bims-indpro 2022-08-28 papers

bims-indpro

Biomed News

on Intrinsically disordered proteins

Issue of 2022–08–28
twenty-two papers selected by
Sara Mingu, Johannes Gutenberg University

Microtubule lattice spacing governs cohesive envelope formation of tau family proteins.
Model biomolecular condensates have heterogeneous structure quantitatively dependent on the interaction profile of their constituent macromolecules.
Machine-learning analysis of intrinsically disordered proteins identifies key factors that contribute to neurodegeneration-related aggregation.
ReSMAP: Web Server for Predicting Residue-Specific Membrane-Association Propensities of Intrinsically Disordered Proteins.
Low Complexity Induces Structure in Protein Regions Predicted as Intrinsically Disordered.
An Interpretable Machine-Learning Algorithm to Predict Disordered Protein Phase Separation Based on Biophysical Interactions.
Peptidyl Prolyl Isomerase A Modulates the Liquid-Liquid Phase Separation of Proline-Rich IDPs.
Molecular Mechanisms of Steric Pressure Generation and Membrane Remodeling by Intrinsically Disordered Proteins.
Measurement of 1Hα transverse relaxation rates in proteins: application to solvent PREs.
HSF1, Aging, and Neurodegeneration.
Correlating multi-functional role of cold shock domain proteins with intrinsically disordered regions.
A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12.
Markov State Models and Molecular Dynamics Simulations Reveal the Conformational Transition of the Intrinsically Disordered Hypervariable Region of K-Ras4B to the Ordered Conformation.
Scaffolding viral protein NC nucleates phase separation of the HIV-1 biomolecular condensate.
The ZSWIM8 ubiquitin ligase regulates neurodevelopment by guarding the protein quality of intrinsically disordered Dab1.
Molecular Dynamics Investigation into pH Dependent Metal Binding of the Intrinsically Disordered Worm Jaw Protein, Nvjp-1.
New Insights into Phase Separation Processes and Membraneless Condensates of EIN2.
Identification of Germinal Neurofibromin Hotspots.
Catechol-Containing Compounds are a Broad Class of Protein Aggregation Inhibitors: Redox State is a Key Determinant of the Inhibitory Activities.
Aberrant liquid-liquid phase separation and amyloid aggregation of proteins related to neurodegenerative diseases.
Hyperphosphorylation-Mimetic TDP-43 Drives Amyloid Formation and Possesses Neuronal Toxicity at the Oligomeric Stage.
Mislocalization of Nup62 Contributes to TDP-43 Proteinopathy in ALS/FTLD.

Nat Chem Biol. 2022 Aug 22.

Microtubule lattice spacing governs cohesive envelope formation of tau family proteins.

Valerie Siahaan, Ruensern Tan, Tereza Humhalova, Lenka Libusova, Samuel E Lacey, Tracy Tan, Mariah Dacy, Kassandra M Ori-McKenney, Richard J McKenney, Marcus Braun, Zdenek Lansky.

Tau is an intrinsically disordered microtubule-associated protein (MAP) implicated in neurodegenerative disease. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive 'envelopes' around microtubules. Envelopes differentially regulate lattice accessibility for other MAPs, but the mechanism of envelope formation remains unclear. Here we find that tau envelopes form cooperatively, locally altering the spacing of tubulin dimers within the microtubule lattice. Envelope formation compacted the underlying lattice, whereas lattice extension induced tau envelope disassembly. Investigating other members of the tau family, we find that MAP2 similarly forms envelopes governed by lattice spacing, whereas MAP4 cannot. Envelopes differentially biased motor protein movement, suggesting that tau family members could spatially divide the microtubule surface into functionally distinct regions. We conclude that the interdependent allostery between lattice spacing and cooperative envelope formation provides the molecular basis for spatial regulation of microtubule-based processes by tau and MAP2.

DOI: https://doi.org/10.1038/s41589-022-01096-2
Soft Matter. 2022 Aug 25.

Model biomolecular condensates have heterogeneous structure quantitatively dependent on the interaction profile of their constituent macromolecules.

Julian C Shillcock, Clément Lagisquet, Jérémy Alexandre, Laurent Vuillon, John H Ipsen.

Biomolecular condensates play numerous roles in cells by selectively concentrating client proteins while excluding others. These functions are likely to be sensitive to the spatial organization of the scaffold proteins forming the condensate. We use coarse-grained molecular simulations to show that model intrinsically-disordered proteins phase separate into a heterogeneous, structured fluid characterized by a well-defined length scale. The proteins are modelled as semi-flexible polymers with punctate, multifunctional binding sites in good solvent conditions. Their dense phase is highly solvated with a spatial structure that is more sensitive to the separation of the binding sites than their affinity. We introduce graph theoretic measures to quantify their heterogeneity, and find that it increases with increasing binding site number, and exhibits multi-timescale dynamics. The model proteins also swell on passing from the dilute solution to the dense phase. The simulations predict that the structure of the dense phase is modulated by the location and affinity of binding sites distant from the termini of the proteins, while sites near the termini more strongly affect its phase behaviour. The relations uncovered between the arrangement of weak interaction sites on disordered proteins and the material properties of their dense phase can be experimentally tested to give insight into the biophysical properties, pathological effects, and rational design of biomolecular condensates.

DOI: https://doi.org/10.1039/d2sm00387b
Front Aging Neurosci. 2022 ;14 938117

Machine-learning analysis of intrinsically disordered proteins identifies key factors that contribute to neurodegeneration-related aggregation.

Akshatha Ganne, Meenakshisundaram Balasubramaniam, Srinivas Ayyadevara, Robert J Shmookler Reis.

  Protein structure is determined by the amino acid sequence and a variety of post-translational modifications, and provides the basis for physiological properties. Not all proteins in the proteome attain a stable conformation; roughly one third of human proteins are unstructured or contain intrinsically disordered regions exceeding 40% of their length. Proteins comprising or containing extensive unstructured regions are termed intrinsically disordered proteins (IDPs). IDPs are known to be overrepresented in protein aggregates of diverse neurodegenerative diseases. We evaluated the importance of disordered proteins in the nematode Caenorhabditis elegans, by RNAi-mediated knockdown of IDPs in disease-model strains that mimic aggregation associated with neurodegenerative pathologies. Not all disordered proteins are sequestered into aggregates, and most of the tested aggregate-protein IDPs contribute to important physiological functions such as stress resistance or reproduction. Despite decades of research, we still do not understand what properties of a disordered protein determine its entry into aggregates. We have employed machine-learning models to identify factors that predict whether a disordered protein is found in sarkosyl-insoluble aggregates isolated from neurodegenerative-disease brains (both AD and PD). Machine-learning predictions, coupled with principal component analysis (PCA), enabled us to identify the physiochemical properties that determine whether a disordered protein will be enriched in neuropathic aggregates.

Keywords:  Alzheimer’s disease; Parkinson’s disease; drug screening and discovery; intrinsically disordered proteins (IDPs); misfolding and aggregation; neural network; proteostasis; support vector machine

DOI:  https://doi.org/10.3389/fnagi.2022.938117
Membranes (Basel). 2022 Aug 11. pii: 773. [Epub ahead of print]12(8):

ReSMAP: Web Server for Predicting Residue-Specific Membrane-Association Propensities of Intrinsically Disordered Proteins.

Sanbo Qin, Alan Hicks, Souvik Dey, Ramesh Prasad, Huan-Xiang Zhou.

  The functional processes of many proteins involve the association of their intrinsically disordered regions (IDRs) with acidic membranes. We have identified the membrane-association characteristics of IDRs using extensive molecular dynamics (MD) simulations and validated them with NMR spectroscopy. These studies have led to not only deep insight into functional mechanisms of IDRs but also to intimate knowledge regarding the sequence determinants of membrane-association propensities. Here we turned this knowledge into a web server called ReSMAP, for predicting the residue-specific membrane-association propensities from IDR sequences. The membrane-association propensities are calculated from a sequence-based partition function, trained on the MD simulation results of seven IDRs. Robustness of the prediction is demonstrated by leaving one IDR out of the training set. We anticipate there will be many applications for the ReSMAP web server, including rapid screening of IDR sequences for membrane association.

Keywords:  amphipathic helix; intrinsically disordered proteins; intrinsically disordered regions; membrane binding; membrane-association propensity

DOI:  https://doi.org/10.3390/membranes12080773
Biomolecules. 2022 Aug 10. pii: 1098. [Epub ahead of print]12(8):

Low Complexity Induces Structure in Protein Regions Predicted as Intrinsically Disordered.

Mariane Gonçalves-Kulik, Pablo Mier, Kristina Kastano, Juan Cortés, Pau Bernadó, Friederike Schmid, Miguel A Andrade-Navarro.

  There is increasing evidence that many intrinsically disordered regions (IDRs) in proteins play key functional roles through interactions with other proteins or nucleic acids. These interactions often exhibit a context-dependent structural behavior. We hypothesize that low complexity regions (LCRs), often found within IDRs, could have a role in inducing local structure in IDRs. To test this, we predicted IDRs in the human proteome and analyzed their structures or those of homologous sequences in the Protein Data Bank (PDB). We then identified two types of simple LCRs within IDRs: regions with only one (polyX or homorepeats) or with only two types of amino acids (polyXY). We were able to assign structural information from the PDB more often to these LCRs than to the surrounding IDRs (polyX 61.8% > polyXY 50.5% > IDRs 39.7%). The most frequently observed polyX and polyXY within IDRs contained E (Glu) or G (Gly). Structural analyses of these sequences and of homologs indicate that polyEK regions induce helical conformations, while the other most frequent LCRs induce coil structures. Our work proposes bioinformatics methods to help in the study of the structural behavior of IDRs and provides a solid basis suggesting a structuring role of LCRs within them.

Keywords:  homorepeats; intrinsically disordered regions; low complexity regions; protein structure

DOI:  https://doi.org/10.3390/biom12081098
Biomolecules. 2022 Aug 17. pii: 1131. [Epub ahead of print]12(8):

An Interpretable Machine-Learning Algorithm to Predict Disordered Protein Phase Separation Based on Biophysical Interactions.

Hao Cai, Robert M Vernon, Julie D Forman-Kay.

  Protein phase separation is increasingly understood to be an important mechanism of biological organization and biomaterial formation. Intrinsically disordered protein regions (IDRs) are often significant drivers of protein phase separation. A number of protein phase-separation-prediction algorithms are available, with many being specific for particular classes of proteins and others providing results that are not amenable to the interpretation of the contributing biophysical interactions. Here, we describe LLPhyScore, a new predictor of IDR-driven phase separation, based on a broad set of physical interactions or features. LLPhyScore uses sequence-based statistics from the RCSB PDB database of folded structures for these interactions, and is trained on a manually curated set of phase-separation-driving proteins with different negative training sets including the PDB and human proteome. Competitive training for a variety of physical chemical interactions shows the greatest contribution of solvent contacts, disorder, hydrogen bonds, pi-pi contacts, and kinked beta-structures to the score, with electrostatics, cation-pi contacts, and the absence of a helical secondary structure also contributing. LLPhyScore has strong phase-separation-prediction recall statistics and enables a breakdown of the contribution from each physical feature to a sequence's phase-separation propensity, while recognizing the interdependence of many of these features. The tool should be a valuable resource for guiding experiments and providing hypotheses for protein function in normal and pathological states, as well as for understanding how specificity emerges in defining individual biomolecular condensates.

Keywords:  biomolecular condensates; intrinsically disordered proteins; machine learning; phase separation; physical interactions; predictor

DOI:  https://doi.org/10.3390/biom12081131
J Am Chem Soc. 2022 Aug 26.

Peptidyl Prolyl Isomerase A Modulates the Liquid-Liquid Phase Separation of Proline-Rich IDPs.

Maria Babu, Filippo Favretto, Marija Rankovic, Markus Zweckstetter.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) and the action of molecular chaperones are tightly connected. An important class of molecular chaperones are peptidyl prolyl isomerases, which enhance the cis/trans-isomerization of proline. However, little is known about the impact of peptidyl prolyl isomerases on the LLPS of IDPs, which often contain many prolines. Here, we demonstrate that the most ubiquitous peptidyl prolyl isomerase, peptidyl prolyl isomerase A (PPIA), concentrates inside liquid-like droplets formed by the Alzheimer's disease-associated protein tau, as well as inside RNA-induced coacervates of a proline-arginine dipeptide repeat protein. We further show that the recruitment of PPIA into the IDP droplets triggers their dissolution and return to a single mixed phase. NMR-based binding and proline isomerization studies provide insights into the mechanism of LLPS modulation. Together, the results establish a regulatory role of proline isomerases on the liquid-liquid phase separation of proline-rich IDPs.

DOI: https://doi.org/10.1021/jacs.2c07149
Biophys J. 2022 Aug 24. pii: S0006-3495(22)00688-9. [Epub ahead of print]

Molecular Mechanisms of Steric Pressure Generation and Membrane Remodeling by Intrinsically Disordered Proteins.

Justin R Houser, Hyun Woo Cho, Carl C Hayden, Noel X Yang, Liping Wang, Eileen M Lafer, D Thirumalai, Jeanne C Stachowiak.

Cellular membranes, which are densely crowded by proteins, take on an elaborate array of highly curved shapes. Steric pressure generated by protein crowding plays a significant role in shaping membrane surfaces. It is increasingly clear that many proteins involved in membrane remodeling contain substantial regions of intrinsic disorder. These domains have large hydrodynamic radii, suggesting that they may contribute significantly to steric congestion on membrane surfaces. However, it has been unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. To address this gap, we use a recently developed sensor based on Förster resonance energy transfer to measure steric pressure generated at membrane surfaces by the intrinsically disordered domain of the endocytic protein, AP180. We find that disordered domains generate substantial steric pressure that arises from both entropic and electrostatic components. Interestingly, this steric pressure is largely invariant with the molecular weight of the disordered domain, provided that coverage of the membrane surface is held constant. Moreover, equivalent levels of steric pressure result in equivalent degrees of membrane remodeling, regardless of protein molecular weight. This result, which is consistent with classical polymer scaling relationships for semi-dilute solutions, helps to explain the molecular and physical origins of steric pressure generation by intrinsically disordered domains. From a physiological perspective, these findings suggest that a broad range of membrane-associated disordered domains are likely to play a significant and previously unknown role in controlling membrane shape.

DOI: https://doi.org/10.1016/j.bpj.2022.08.028
J Biomol NMR. 2022 Aug 26.

Measurement of 1Hα transverse relaxation rates in proteins: application to solvent PREs.

Yuki Toyama, Atul Kaushik Rangadurai, Lewis E Kay.

  It has recently been demonstrated that accurate near surface electrostatic potentials can be calculated for proteins from solvent paramagnetic relaxation enhancements (PREs) of amide protons measured using spin labels of similar structures but different charges (Yu et al. in Proc Natl Acad Sci 118(25):e2104020118, 2021). Here we develop methodology for extending such measurements to intrinsically disordered proteins at neutral pH where amide spectra are of very poor quality. Under these conditions it is shown that accurate PRE values can be measured using the haCONHA experiment that has been modified for recording 1Hα transverse relaxation rates. The optimal pulse scheme includes a spin-lock relaxation element for suppression of homonuclear scalar coupled evolution for all 1Hα protons, except those derived from Ser and Thr residues, and minimizes the radiation damping field from water magnetization that would otherwise increase measured relaxation rates. The robustness of the experiment is verified by developing a second approach using a band selective adiabatic decoupling scheme for suppression of scalar coupling modulations during 1Hα relaxation and showing that the measured PRE values from the two methods are in excellent agreement. The near surface electrostatic potential of a 103-residue construct comprising the C-terminal intrinsically disordered region of the RNA-binding protein CAPRIN1 is obtained at pH 5.5 using both 1HN and 1Hα-based relaxation rates, and at pH 7.4 where only 1Hα rates can be quantified, with very good agreement between potentials obtained under all experimental conditions.

Keywords:  1H relaxation; CAPRIN1; Electrostatic potential; Intrinsically disordered proteins; Scalar coupled modulation

DOI:  https://doi.org/10.1007/s10858-022-00401-4
Adv Exp Med Biol. 2022 Aug 23.

HSF1, Aging, and Neurodegeneration.

Alice Y Liu, Conceição A Minetti, David P Remeta, Kenneth J Breslauer, Kuang Yu Chen.

  Heat shock factor 1 (HSF1) is a master transcription regulator that mediates the induction of heat shock protein chaperones for quality control (QC) of the proteome and maintenance of proteostasis as a protective mechanism in response to stress. Research in this particular area has accelerated dramatically over the past three decades following successful isolation, cloning, and characterization of HSF1. The intricate multi-protein complexes and transcriptional activation orchestrated by HSF1 are fundamental processes within the cellular QC machinery. Our primary focus is on the regulation and function of HSF1 in aging and neurodegenerative diseases (ND) which represent physiological and pathological states of dysfunction in protein QC. This chapter presents an overview of HSF1 structural, functional, and energetic properties in healthy cells while addressing the deterioration of HSF1 function viz-à-viz age-dependent and neuron-specific vulnerability to ND. We discuss the structural domains of HSF1 with emphasis on the intrinsically disordered regions and note that disease proteins associated with ND are often structurally disordered and exquisitely sensitive to changes in cellular environment as may occur during aging. We propose a hypothesis that age-dependent changes of the intrinsically disordered proteome likely hold answers to understand many of the functional, structural, and organizational changes of proteins and signaling pathways in aging - dysfunction of HSF1 and accumulation of disease protein aggregates in ND included.Structured AbstractsIntroduction: Heat shock factor 1 (HSF1) is a master transcription regulator that mediates the induction of heat shock protein chaperones for quality control (QC) of the proteome as a cyto-protective mechanism in response to stress. There is cumulative evidence of age-related deterioration of this QC mechanism that contributes to disease vulnerability.
OBJECTIVES: Herein we discuss the regulation and function of HSF1 as they relate to the pathophysiological changes of protein quality control in aging and neurodegenerative diseases (ND).
METHODS: We present an overview of HSF1 structural, functional, and energetic properties in healthy cells while addressing the deterioration of HSF1 function vis-à-vis age-dependent and neuron-specific vulnerability to neurodegenerative diseases.
RESULTS: We examine the impact of intrinsically disordered regions on the function of HSF1 and note that proteins associated with neurodegeneration are natively unstructured and exquisitely sensitive to changes in cellular environment as may occur during aging.
CONCLUSIONS: We put forth a hypothesis that age-dependent changes of the intrinsically disordered proteome hold answers to understanding many of the functional, structural, and organizational changes of proteins - dysfunction of HSF1 in aging and appearance of disease protein aggregates in neurodegenerative diseases included.

Keywords:  Aging; HSF1, Heat shock factor 1; HSP, heat shock protein family; Hsp, Specific heat shock protein; Intrinsically disordered proteome; Neurodegeneration; Protein homeostasis

DOI:  https://doi.org/10.1007/5584_2022_733
Int J Biol Macromol. 2022 Aug 18. pii: S0141-8130(22)01803-7. [Epub ahead of print]220 743-753

Correlating multi-functional role of cold shock domain proteins with intrinsically disordered regions.

Amit Chaudhary, Pankaj Kumar Chaurasia, Sandeep Kushwaha, Pallavi Chauhan, Aakash Chawade, Ashutosh Mani.

  Cold shock proteins (CSPs) are an ancient and conserved family of proteins. They are renowned for their role in response to low-temperature stress in bacteria and nucleic acid binding activities. In prokaryotes, cold and non-cold inducible CSPs are involved in various cellular and metabolic processes such as growth and development, osmotic oxidation, starvation, stress tolerance, and host cell invasion. In prokaryotes, cold shock condition reduces cell transcription and translation efficiency. Eukaryotic cold shock domain (CSD) proteins are evolved form of prokaryotic CSPs where CSD is flanked by N- and C-terminal domains. Eukaryotic CSPs are multi-functional proteins. CSPs also act as nucleic acid chaperons by preventing the formation of secondary structures in mRNA at low temperatures. In human, CSD proteins play a crucial role in the progression of breast cancer, colon cancer, lung cancer, and Alzheimer's disease. A well-defined three-dimensional structure of intrinsically disordered regions of CSPs family members is still undetermined. In this article, intrinsic disorder regions of CSPs have been explored systematically to understand the pleiotropic role of the cold shock family of proteins.

Keywords:  CSPs; Cold shock domain; Cold shock protein; Intrinsically disordered regions; Stress protein

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.08.100
Biomolecules. 2022 Jul 28. pii: 1047. [Epub ahead of print]12(8):

A Trajectory of Discovery: Metabolic Regulation by the Conditionally Disordered Chloroplast Protein, CP12.

Cassy Gérard, Frédéric Carrière, Véronique Receveur-Bréchot, Hélène Launay, Brigitte Gontero.

  The chloroplast protein CP12, which is widespread in photosynthetic organisms, belongs to the intrinsically disordered proteins family. This small protein (80 amino acid residues long) presents a bias in its composition; it is enriched in charged amino acids, has a small number of hydrophobic residues, and has a high proportion of disorder-promoting residues. More precisely, CP12 is a conditionally disordered proteins (CDP) dependent upon the redox state of its four cysteine residues. During the day, reducing conditions prevail in the chloroplast, and CP12 is fully disordered. Under oxidizing conditions (night), its cysteine residues form two disulfide bridges that confer some stability to some structural elements. Like many CDPs, CP12 plays key roles, and its redox-dependent conditional disorder is important for the main function of CP12: the dark/light regulation of the Calvin-Benson-Bassham (CBB) cycle responsible for CO2 assimilation. Oxidized CP12 binds to glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase and thereby inhibits their activity. However, recent studies reveal that CP12 may have other functions beyond the CBB cycle regulation. In this review, we report the discovery of this protein, its features as a disordered protein, and the many functions this small protein can have.

Keywords:  Calvin-Benson-Bassham cycle; conditionally disordered protein; history of modern science; metabolism regulation; moonlighting protein; protein-protein interaction

DOI:  https://doi.org/10.3390/biom12081047
J Chem Inf Model. 2022 Aug 22.

Markov State Models and Molecular Dynamics Simulations Reveal the Conformational Transition of the Intrinsically Disordered Hypervariable Region of K-Ras4B to the Ordered Conformation.

Hao Zhang, Duan Ni, Jigang Fan, Minyu Li, Jian Zhang, Chen Hua, Ruth Nussinov, Shaoyong Lu.

K-Ras4B, the most frequently mutated Ras isoform in human tumors, plays a vital part in cell growth, differentiation, and survival. Its tail, the C-terminal hypervariable region (HVR), is involved in anchoring K-Ras4B at the cellular plasma membrane and in isoform-specific protein-protein interactions and signaling. In the inactive guanosine diphosphate-bound state, the intrinsically disordered HVR interacts with the catalytic domain at the effector-binding region, rendering K-Ras4B in its autoinhibited state. Activation releases the HVR from the catalytic domain, with its ensemble favoring an ordered α-helical structure. The large-scale conformational transition of the HVR from the intrinsically disordered to the ordered conformation remains poorly understood. Here, we deploy a computational scheme that integrates a transition path-generation algorithm, extensive molecular dynamics simulation, and Markov state model analysis to investigate the conformational landscape of the HVR transition pathway. Our findings reveal a stepwise pathway for the HVR transition and uncover several key conformational substates along the transition pathway. Importantly, key interactions between the HVR and the catalytic domain are unraveled, highlighting the pathogenesis of K-Ras4B mild mutations in several congenital developmental anomaly syndromes. Together, these findings provide a deeper understanding of the HVR transition mechanism and the regulation of K-Ras4B activity at an atomic level.

DOI: https://doi.org/10.1021/acs.jcim.2c00591
Cell Rep. 2022 Aug 23. pii: S2211-1247(22)01069-5. [Epub ahead of print]40(8): 111251

Scaffolding viral protein NC nucleates phase separation of the HIV-1 biomolecular condensate.

Anne Monette, Meijuan Niu, Maya Nijhoff Asser, Robert J Gorelick, Andrew J Mouland.

  Membraneless biomolecular condensates (BMCs) contribute to the replication of a growing number of viruses but remain to be functionally characterized. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) proteins phase separated into condensates regulating virus assembly. Here we discover that intrinsically disordered human immunodeficiency virus-type 1 (HIV-1) core proteins condense with the viral genomic RNA (vRNA) to assemble as BMCs attaining a geometry characteristic of viral reverse transcription complexes. We explore the predisposition, mechanisms, and pharmacologic sensitivity of HIV-1 core BMCs in living cells. HIV-1 vRNA-interacting NC condensates were found to be scaffolds onto which client capsid, reverse transcriptase, and integrase condensates assemble. HIV-1 core BMCs exhibit fundamental characteristics of BMCs and are drug-sensitive. Lastly, protease-mediated maturation of Gag and Gag-Pol precursor proteins yield abundant and visible BMCs in cells. This study redefines HIV-1 core components as fluid BMCs and advances our understanding of the nature of viral cores during ingress.

Keywords:  anti-retroviral drugs; capsid; cytoskeleton; human immunodeficiency virus-type 1; integrase; liquid-liquid phase separation; nucleocapsid; protease; reverse transcriptase; viral genomic RNA; virus-engineered biomolecular condensates

DOI:  https://doi.org/10.1016/j.celrep.2022.111251
Cereb Cortex. 2022 Aug 20. pii: bhac313. [Epub ahead of print]

The ZSWIM8 ubiquitin ligase regulates neurodevelopment by guarding the protein quality of intrinsically disordered Dab1.

Guan Wang, Jing Lei, Yifeng Wang, Jiahui Yu, Yinghui He, Weiqi Zhao, Zhechun Hu, Zhenzhong Xu, Yishi Jin, Yan Gu, Xing Guo, Bing Yang, Zhihua Gao, Zhiping Wang.

  Protein quality control (PQC) is essential for maintaining protein homeostasis and guarding the accuracy of neurodevelopment. Previously, we found that a conserved EBAX-type CRL regulates the protein quality of SAX-3/ROBO guidance receptors in Caenorhabditis elegans. Here, we report that ZSWIM8, the mammalian homolog of EBAX-1, is essential for developmental stability of mammalian brains. Conditional deletion of Zswim8 in the embryonic nervous system causes global cellular stress, partial perinatal lethality and defective migration of neural progenitor cells. CRISPR-mediated knockout of ZSWIM8 impairs spine formation and synaptogenesis in hippocampal neurons. Mechanistic studies reveal that ZSWIM8 controls protein quality of Disabled 1 (Dab1), a key signal molecule for brain development, thus protecting the signaling strength of Dab1. As a ubiquitin ligase enriched with intrinsically disordered regions (IDRs), ZSWIM8 specifically recognizes IDRs of Dab1 through a "disorder targets misorder" mechanism and eliminates misfolded Dab1 that cannot be properly phosphorylated. Adult survivors of ZSWIM8 CKO show permanent hippocampal abnormality and display severely impaired learning and memory behaviors. Altogether, our results demonstrate that ZSWIM8-mediated PQC is critical for the stability of mammalian brain development.

Keywords:  Cullin-RING E3 ubiquitin ligase; Reelin and Dab1 signaling; brain development; intrinsically disordered region; protein quality control

DOI:  https://doi.org/10.1093/cercor/bhac313
J Phys Chem B. 2022 Aug 25.

Molecular Dynamics Investigation into pH Dependent Metal Binding of the Intrinsically Disordered Worm Jaw Protein, Nvjp-1.

Selemon Bekele, Kristi Singh, Evan Helton, Sanaz Farajollahi, Rajesh R Naik, Patrick Dennis, Nancy Kelley-Loughnane, Rajiv Berry.

Sclerotization of the Nereis virens jaw is mediated by metal binding to the histidine-rich jaw protein, Nvjp-1. Previous studies showed that the mechanical properties of Nvjp-1 hydrogels could be modulated with zinc binding as well as the associated anion. Here, we show that the mechanical properties of Nvjp-1 hydrogels can be modulated by pH and that zinc binding to Nvjp-1 is stable at both acidic and alkaline pH conditions. To probe the mechanism of Zn2+ binding to Nvjp-1 at different pH conditions, we utilized all atom molecular dynamics simulations employing a polarizable force field. At low pH conditions, polar residues predominantly interacted with Zn2+, with at most two residues interacting with a given zinc ion. Surprisingly, little to no Zn2+ binding was observed with the abundant Nvjp-1 acidic residues, which form salt-bridges with the protonated histidines to effectively block their binding to Zn2+ ions. As the pH was shifted to alkaline conditions, Zn2+ binding residues reconfigured to form additional coordination bonds with histidine, resulting in a reduction in the radius of gyration that correlated with hydrogel sclerotization. Furthermore, acetate ions were shown to facilitate the capture of zinc ions through association with protonated histidines at low pH, freeing acidic residues to interact with Zn2+ ions and increasing the number of Zn2+ ions that diffuse into the Nvjp-1 interior. Thus, these studies provide valuable molecular insights into how amino acid residues in Nvjp-1 manage metal salt binding and coordination in hydrogels as a function of the pH and ionic environments.

DOI: https://doi.org/10.1021/acs.jpcb.2c02807
Plants (Basel). 2022 Aug 18. pii: 2149. [Epub ahead of print]11(16):

New Insights into Phase Separation Processes and Membraneless Condensates of EIN2.

Jian Lu, Chi-Kuang Wen, Georg Groth.

  Recent technological advances allow us to resolve molecular processes in living cells with high spatial and temporal resolution. Based on these technological advances, membraneless intracellular condensates formed by reversible functional aggregation and phase separation have been identified as important regulatory modules in diverse biological processes. Here, we present bioinformatic and cellular studies highlighting the possibility of the involvement of the central activator of ethylene responses EIN2 in such cellular condensates and phase separation processes. Our work provides insight into the molecular type (identity) of the observed EIN2 condensates and on potential intrinsic elements and sequence motifs in EIN2-C that may regulate condensate formation and dynamics.

Keywords:  EIN2; ethylene signaling; intrinsically disordered proteins; liquid–liquid phase separation; membraneless condensates

DOI:  https://doi.org/10.3390/plants11162149
Biomedicines. 2022 Aug 21. pii: 2044. [Epub ahead of print]10(8):

Identification of Germinal Neurofibromin Hotspots.

Sergio Lois, Juan Báez-Flores, María Isidoro-García, Jesus Lacal, Juan Carlos Triviño.

  Neurofibromin is engaged in many cellular processes and when the proper protein functioning is impaired, it causes neurofibromatosis type 1 (NF1), one of the most common inherited neurological disorders. Recent advances in sequencing and screening of the NF1 gene have increased the number of detected variants. However, the correlation of these variants with the clinic remains poorly understood. In this study, we analyzed 4610 germinal NF1 variants annotated in ClinVar and determined on exon level the mutational spectrum and potential pathogenic regions. Then, a binomial and sliding windows test using 783 benign and 938 pathogenic NF1 variants were analyzed against functional and structural regions of neurofibromin. The distribution of synonymous, missense, and frameshift variants are statistically significant in certain regions of neurofibromin suggesting that the type of variant and its associated phenotype may depend on protein disorder. Indeed, there is a negative correlation between the pathogenic fraction prediction and the disorder data, suggesting that the higher an intrinsically disordered region is, the lower the pathogenic fraction is and vice versa. Most pathogenic variants are associated to NF1 and our analysis suggests that GRD, CSRD, TBD, and Armadillo1 domains are hotspots in neurofibromin. Knowledge about NF1 genotype-phenotype correlations can provide prognostic guidance and aid in organ-specific surveillance.

Keywords:  NF1; germinal variants; neurofibromatosis type 1; neurofibromin; pathogenic variants

DOI:  https://doi.org/10.3390/biomedicines10082044
Pharmacol Res. 2022 Aug 19. pii: S1043-6618(22)00354-1. [Epub ahead of print] 106409

Catechol-Containing Compounds are a Broad Class of Protein Aggregation Inhibitors: Redox State is a Key Determinant of the Inhibitory Activities.

Paul Velander, Ling Wu, Sherry B Hildreth, Nancy J Vogelaar, Biswarup Mukhopadhyay, Richard F Helm, Shijun Zhang, Bin Xu.

  A range of neurodegenerative and related aging diseases, such as Alzheimer's disease and type 2 diabetes, are linked to toxic protein aggregation. Yet the mechanisms of protein aggregation inhibition by small molecule inhibitors remain poorly understood, in part because most protein targets of aggregation assembly are partially unfolded or intrinsically disordered, which hinders detailed structural characterization of protein-inhibitor complexes and structural-based inhibitor design. Herein we employed a parallel small molecule library-screening approach to identify inhibitors against three prototype amyloidogenic proteins in neurodegeneration and related proteinopathies: amylin, Aβ and tau. One remarkable class of inhibitors identified from these screens against different amyloidogenic proteins was catechol-containing compounds and redox-related quinones/anthraquinones. Secondary assays validated most of the identified inhibitors. In vivo efficacy evaluation of a selected catechol-containing compound, rosmarinic acid, demonstrated its strong mitigating effects of amylin amyloid deposition and related diabetic pathology in transgenic HIP rats. Further systematic investigation of selected class of inhibitors under aerobic and anaerobic conditions revealed that the redox state of the broad class of catechol-containing compounds is a key determinant of the amyloid inhibitor activities. The molecular insights we gained not only explain why a large number of catechol-containing polyphenolic natural compounds, often enriched in healthy diet, have anti-neurodegeneration and anti-aging activities, but also could guide the rational design of therapeutic or nutraceutical strategies to target a broad range of neurodegenerative and related aging diseases.

Keywords:  HIP rats; Protein misfolding diseases; aggregation modifying inhibitors; catechol-containing compounds; drug library screening; redox regulation

DOI:  https://doi.org/10.1016/j.phrs.2022.106409
Int J Biol Macromol. 2022 Aug 23. pii: S0141-8130(22)01835-9. [Epub ahead of print]220 703-720

Aberrant liquid-liquid phase separation and amyloid aggregation of proteins related to neurodegenerative diseases.

Azeem Ahmad, Vladimir N Uversky, Rizwan Hasan Khan.

  Recent evidence has shown that the processes of liquid-liquid phase separation (LLPS) or liquid-liquid phase transitions (LLPTs) are a crucial and prevalent phenomenon that underlies the biogenesis of numerous membrane-less organelles (MLOs) and biomolecular condensates within the cells. Findings show that processes associated with LLPS play an essential role in physiology and disease. In this review, we discuss the physical and biomolecular factors that contribute to the development of LLPS, the associated functions, as well as their consequences for cell physiology and neurological disorders. Additionally, the finding of mis-regulated proteins, which have long been linked to aggregates in neuropathology, are also known to induce LLPS/LLPTs, prompting a lot of interest in understanding the connection between aberrant phase separation and disorder conditions. Moreover, the methods used in recent and ongoing studies in this field are also explored, as is the possibility that these findings will encourage new lines of inquiry into the molecular causes of neurodegenerative diseases.

Keywords:  Biomolecular condensates; Liquid-liquid phase separation; Liquid-solid transition; Membrane-less organelles

DOI:  https://doi.org/10.1016/j.ijbiomac.2022.08.132
ACS Chem Neurosci. 2022 Aug 25.

Hyperphosphorylation-Mimetic TDP-43 Drives Amyloid Formation and Possesses Neuronal Toxicity at the Oligomeric Stage.

Wan-Chin Chiang, Yu-Sheng Fang, Yuh Shen Lye, Tzu-Yu Weng, Kiruthika Ganesan, Shih-Han Huang, Lan-Yun Chang, Shih-Chieh Chou, Yun-Ru Chen.

  TDP-43 proteinopathies cover a range of neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Hyperphosphorylated TDP-43 was found within the inclusion bodies in disease lesions; however, the role of hyperphosphorylation and the toxic species are still ambiguous. To characterize the hyperphosphorylation effect of TDP-43, here, we employed five serine mutations implicated in the diseases at serine locations 379, 403, 404, 409, and 410 in the C-terminus to aspartate (S5D) and to alanine (S5A). We systematically characterized the conformation, liquid-liquid phase separation, oligomerization, and fibrillization of TDP-43 variants. Results revealed that the recombinant TDP-43 variants readily formed structurally similar spherical oligomers, as evidenced by circular dichroism spectroscopy, fluorescence spectroscopy, the TDP-43 oligomer-specific antibody assay, dynamic light scattering, and transmission electron microscopy. After incubation, only the phosphor-mimic S5D TDP-43 formed thioflavin-positive amyloid fibrils, whereas wild-type and S5A TDP-43 formed amorphous aggregates. We also examined membrane disruption, the cytotoxicity of human neuroblastoma, and the synaptic loss of primary neurons induced by oligomers and large aggregates of TDP-43. The results showed that all oligomeric TDP-43 variants were toxic regardless of hyperphosphorylation, but the fibrils and amorphous aggregates were not. Overall, our results demonstrated the hyperphosphorylation effect on fibril formation and the toxicity attributed from TDP-43 oligomers. This study facilitates the understanding and therapeutic development for TDP-43 proteinopathies.

Keywords:  TDP-43; amyloid; fibril; hyperphosphorylation; neurodegenerative disease; oligomer; toxicity

DOI:  https://doi.org/10.1021/acschemneuro.1c00873
ACS Chem Neurosci. 2022 Aug 24.

Mislocalization of Nup62 Contributes to TDP-43 Proteinopathy in ALS/FTLD.

Niharika Nag, Timir Tripathi.

  The nucleocytoplasmic transport (NCT) is impaired in C9-ALS/FTLD, a common genetically caused form of ALS and FTLD. The NCT is regulated by proteins called FG-nucleoporins (FG-Nups), with domains enriched in phenylalanine-glycine repeats. However, the relationship between FG-Nups and TDP-43, an RBP found to be mislocalized in ALS/FTLD patients, has not been defined. A recent study found that a critical protein, FG-Nup62, is mislocalized both in vivo and in vitro in diseased states. The mislocalized Nup62 was colocalized with TDP-43 in cytoplasmic inclusions and promoted its liquid-to-solid transition. The work highlights the involvement of Nup62 in the pathogenesis of ALS/FTLD and the interaction between Nup62 and TDP-43.

Keywords:  ALS/FTLD; FG-Nups; Nucleocytoplasmic transport; Nup62; TDP-43; TDP-43 condensates; amyotrophic lateral sclerosis; proteinopathy

DOI:  https://doi.org/10.1021/acschemneuro.2c00480