24-h continuous non-invasive multiparameter home monitoring of vitals in patients with Rett syndrome by an innovative wearable technology: evidence of an overlooked chronic fatigue status.

Background: Sleep is disturbed in Rett syndrome (RTT), a rare and progressive neurodevelopmental disorder primarily affecting female patients (prevalence 7.1/100,000 female patients) linked to pathogenic variations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Autonomic nervous system dysfunction with a predominance of the sympathetic nervous system (SNS) over the parasympathetic nervous system (PSNS) is reported in RTT, along with exercise fatigue and increased sudden death risk. The aim of the present study was to test the feasibility of a continuous 24 h non-invasive home monitoring of the biological vitals (biovitals) by an innovative wearable sensor device in pediatric and adolescent/adult RTT patients. Methods: A total of 10 female patients (mean age 18.3 ± 9.4 years, range 4.7-35.5 years) with typical RTT and MECP2 pathogenic variations were enrolled. Clinical severity was assessed by validated scales. Heart rate (HR), respiratory rate (RR), and skin temperature (SkT) were monitored by the YouCare Wearable Medical Device (Accyourate Group SpA, L'Aquila, Italy). The average percentage of maximum HR (HRmax%) was calculated. Heart rate variability (HRV) was expressed by consolidated time-domain and frequency-domain parameters. The HR/LF (low frequency) ratio, indicating SNS activation under dynamic exercise, was calculated. Simultaneous continuous measurement of indoor air quality variables was performed and the patients' contributions to the surrounding water vapor partial pressure [PH2O (pt)] and carbon dioxide [PCO2 (pt)] were indirectly estimated. Results: Of the 6,559.79 h of biovital recordings, 5051.03 h (77%) were valid for data interpretation. Sleep and wake hours were 9.0 ± 1.1 h and 14.9 ± 1.1 h, respectively. HRmax % [median: 71.86% (interquartile range 61.03-82%)] and HR/LF [median: 3.75 (interquartile range 3.19-5.05)] were elevated, independent from the wake-sleep cycle. The majority of HRV time- and frequency-domain parameters were significantly higher in the pediatric patients (p ≤ 0.031). The HRV HR/LF ratio was associated with phenotype severity, disease progression, clinical sleep disorder, subclinical hypoxia, and electroencephalographic observations of multifocal epileptic activity and general background slowing. Conclusion: Our findings indicate the feasibility of a continuous 24-h non-invasive home monitoring of biovital parameters in RTT. Moreover, for the first time, HRmax% and the HR/LF ratio were identified as potential objective markers of fatigue, illness severity, and disease progression.

Structural Insights into the Activation of Human Aryl Hydrocarbon Receptor by the Environmental Contaminant Benzo[a]pyrene and Structurally Related Compounds.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor belonging to the bHLH/PAS protein family and responding to hundreds of natural and chemical substances. It is primarily involved in the defense against chemical insults and bacterial infections or in the adaptive immune response, but also in the development of pathological conditions ranging from inflammatory to neoplastic disorders. Despite its prominent roles in many (patho)physiological processes, the lack of high-resolution structural data has precluded for thirty years an in-depth understanding of the structural mechanisms underlying ligand-binding specificity, promiscuity and activation of AHR. We recently reported a cryogenic electron microscopy (cryo-EM) structure of human AHR bound to the natural ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2 that provided the first experimental visualization of its ligand-binding PAS-B domain. Here, we report a 2.75 Å resolution structure of the AHR complex bound to the environmental pollutant benzo[a]pyrene (B[a]P). The structure substantiates the existence of a bipartite PAS-B ligand-binding pocket with a geometrically constrained primary binding site controlling ligand binding specificity and affinity, and a secondary binding site contributing to the binding promiscuity of AHR. We also report a docking study of B[a]P congeners that validates the B[a]P-bound PAS-B structure as a suitable model for accurate computational ligand binding assessment. Finally, comparison of our agonist-bound complex with the recently reported structures of mouse and fruit fly AHR PAS-B in different activation states suggests a ligand-induced loop conformational change potentially involved in the regulation of AHR function.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation.

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins.

The 70-kD heat shock protein (Hsp70) chaperone system is a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform different and specific cellular functions is regulated by the J-domain protein (JDP) co-chaperone family carrying the small namesake J-domain, required to interact and drive the ATPase cycle of Hsp70s. Besides the J-domain, prokaryotic and eukaryotic JDPs display a staggering diversity in domain architecture, function, and cellular localization. Very little is known about the overall JDP family, despite their essential role in cellular proteostasis, development, and its link to a broad range of human diseases. In this work, we leverage the exponentially increasing number of JDP gene sequences identified across all kingdoms owing to the advancements in sequencing technology and provide a broad overview of the JDP repertoire. Using an automated classification scheme based on artificial neural networks (ANNs), we demonstrate that the sequences of J-domains carry sufficient discriminatory information to reliably recover the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.

Compartmentalization of the SUMO/RNF4 pathway by SLX4 drives DNA repair.

SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.

The structure of pathogenic huntingtin exon 1 defines the bases of its aggregation propensity.

Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.

Nucleation of Biomolecular Condensates from Finite-Sized Simulations.

The nucleation of protein condensates is a concentration-driven process of assembly. When modeled in the canonical ensemble, condensation is affected by finite-size effects. Here, we present a general and efficient route for obtaining ensemble properties of protein condensates in the macroscopic limit from finite-sized nucleation simulations. The approach is based on a theoretical description of droplet nucleation in the canonical ensemble and enables estimation of thermodynamic and kinetic parameters, such as the macroscopic equilibrium density of the dilute protein phase, the surface tension of the condensates, and nucleation free energy barriers. We apply the method to coarse-grained simulations of NDDX4 and FUS-LC, two phase-separating disordered proteins with different physicochemical characteristics. Our results show that NDDX4 condensate droplets, characterized by lower surface tension, higher solubility, and faster monomer exchange dynamics compared to those of FUS-LC, form with negligible nucleation barriers. In contrast, FUS-LC condensates form via an activated process over a wide range of concentrations.

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR cytosolic complex.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites. However, the scarcity of structural data precludes understanding of how AHR is activated by such diverse compounds. Our 2.85 Å structure of the human indirubin-bound AHR complex with the chaperone Hsp90 and the co-chaperone XAP2, reported herein, reveals a closed conformation Hsp90 dimer with AHR threaded through its lumen and XAP2 serving as a brace. Importantly, we disclose the long-awaited structure of the AHR PAS-B domain revealing a unique organisation of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing structural details of the molecular initiating event leading to AHR activation, our study rationalises almost forty years of biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR.

Large oligomeric enzymes control a myriad of cellular processes, from protein synthesis and degradation to metabolism. The 0.5 MDa large TET2 aminopeptidase, a prototypical protease important for cellular homeostasis, degrades peptides within a ca. 60 Å wide tetrahedral chamber with four lateral openings. The mechanisms of substrate trafficking and processing remain debated. Here, we integrate magic-angle spinning (MAS) NMR, mutagenesis, co-evolution analysis and molecular dynamics simulations and reveal that a loop in the catalytic chamber is a key element for enzymatic function. The loop is able to stabilize ligands in the active site and may additionally have a direct role in activating the catalytic water molecule whereby a conserved histidine plays a key role. Our data provide a strong case for the functional importance of highly dynamic - and often overlooked - parts of an enzyme, and the potential of MAS NMR to investigate their dynamics at atomic resolution.

The diversity of molecular interactions involving intrinsically disordered proteins: A molecular modeling perspective.

Intrinsically Disordered Proteins and Regions (IDPs/IDRs) are key components of a multitude of biological processes. Conformational malleability enables IDPs/IDRs to perform very specialized functions that cannot be accomplished by globular proteins. The functional role for most of these proteins is related to the recognition of other biomolecules to regulate biological processes or as a part of signaling pathways. Depending on the extent of disorder, the number of interacting sites and the type of partner, very different architectures for the resulting assemblies are possible. More recently, molecular condensates with liquid-like properties composed of multiple copies of IDPs and nucleic acids have been proven to regulate key processes in eukaryotic cells. The structural and kinetic details of disordered biomolecular complexes are difficult to unveil experimentally due to their inherent conformational heterogeneity. Computational approaches, alone or in combination with experimental data, have emerged as unavoidable tools to understand the functional mechanisms of this elusive type of assemblies. The level of description used, all-atom or coarse-grained, strongly depends on the size of the molecular systems and on the timescale of the investigated mechanism. In this mini-review, we describe the most relevant architectures found for molecular interactions involving IDPs/IDRs and the computational strategies applied for their investigation.

Arginine multivalency stabilizes protein/RNA condensates.

Biomolecular condensates assembled through liquid-liquid phase separation (LLPS) of proteins and RNAs are currently recognized to play an important role in cellular organization. Their assembly depends on the formation of a network of transient, multivalent interactions between flexible scaffold biomolecules. Understanding how protein and RNA sequences determine these interactions and ultimately regulate the phase separation is an open key challenge. Recent in vitro studies have revealed that arginine and lysine residues, which are enriched in most cellular condensates, have markedly distinct propensities to drive the LLPS of protein/RNA mixtures. Here, we employ explicit-solvent atomistic molecular dynamics simulations to shed light on the microscopic origin of this difference by investigating mixtures of polyU oligonucleotides with either polyR/polyK peptides. In agreement with experiments, our simulations indicate that arginine has a higher affinity for polyU than lysine both in highly diluted conditions and in concentrated solutions with a biomolecular density comparable to cellular condensate. The analysis of intermolecular contacts suggests that this differential behavior is due to the propensity of arginine side chains to simultaneously form a higher number of specific interactions with oligonucleotides, including hydrogen bonds and stacking interactions. Our results provide a molecular description of how the multivalency of the guanidinium group enables the coordination of multiple RNA groups by a single arginine residue, thus ultimately stabilizing protein/RNA condensates.

Molecular dissection of amyloid disaggregation by human HSP70.

The deposition of highly ordered fibrillar-type aggregates into inclusion bodies is a hallmark of neurodegenerative diseases such as Parkinson's disease. The high stability of such amyloid fibril aggregates makes them challenging substrates for the cellular protein quality-control machinery1,2. However, the human HSP70 chaperone and its co-chaperones DNAJB1 and HSP110 can dissolve preformed fibrils of the Parkinson's disease-linked presynaptic protein α-synuclein in vitro3,4. The underlying mechanisms of this unique activity remain poorly understood. Here we use biochemical tools and nuclear magnetic resonance spectroscopy to determine the crucial steps of the disaggregation process of amyloid fibrils. We find that DNAJB1 specifically recognizes the oligomeric form of α-synuclein via multivalent interactions, and selectively targets HSP70 to fibrils. HSP70 and DNAJB1 interact with the fibril through exposed, flexible amino and carboxy termini of α-synuclein rather than the amyloid core itself. The synergistic action of DNAJB1 and HSP110 strongly accelerates disaggregation by facilitating the loading of several HSP70 molecules in a densely packed arrangement at the fibril surface, which is ideal for the generation of 'entropic pulling' forces. The cooperation of DNAJB1 and HSP110 in amyloid disaggregation goes beyond the classical substrate targeting and recycling functions that are attributed to these HSP70 co-chaperones and constitutes an active and essential contribution to the remodelling of the amyloid substrate. These mechanistic insights into the essential prerequisites for amyloid disaggregation may provide a basis for new therapeutic interventions in neurodegeneration.

Unraveling Molecular Interactions in Liquid-Liquid Phase Separation of Disordered Proteins by Atomistic Simulations.

Membraneless organelles are dynamical cellular condensates formed via biomolecular liquid-liquid phase separation of proteins and RNA molecules. Multiple evidence suggests that in several cases disordered proteins are structural scaffolds that drive the condensation by forming a dynamic network of inter- and intramolecular contacts. Despite the blooming research activity in this field, the structural characterization of these entities is very limited, and we still do not understand how the phase behavior is encoded in the amino acid sequences of the scaffolding proteins. Here we exploited explicit-solvent atomistic simulations to investigate the N-terminal disordered region of DEAD-box helicase 4 (NDDX4), which is a well-established model for phase separation. Notably, we determined NDDX4 conformational ensemble at the single-molecule level, and we relied on a "divide-and-conquer" strategy, based on simulations of various protein fragments at high concentration, to probe intermolecular interactions in conditions mimicking real condensates. Our results provide a high-resolution picture of the molecular mechanisms underlying phase separation in agreement with NMR and mutagenesis data and suggest that clusters of arginine and aromatic residues may stabilize the assembly of several condensates.

Coevolutionary Analysis of Protein Subfamilies by Sequence Reweighting.

Extracting structural information from sequence co-variation has become a common computational biology practice in the recent years, mainly due to the availability of large sequence alignments of protein families. However, identifying features that are specific to sub-classes and not shared by all members of the family using sequence-based approaches has remained an elusive problem. We here present a coevolutionary-based method to differentially analyze subfamily specific structural features by a continuous sequence reweighting (SR) approach. We introduce the underlying principles and test its predictive capabilities on the Response Regulator family, whose subfamilies have been previously shown to display distinct, specific homo-dimerization patterns. Our results show that this reweighting scheme is effective in assigning structural features known a priori to subfamilies, even when sequence data is relatively scarce. Furthermore, sequence reweighting allows assessing if individual structural contacts pertain to specific subfamilies and it thus paves the way for the identification specificity-determining contacts from sequence variation data.

Efficient conversion of chemical energy into mechanical work by Hsp70 chaperones.

Hsp70 molecular chaperones are abundant ATP-dependent nanomachines that actively reshape non-native, misfolded proteins and assist a wide variety of essential cellular processes. Here, we combine complementary theoretical approaches to elucidate the structural and thermodynamic details of the chaperone-induced expansion of a substrate protein, with a particular emphasis on the critical role played by ATP hydrolysis. We first determine the conformational free-energy cost of the substrate expansion due to the binding of multiple chaperones using coarse-grained molecular simulations. We then exploit this result to implement a non-equilibrium rate model which estimates the degree of expansion as a function of the free energy provided by ATP hydrolysis. Our results are in quantitative agreement with recent single-molecule FRET experiments and highlight the stark non-equilibrium nature of the process, showing that Hsp70s are optimized to effectively convert chemical energy into mechanical work close to physiological conditions.

Coevolutionary Analysis of Protein Sequences for Molecular Modeling.

Thanks to the explosion of genomic sequencing, coevolutionary analysis of protein sequences has gained great and ever-increasing popularity in the last decade, and it is currently an important and well-established tool in structural bioinformatics and computational biology. This chapter concisely introduces the theoretical foundation and the practical aspects of coevolutionary analysis, as well as discusses the molecular modeling strategies to exploit its results in the study of protein structure, dynamics, and interactions. We present here a complete pipeline from sequence extraction to contact prediction through two examples, focusing on the predictions of inter-residue contacts in a single protein domain and on the analysis of a multi-domain protein that undergoes functional, large-scale conformational transitions.

Interplay of Protein Disorder in Retinoic Acid Receptor Heterodimer and Its Corepressor Regulates Gene Expression.

In its unliganded form, the retinoic acid receptor (RAR) in heterodimer with the retinoid X receptor (RXR) exerts a strong repressive activity facilitated by the recruitment of transcriptional corepressors in the promoter region of target genes. By integrating complementary structural, biophysical, and computational information, we demonstrate that intrinsic disorder is a required feature for the precise regulation of RAR activity. We show that structural dynamics of RAR and RXR H12 regions is an essential mechanism for RAR regulation. Unexpectedly we found that, while mainly disordered, the corepressor N-CoR presents evolutionary conserved structured regions involved in transient intramolecular contacts. In the presence of RXR/RAR, N-CoR exploits its multivalency to form a cooperative multisite complex that displays equilibrium between different conformational states that can be tuned by cognate ligands and receptor mutations. This equilibrium is key to preserving the repressive basal state while allowing the conversion to a transcriptionally active form.

Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific 1H-13C Labeling and Fast Magic-Angle Spinning NMR.

Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize the dynamics of phenylalanine residues using 1H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme. Our approach yields isolated two-spin systems that are ideally suited for artifact-free dynamics measurements, and allows probing motions effectively without molecular weight limitations. The application to the TET2 enzyme assembly of ∼0.5 MDa size, the currently largest protein assigned by MAS NMR, provides insights into motions occurring on a wide range of time scales (picoseconds to milliseconds). We quantitatively probe ring-flip motions and show the temperature dependence by MAS NMR measurements down to 100 K. Interestingly, favorable line widths are observed down to 100 K, with potential implications for DNP NMR. Furthermore, we report the first 13C R1ρ MAS NMR relaxation-dispersion measurements and detect structural excursions occurring on a microsecond time scale in the entry pore to the catalytic chamber and at a trimer interface that was proposed as the exit pore. We show that the labeling scheme with deuteration at ca. 50 kHz MAS provides superior resolution compared to 100 kHz MAS experiments with protonated, uniformly 13C-labeled samples.