Cellular labelling favours unfolded proteins

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.

The Mysterious Unfoldome: Structureless, Underappreciated, Yet Vital Part of Any Given Proteome

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/568068 ◽

2010 ◽

Vol 2010 ◽

pp. 1-14 ◽

Cited By ~ 146

Author(s):

Vladimir N. Uversky

Keyword(s):

Large Scale ◽

Intrinsically Disordered Proteins ◽

Biologically Active ◽

Disordered Proteins ◽

Unfolded Proteins ◽

Proteome Level ◽

Natively Unfolded ◽

Natively Unfolded Proteins

Contrarily to the general believe, many biologically active proteins lack stable tertiary and/or secondary structure under physiological conditions in vitro. These intrinsically disordered proteins (IDPs) are highly abundant in nature and many of them are associated with various human diseases. The functional repertoire of IDPs complements the functions of ordered proteins. Since IDPs constitute a significant portion of any given proteome, they can be combined in an unfoldome; which is a portion of the proteome including all IDPs (also known as natively unfolded proteins, therefore, unfoldome), and describing their functions, structures, interactions, evolution, and so forth. Amino acid sequence and compositions of IDPs are very different from those of ordered proteins, making possible reliable identification of IDPs at the proteome level by various computational means. Furthermore, IDPs possess a number of unique structural properties and are characterized by a peculiar conformational behavior, including their high stability against low pH and high temperature and their structural indifference toward the unfolding by strong denaturants. These peculiarities were shown to be useful for elaboration of the experimental techniques for the large-scale identification of IDPs in various organisms. Some of the computational and experimental tools for the unfoldome discovery are discussed in this review.

Flexibility and mobility of SARS-CoV-2-related protein structures

Scientific Reports ◽

10.1038/s41598-021-82849-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Rudolf A. Römer ◽

Navodya S. Römer ◽

A. Katrine Wallis

Keyword(s):

In Silico ◽

Drug Targets ◽

Large Scale ◽

Structural Information ◽

Protein Structures ◽

Antiviral Drug ◽

Docking Studies ◽

Related Protein

AbstractThe worldwide CoVid-19 pandemic has led to an unprecedented push across the whole of the scientific community to develop a potent antiviral drug and vaccine as soon as possible. Existing academic, governmental and industrial institutions and companies have engaged in large-scale screening of existing drugs, in vitro, in vivo and in silico. Here, we are using in silico modelling of possible SARS-CoV-2 drug targets, as deposited on the Protein Databank (PDB), and ascertain their dynamics, flexibility and rigidity. For example, for the SARS-CoV-2 spike protein—using its complete homo-trimer configuration with 2905 residues—our method identifies a large-scale opening and closing of the S1 subunit through movement of the S$${}^\text{B}$$ B domain. We compute the full structural information of this process, allowing for docking studies with possible drug structures. In a dedicated database, we present similarly detailed results for the further, nearly 300, thus far resolved SARS-CoV-2-related protein structures in the PDB.

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 ◽

Advantages And Disadvantages ◽

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.

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 ◽

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.

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

Biochemical Journal ◽

10.1042/bcj20200026 ◽

2020 ◽

Vol 477 (5) ◽

pp. 971-983 ◽

Cited By ~ 1

Author(s):

Prakash Kalwani ◽

Devashish Rath ◽

Anand Ballal

Keyword(s):

Rna Recognition Motif ◽

Type I ◽

Mutant Proteins ◽

Oligomeric Form ◽

Photosynthetic Organism ◽

Cyanobacterium Anabaena

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.

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 ◽

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.

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 ◽

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.

FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

10.1101/071332 ◽

2016 ◽

Cited By ~ 1

Author(s):

Steffen Preissler ◽

Claudia Rato ◽

Luke Perera ◽

Vladimir Saudek ◽

David Ron

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Active Site ◽

Enzymatic Reaction ◽

Covalent Modification ◽

Unfolded Proteins ◽

Unfolded Protein ◽

Er Chaperone

Significance statementSome 25 years ago it was discovered that the activity of the ER chaperone BiP is regulated by covalent modification, the nature of which, AMPylation (not ADPribosylation, as had long been thought) and the enzyme responsible, FICD, have only recently been identified. Genetic inactivation of FICD and in vitro studies of the purified enzyme and substrate have done much to clarify the biochemical consequences of the modification and its underlying logic: As ER stress wanes, FICD uses ATP to AMPylate Thr518 of BiP locking BiP in a relatively inactive conformation. As ER stress levels re-mount the cells draw on this pool of inactive chaperone, which is de-AMPylated and restored to its fully active state.Here we report on the identity of the de-AMPylating enzyme - and with it on the surprising finding that both AMPylation and de-AMPylation of BiP are carried out by the same polypeptide (FICD) using the same active site, both in vivo and in vitro. Analysis of the reaction products reveals that de-AMPylation does not involve trivial concentration-dependent micro-reversibility of an enzymatic reaction, but rather a switch in the active site of FICD that facilitates two antagonistic thermodynamically favored reactions.Surprisingly BiP de-AMPylation (not AMPylation) is the default activity of FICD. The side-chain of a single regulatory residue, E234, toggles the enzyme between de-AMPylation and AMPylation in vitro. Our studies thereby uncover an active mechanism that must exist in the ER for coupling waning levels of unfolded protein stress to the conversion of FICD from its default de-AMPylation mode to BiP AMPylation. Whilst the details of this active switch remain to be discovered, we are able to suggest a plausible mechanism by which it may come about.Identification of the enzyme that de-modifies BiP to reactivate it will be of interest to cell biologists, whereas the novel features of FICD as a dualfunctioning enzyme with a single bi-functional active site will be of broad interest to enzymologists and molecular biologists.AbstractProtein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response (UPR) that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoanspecific FIC-domain containing ER-localized enzyme, FICD/HYPE, rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here it is shown that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD−/− cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.

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 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.

Protein arginylation is regulated during SARS-CoV-2 infection

10.1101/2021.11.02.466971 ◽

2021 ◽

Author(s):

Janaina Macedo-da-Silva ◽

Livia Rosa-Fernandes ◽

Vinicius de Moraes Gomes ◽

Veronica Feijoli Santiago ◽

Catarina Maria Stanischesk Molnar ◽

...

Keyword(s):

Viral Load ◽

Essential Element ◽

Unfolded Proteins ◽

Time Data ◽

Unfolded Protein ◽

Infected Cells ◽

Protein Response

In 2019, the world witnessed the onset of an unprecedented pandemic. In September 2021, the infection by SARS-CoV-2 had already been responsible for the death of more than 4 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER-stress and activation of unfolded protein response (UPR) pathway. The degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-end rule pathway. Here we present, for the first time, data on the role of arginylation during SARS-CoV-2 infection. We studied the modulation of protein arginylation in Vero CCL-81 and Calu-3 cells infected after 2h, 6h, 12h, 24h, and 48h. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure the levels of ATE1 and arginylated proteins. This regulation is seen specifically during infections by coronaviruses. We demonstrate that during SARS-CoV-2 infection there is an increase in the expression of the ATE1 enzyme associated with regulated levels of specific arginylated proteins. On the other hand, infected macrophages showed no ATE1 regulation. An important finding revealed that modulation of the N-end rule pathway differs between different types of infected cells. We also confirmed the potential of tannic acid to reduce viral load, and furthermore, to modulate ATE1 levels during infection. In addition, the arginylation inhibitor merbromin (MER) is also capable of both reducing viral load and reducing ATE1 levels. Taken together, these data show the importance of arginylation during the progression of SARS-CoV-2 infection and open the door for future studies that may unravel the role of ATE1 and its inhibitors in pathogen infection.