Hidden structure in disordered proteins is adaptive to intracellular changes

Intrinsically disordered protein regions (IDRs) are ubiquitous in all proteomes and essential to cellular function. Unlike folded domains, IDRs exist in an ensemble of rapidly changing conformations. The sequence-encoded structural biases in IDR ensembles are important for function, but are difficult to resolve. Here, we reveal hidden structural preferences in IDR ensembles in vitro with two orthogonal structural methods (SAXS and FRET), and demonstrate that these structural preferences persist in cells using live cell microscopy. Importantly, we demonstrate that some IDRs have structural preferences that can adaptively respond to even mild intracellular environment changes, while other IDRs may display a remarkable structural resilience. We propose that the ability to sense and respond to changes in cellular physicochemical composition, or to resist such changes, is a sequence-dependent property of IDRs in organisms across all kingdoms of life.

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Degradation of Intrinsically Disordered Proteins by the NADH 26S Proteasome

Biomolecules ◽

10.3390/biom10121642 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1642

Author(s):

Peter Tsvetkov ◽

Nadav Myers ◽

Julia Adler ◽

Yosef Shaul

Keyword(s):

Substrate Specificity ◽

Structural Integrity ◽

Intrinsically Disordered Proteins ◽

Intrinsically Disordered Protein ◽

Degradation Pathway ◽

26S Proteasome ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Disordered Protein

The 26S proteasome is the endpoint of the ubiquitin- and ATP-dependent degradation pathway. Over the years, ATP was regarded as completely essential for 26S proteasome function due to its role in ubiquitin-signaling, substrate unfolding and ensuring its structural integrity. We have previously reported that physiological concentrations of NADH are efficient in replacing ATP to maintain the integrity of an enzymatically functional 26S PC. However, the substrate specificity of the NADH-stabilized 26S proteasome complex (26S PC) was never assessed. Here, we show that the binding of NADH to the 26S PC inhibits the ATP-dependent and ubiquitin-independent degradation of the structured ODC enzyme. Moreover, the NADH-stabilized 26S PC is efficient in degrading intrinsically disordered protein (IDP) substrates that might not require ATP-dependent unfolding, such as p27, Tau, c-Fos and more. In some cases, NADH-26S proteasomes were more efficient in processing IDPs than the ATP-26S PC. These results indicate that in vitro, physiological concentrations of NADH can alter the processivity of ATP-dependent 26S PC substrates such as ODC and, more importantly, the NADH-stabilized 26S PCs promote the efficient degradation of many IDPs. Thus, ATP-independent, NADH-dependent 26S proteasome activity exemplifies a new principle of how mitochondria might directly regulate 26S proteasome substrate specificity.

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Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

Nature Communications ◽

10.1038/s41467-021-25736-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Cesar L. Cuevas-Velazquez ◽

Tamara Vellosillo ◽

Karina Guadalupe ◽

Hermann Broder Schmidt ◽

Feng Yu ◽

...

Keyword(s):

Osmotic Stress ◽

Chemical Properties ◽

Intrinsically Disordered Protein ◽

Intrinsically Disordered ◽

Disordered Protein ◽

Physical Chemical ◽

Cellular Environment ◽

Experimental Approaches ◽

Intracellular Changes

AbstractCell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools.

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Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells

10.1101/2021.02.17.431712 ◽

2021 ◽

Author(s):

Cesar L Cuevas-Velazquez ◽

Tamara Vellosillo ◽

Karina Guadalupe ◽

H Broder Schmidt ◽

Feng Yu ◽

...

Keyword(s):

Osmotic Stress ◽

Chemical Properties ◽

Intrinsically Disordered Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Physical Chemical ◽

Cellular Environment ◽

Highly Sensitive ◽

Intracellular Changes

SUMMARYCell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Methods that dynamically monitor these intracellular effects are currently lacking. Here, we leveraged the environmental sensitivity and structural plasticity of intrinsically disordered regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use in environmentally-responsive molecular tools.

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Baicalein inhibits heparin-induced Tau aggregation by initializing non-toxic Tau oligomer formation

Cell Communication and Signaling ◽

10.1186/s12964-021-00704-3 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Shweta Kishor Sonawane ◽

Vladimir N. Uversky ◽

Subashchandrabose Chinnathambi

Keyword(s):

Intrinsically Disordered Proteins ◽

Senile Plaques ◽

Neuroblastoma Cells ◽

Memory Loss ◽

Disordered Proteins ◽

Protein Accumulation ◽

Scutellaria Baicalensis Georgi ◽

Intrinsically Disordered ◽

Tau Aggregation

Abstract Background Amyloid aggregate deposition is the key feature of Alzheimer’s disease. The proteinaceous aggregates found in the afflicted brain are the intra-neuronal neurofibrillary tangles formed by the microtubule-associated protein Tau and extracellular deposits, senile plaques, of amyloid beta (Aβ) peptide proteolytically derived from the amyloid precursor protein. Accumulation of these aggregates has manifestations in the later stages of the disease, such as memory loss and cognitive inabilities originating from the neuronal dysfunction, neurodegeneration, and brain atrophy. Treatment of this disease at the late stages is difficult, and many clinical trials have failed. Hence, the goal is to find means capable of preventing the aggregation of these intrinsically disordered proteins by inhibiting the early stages of their pathological transformations. Polyphenols are known to be neuroprotective agents with the noticeable potential against many neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Prion diseases. Methods We analyzed the capability of Baicalein to inhibit aggregation of human Tau protein by a multifactorial analysis that included several biophysical and biochemical techniques. Results The potency of Baicalein, a polyphenol from the Scutellaria baicalensis Georgi, against in vitro Tau aggregation and PHF dissolution has been screened and validated. ThS fluorescence assay revealed the potent inhibitory activity of Baicalein, whereas ANS revealed its mechanism of Tau inhibition viz. by oligomer capture and dissociation. In addition, Baicalein dissolved the preformed mature fibrils of Tau thereby possessing a dual target action. Tau oligomers formed by Baicalein were non-toxic to neuronal cells, highlighting its role as a potent molecule to be screened against AD. Conclusion In conclusion, Baicalein inhibits aggregation of hTau40 by enhancing the formation of SDS-stable oligomers and preventing fibril formation. Baicalein-induced oligomers do not affect the viability of the neuroblastoma cells. Therefore, Baicalein can be considered as a lead molecule against Tau pathology in AD.

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Generation of the configurational ensemble of an intrinsically disordered protein from unbiased molecular dynamics simulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907251116 ◽

2019 ◽

Vol 116 (41) ◽

pp. 20446-20452 ◽

Cited By ~ 9

Author(s):

Utsab R. Shrestha ◽

Puneet Juneja ◽

Qiu Zhang ◽

Viswanathan Gurumoorthy ◽

Jose M. Borreguero ◽

...

Keyword(s):

Molecular Dynamics ◽

Intrinsically Disordered Proteins ◽

Chemical Shifts ◽

Intrinsically Disordered Protein ◽

Physical Models ◽

Dynamics Simulation ◽

Disordered Proteins ◽

Intrinsically Disordered ◽

Eukaryotic Proteomes ◽

Heterogeneous Ensemble

Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes, play a major role in cell signaling, and are associated with human diseases. To understand IDP function it is critical to determine their configurational ensemble, i.e., the collection of 3-dimensional structures they adopt, and this remains an immense challenge in structural biology. Attempts to determine this ensemble computationally have been hitherto hampered by the necessity of reweighting molecular dynamics (MD) results or biasing simulation in order to match ensemble-averaged experimental observables, operations that reduce the precision of the generated model because different structural ensembles may yield the same experimental observable. Here, by employing enhanced sampling MD we reproduce the experimental small-angle neutron and X-ray scattering profiles and the NMR chemical shifts of the disordered N terminal (SH4UD) of c-Src kinase without reweighting or constraining the simulations. The unbiased simulation results reveal a weakly funneled and rugged free energy landscape of SH4UD, which gives rise to a heterogeneous ensemble of structures that cannot be described by simple polymer theory. SH4UD adopts transient helices, which are found away from known phosphorylation sites and could play a key role in the stabilization of structural regions necessary for phosphorylation. Our findings indicate that adequately sampled molecular simulations can be performed to provide accurate physical models of flexible biosystems, thus rationalizing their biological function.

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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 ◽

Intrinsically Disordered ◽

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.

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Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽

10.7554/elife.10785 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Andrei Vovk ◽

Chad Gu ◽

Michael G Opferman ◽

Larisa E Kapinos ◽

Roderick YH Lim ◽

...

Keyword(s):

Spatial Organization ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Transport Proteins ◽

Disordered Proteins ◽

Biophysical Model ◽

Nuclear Pore ◽

Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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Enhancing Binding Affinity of an Intrinsically Disordered Protein by α-Methylation of Key Amino Acid Residues

10.26434/chemrxiv.10113128 ◽

2019 ◽

Author(s):

Valentin Bauer ◽

Boris Schmidtgall ◽

Gergő Gógl ◽

Jozica Dolenc ◽

Judit Osz ◽

...

Keyword(s):

Amino Acids ◽

Protein Interactions ◽

Intrinsically Disordered Proteins ◽

Intrinsically Disordered Protein ◽

Selective Inhibition ◽

Disordered Proteins ◽

Creb Binding Protein ◽

Alpha Helices ◽

Intrinsically Disordered ◽

Order Of Magnitude

Intrinsically disordered proteins (IDPs), which undergo folding upon binding to their targets, are critical players in protein interaction networks. Here we demonstrate that incorporation of non-canonical alpha-methylated amino acids into the unstructured activation domain of the transcriptional coactivator ACTR can stabilize helical conformations and strengthen binding interactions with the nuclear coactivator binding domain (NCBD) of CREB-binding protein (CBP). A combinatorial alpha-methylation scan of the ACTR sequence converged on two substitutions at positions 1055 and 1076 that increase affinity for both NCBD and the full length 270 kDa CBP by one order of magnitude. The first X-ray structure of the modified ACTR domain bound to NCBD revealed that the key alpha-methylated amino acids were localized within alpha-helices. Biophysical studies showed that the observed changes in binding energy are the result of long-range interactions and redistribution of enthalpy and entropy. This proof-of-concept study establishes a potential strategy for selective inhibition of protein-protein interactions involving IDPs in cells.<br>

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An intrinsically disordered protein, CP12: jack of all trades and master of the Calvin cycle

Biochemical Society Transactions ◽

10.1042/bst20120097 ◽

2012 ◽

Vol 40 (5) ◽

pp. 995-999 ◽

Cited By ~ 41

Author(s):

Brigitte Gontero ◽

Stephen C. Maberly

Keyword(s):

Amino Acids ◽

Stress Responses ◽

Intrinsically Disordered Proteins ◽

Calvin Cycle ◽

Intrinsically Disordered Protein ◽

Disulfide Bridge ◽

Co2 Assimilation ◽

Disordered Proteins ◽

Dimensional Structure ◽

Intrinsically Disordered

Many proteins contain disordered regions under physiological conditions and lack specific three-dimensional structure. These are referred to as IDPs (intrinsically disordered proteins). CP12 is a chloroplast protein of approximately 80 amino acids and has a molecular mass of approximately 8.2–8.5 kDa. It is enriched in charged amino acids and has a small number of hydrophobic residues. It has a high proportion of disorder-promoting residues, but has at least two (often four) cysteine residues forming one (or two) disulfide bridge(s) under oxidizing conditions that confers some order. However, CP12 behaves like an IDP. It appears to be universally distributed in oxygenic photosynthetic organisms and has recently been detected in a cyanophage. The best studied role of CP12 is its regulation of the Calvin cycle responsible for CO2 assimilation. Oxidized CP12 forms a supramolecular complex with two key Calvin cycle enzymes, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and PRK (phosphoribulokinase), down-regulating their activity. Association–dissociation of this complex, induced by the redox state of CP12, allows the Calvin cycle to be inactive in the dark and active in the light. CP12 is promiscuous and interacts with other enzymes such as aldolase and malate dehydrogenase. It also plays other roles in plant metabolism such as protecting GAPDH from inactivation and scavenging metal ions such as copper and nickel, and it is also linked to stress responses. Thus CP12 seems to be involved in many functions in photosynthetic cells and behaves like a jack of all trades as well as being a master of the Calvin cycle.

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Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein

mBio ◽

10.1128/mbio.01712-17 ◽

2018 ◽

Vol 9 (1) ◽

Cited By ~ 16

Author(s):

Javeed Ahmad ◽

Aisha Farhana ◽

Rita Pancsa ◽

Simran Kaur Arora ◽

Alagiri Srinivasan ◽

...

Keyword(s):

Dendritic Cells ◽

Mycobacterium Tuberculosis ◽

Host Cell ◽

Terminal Segment ◽

Host Protein ◽

Host Cells ◽

Productive Infection ◽

Disordered Proteins ◽

Intrinsically Disordered

ABSTRACT Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis. IMPORTANCE To hijack the human host cell machinery to enable survival inside macrophages, the pathogen Mycobacterium tuberculosis requires a repertoire of proteins that can mimic host protein function and modulate host cell machinery. Here, we have shown how a single protein can play multiple functions and hijack the host cell for the benefit of the pathogen. Full-length membrane-anchored PPE37 protein is cleaved into N- and C-terminal domains under iron-depleted conditions. The N-terminal domain facilitates the propathogen semimature tolerogenic state of dendritic cells, whereas the C-terminal segment is localized into host cell nucleus and induces apoptosis. The immune implications of these in vitro observations were assessed and validated in mice and also human TB patients. This study presents novel mechanistic insight adopted by M. tuberculosis to survive inside host cells.

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