):

Yu Chen ◽

Claudia Cattoglio ◽

Gina Dailey ◽

Qiulin Zhu ◽

Robert Tjian ◽

...

Keyword(s):

Dna Binding ◽

Transcription Initiation ◽

Specific Inhibitor ◽

Single Particle Tracking ◽

Specific Gene ◽

Hypoxia Inducible Factors ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions

Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct non-overlapping binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro — the hypoxia response element (HRE) — but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, a major determinant of chromatin binding and diffusion behavior is dictated by the AD-containing intrinsically disordered regions. These findings reveal a previously unappreciated role of IDRs in regulating the TF search process that may play a role in selective functional target site binding on chromatin.

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Chimeric 14-3-3 proteins for unravelling interactions with intrinsically disordered partners

10.1101/130245 ◽

2017 ◽

Author(s):

Nikolai N. Sluchanko ◽

Kristina V. Tugaeva ◽

Alfred A. Antson

Keyword(s):

Structural Information ◽

Protein Complexes ◽

Cellular Processes ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

C Terminus ◽

Transient Interactions ◽

Partner Proteins

ABSTRACTIn eukaryotes, several proteins act as “hubs”, integrating signals from a variety of interacting partners that bind to the hub through intrinsically disordered regions. Not surprisingly, one of the major hubs, the 14-3-3 protein, that plays wide-ranging roles in cellular processes, has been linked with a number of disorders including neurodegenerative diseases and cancer. A partner protein usually binds with its phosphopeptide accommodated in an amphipathic groove (AG) of 14-3-3, a promising platform for therapeutic intervention. Protein plasticity in the groove allows to accommodate a range of phosphopeptides with different sequences. So far, in spite of mammoth effort, accurate structural information has been derived only for few 14-3-3 complexes with phosphopeptide-containing proteins or various short synthetic peptides. The progress has been prevented by intrinsic disorder of partner proteins and, in case of transient interactions, by the low affinity of phosphopeptides. We reasoned that these problems could be resolved by using chimeric 14-3-3 proteins with incorporated peptide sequences. We tested this hypothesis and found that such chimeric proteins are easy to design, express, purify and crystallize. We show that when attached to the C terminus of 14-3-3 via an optimal linker, peptides become stoichiometrically phosphorylated by protein kinase A during bacterial co-expression. We determined crystal structures for complexes of chimeric 14-3-3 protein fused with three different peptides. In most of the cases, the phosphopeptide is bound inside the AG, providing invaluable information on its interaction with the protein. This approach can reinvigorate studies of 14-3-3 protein complexes, including those with otherwise challenging low affinity phosphopeptides. Furthermore, 14-3-3-phosphopeptide chimeras can be useful for the design of novel biosensors for in vitro and in vivo imaging experiments.

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Peculiarities of the course of type I allergic reactions in patients with blood diseases

Terapevt (General Physician) ◽

10.33920/med-12-2004-06 ◽

2020 ◽

pp. 40-50

Author(s):

A. Nikitina

Keyword(s):

Search Engines ◽

Anaphylactic Shock ◽

Type I ◽

Allergic Reactions ◽

Common Cause ◽

Blood Diseases ◽

Treatment Regimens ◽

Modern Methods

Analysis of literature data presented in search engines — Elibrary, PubMed, Cochrane — concerning the risk of developing type I allergic reactions in patients with blood diseases is presented. It is shown that the most common cause of type I allergic reactions is drugs included in the treatment regimens of this category of patients. The article presents statistics on the increase in the number of drug allergies leading to cases of anaphylactic shock in patients with blood diseases. Modern methods for the diagnosis of type I allergic reactions in vivo and in vitro are considered.

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