Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited evidence of lipid dysmetabolism, autophagy dysregulation and deficient phagocytosis, a canonical microglia function. Mutant PFN1 also displayed enhanced binding affinity for PI3P, a critical signaling molecule involved in autophagic and endocytic processing. Our cumulative data implicate a gain-of-toxic function for mutant PFN1 within the autophagic and endo-lysosomal pathways, as administration of rapamycin rescued phagocytic dysfunction in ALS-PFN1 iMGs. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and implicate microglial vesicular degradation pathways in the pathogenesis of these disorders.

A nematode model to evaluate microdeletion phenotype expression.

Microdeletion syndromes are genetic diseases caused by multilocus chromosomal deletions too small to be detected by karyotyping. They are typified by complex pleiotropic developmental phenotypes that depend both on the extent of the deletion and variations in genetic background. Microdeletion alleles cause a wide array of consequences involving multiple pathways. How simultaneous haploinsufficiency of numerous adjacent genes leads to complex and variable pleiotropic phenotypes is not well understood. CRISPR/Cas9 genome editing has been shown to induce microdeletion-like alleles at a meaningful rate. Here, we describe a microdeletion allele in Caenorhabditis elegans recovered during a CRISPR/Cas9 genome editing experiment. We mapped the allele to chromosome V, balanced it with a reciprocal translocation crossover suppressor, and precisely defined the breakpoint junction. The allele simultaneously removes 32 protein-coding genes, yet animals homozygous for this mutation are viable as adults. Homozygous animals display a complex phenotype including maternal effect lethality, producing polynucleated embryos that grow into uterine tumors, vulva morphogenesis defects, body wall distensions, uncoordinated movement, and a shortened life span typified by death by bursting. Our work provides an opportunity to explore the complexity and penetrance of microdeletion phenotypes in a simple genetic model system.

Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.

Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be fully elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited lipid dysmetabolism and deficits in phagocytosis, a critical microglia function. Our cumulative data implicate an effect of ALS-linked PFN1 on the autophagy pathway, including enhanced binding of mutant PFN1 to the autophagy signaling molecule PI3P, as an underlying cause of defective phagocytosis in ALS-PFN1 iMGs. Indeed, phagocytic processing was restored in ALS-PFN1 iMGs with Rapamycin, an inducer of autophagic flux. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and highlight microglia vesicular degradation pathways as potential therapeutic targets for these disorders.

The Role of Substrate Mediated Allostery in the Catalytic Competency of the Bacterial Oligosaccharyltransferase PglB.

The oligosaccharyltransferase of Campylobacter lari (PglB) catalyzes the glycosylation of asparagine in the consensus sequence N-X-S/T, where X is any residue except proline. Molecular dynamics simulations of PglB bound to two different substrates were used to characterize the differences in the structure and dynamics of the substrate-enzyme complexes that can explain the higher catalytic efficiency observed for substrates containing threonine at the +2 position rather than serine. We observed that a threonine-containing substrate is more tightly bound than a serine-containing substrate. Because serine lacks a methyl group relative to threonine, the serine-containing peptide cannot stably form simultaneous van der Waals interactions with T316 and I572 as the threonine-containing substrate can. As a result, the peptide-PglB interaction is destabilized and the allosteric communication between the periplasmic domain and external loop EL5 is disrupted. These changes ultimately lead to the reorientation of the periplasmic domain relative to the transmembrane domain such that the two domains are further apart compared to PglB bound to the threonine-containing peptide. The crystal structure of PglB bound to the peptide and a lipid-linked oligosaccharide analog shows a pronounced closing of the periplasmic domain over the transmembrane domain in comparison to structures of PglB with peptide only, indicating that a closed conformation of the domains is needed for catalysis. The results of our studies suggest that lower enzymatic activity observed for serine versus threonine results from a combination of less stable binding and structural changes in PglB that influence the ability to form a catalytically competent state. This study illustrates a mechanism for substrate specificity via modulation of dynamic allosteric pathways.

ALS-linked PFN1 variants exhibit loss and gain of functions in the context of formin-induced actin polymerization.

Profilin-1 (PFN1) plays important roles in modulating actin dynamics through binding both monomeric actin and proteins enriched with polyproline motifs. Mutations in PFN1 have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). However, whether ALS-linked mutations affect PFN1 function has remained unclear. To address this question, we employed an unbiased proteomics analysis in mammalian cells to identify proteins that differentially interact with mutant and wild-type (WT) PFN1. These studies uncovered differential binding between two ALS-linked PFN1 variants, G118V and M114T, and select formin proteins. Furthermore, both variants augmented formin-mediated actin assembly relative to PFN1 WT. Molecular dynamics simulations revealed mutation-induced changes in the internal dynamic couplings within an alpha helix of PFN1 that directly contacts both actin and polyproline, as well as structural fluctuations within the actin- and polyproline-binding regions of PFN1. These data indicate that ALS-PFN1 variants have the potential for heightened flexibility in the context of the ternary actin-PFN1-polyproline complex during actin assembly. Conversely, PFN1 C71G was more severely destabilized than the other PFN1 variants, resulting in reduced protein expression in both transfected and ALS patient lymphoblast cell lines. Moreover, this variant exhibited loss-of-function phenotypes in the context of actin assembly. Perturbations in actin dynamics and assembly can therefore result from ALS-linked mutations in PFN1. However, ALS-PFN1 variants may dysregulate actin polymerization through different mechanisms that depend upon the solubility and stability of the mutant protein.

Analysis of Emerging Variants in Structured Regions of the SARS-CoV-2 Genome.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two sets of SARS-CoV-2 genomic sequences to identify emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, 20 variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5' untranslated region (UTR), including a group of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-ss molecular switch in the 3'UTR. Finally, 5 variants destabilize structured elements within the 3'UTR hypervariable region, including the S2M (stem loop 2 m) selfish genetic element, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. Our analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences.

Analysis of Rapidly Emerging Variants in Structured Regions of the SARS-CoV-2 Genome.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has motivated a widespread effort to understand its epidemiology and pathogenic mechanisms. Modern high-throughput sequencing technology has led to the deposition of vast numbers of SARS-CoV-2 genome sequences in curated repositories, which have been useful in mapping the spread of the virus around the globe. They also provide a unique opportunity to observe virus evolution in real time. Here, we evaluate two cohorts of SARS-CoV-2 genomic sequences to identify rapidly emerging variants within structured cis-regulatory elements of the SARS-CoV-2 genome. Overall, twenty variants are present at a minor allele frequency of at least 0.5%. Several enhance the stability of Stem Loop 1 in the 5'UTR, including a set of co-occurring variants that extend its length. One appears to modulate the stability of the frameshifting pseudoknot between ORF1a and ORF1b, and another perturbs a bi-stable molecular switch in the 3'UTR. Finally, five variants destabilize structured elements within the 3'UTR hypervariable region, including the S2M stem loop, raising questions as to the functional relevance of these structures in viral replication. Two of the most abundant variants appear to be caused by RNA editing, suggesting host-viral defense contributes to SARS-CoV-2 genome heterogeneity. This analysis has implications for the development of therapeutics that target viral cis-regulatory RNA structures or sequences, as rapidly emerging variations in these regions could lead to drug resistance.

A Disorder-to-Order Transition Mediates RNA Binding of the Caenorhabditis elegans Protein MEX-5.

CCCH-type tandem zinc finger (TZF) domains are found in many RNA-binding proteins (RBPs) that regulate the essential processes of post-transcriptional gene expression and splicing through direct protein-RNA interactions. In Caenorhabditis elegans, RBPs control the translation, stability, or localization of maternal messenger RNAs required for patterning decisions before zygotic gene activation. MEX-5 (Muscle EXcess) is a C. elegans protein that leads a cascade of RBP localization events that is essential for axis polarization and germline differentiation after fertilization. Here, we report that at room temperature, the CCCH-type TZF domain of MEX-5 contains an unstructured zinc finger that folds upon binding of its RNA target. We have characterized the structure and dynamics of the TZF domain of MEX-5 and designed a variant MEX-5 in which both fingers are fully folded in the absence of RNA. Within the thermal range experienced by C. elegans, the population of the unfolded state of the TZF domain of MEX-5 varies. We observe that the TZF domain becomes less disordered at lower temperatures and more disordered at higher temperatures. However, in the temperature range in which C. elegans is fertile, when MEX-5 needs to be functional, only one of the two zinc fingers is folded.

Networks of electrostatic and hydrophobic interactions modulate the complex folding free energy surface of a designed ßα protein.

The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring ßα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable ßα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.

Characterization of TDP-43 RRM2 Partially Folded States and Their Significance to ALS Pathogenesis.

The human protein TDP-43 is a major component of the cellular aggregates found in amyotrophic lateral sclerosis and other neurodegenerative diseases. Insoluble cytoplasmic aggregates isolated from the brain of amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients contain ubiquitinated, hyperphosphorylated, and N-terminally truncated TDP-43. Truncated fragments of TDP-43 identified from patient tissues contain part of the second RNA recognition motif (RRM2) and the disordered C-terminus, indicating that both domains can be involved in aggregation and toxicity. Here, we focus on RRM2. Using all-atom replica-averaged metadynamics simulations with NMR chemical shift restraints, we characterized the atomic structure of non-native states of RRM2, sparsely populated under native conditions. These structures reveal the exposure to the solvent of aggregation-prone peptide regions, normally buried in the native state, supporting a role in aggregation for the partially folded states of RRM2.

Characterizing Protein Dynamics with NMR R 1ρ Relaxation Experiments.

The measurement of R1ρ , the longitudinal relaxation rate constant in the rotating frame, is one of the few available methods to characterize the µs-ms functional dynamics of biomolecules. Here, we focus on 15N R1ρ experiments for protein NH groups. We present protocols for both on- and off-resonance 15N R1ρ measurements needed for relaxation dispersion studies, and describe the data analysis for extracting kinetic and thermodynamic parameters characterizing the motional processes.

Structural Rearrangement upon Fragmentation of the Stability Core of the ALS-Linked Protein TDP-43.

Amyotrophic lateral sclerosis (ALS) is the most common adult degenerative motor neuron disease. Experimental evidence indicates a direct role of transactive-response DNA-binding protein 43 (TDP-43) in the pathology of ALS and other neurodegenerative diseases. TDP-43 has been identified as a major component of cytoplasmic inclusions in patients with sporadic ALS; however, the molecular basis of the disease mechanism is not yet fully understood. Fragmentation within the second RNA recognition motif (RRM2) of TDP-43 has been observed in patient tissues and may play a role in the formation of aggregates in disease. To determine the structural and dynamical changes resulting from the truncation that could lead to aggregation and toxicity, we performed molecular dynamics simulations of the full-length RRM2 domain (the stability core of TDP-43) and of a truncated variant (where residues 189-207 are deleted to mimic a site of cleavage within RRM2 found in ALS patients). Our simulations show heterogeneous structural reorganization and decreased stability of the truncated RRM2 domain compared to the full-length domain, consistent with previous experimental results. The decreased stability and structural reorganization in the truncated RRM2 result in a higher probability of protein-protein interactions through altered electrostatic surface charges and increased accessibility of hydrophobic residues (including the nuclear export sequence), providing a rationale for the increased cytoplasmic aggregation of RRM2 fragments seen in sporadic ALS patients.

Structural Basis of the Disorder in the Tandem Zinc Finger Domain of the RNA-Binding Protein Tristetraprolin.

Tristetraprolin (TTP) and TIS11d are two human RNA-binding proteins that belong to the CCCH-type tandem zinc finger family. In the RNA-free state, TIS11d coordinates a zinc ion in each of its two fingers, while TTP coordinates a single zinc ion with the N-terminal zinc finger. We have previously identified three residues, located in the C-terminal half of a short α-helix in the second zinc finger, that control how structured the RNA-binding domain is in these two proteins: Y151, L152, and Q153 in TTP and H201, T202, and I203 in TIS11d. Here, we have used molecular dynamics, NMR spectroscopy, and other biochemical methods to investigate the role of these three residues in the stability of the RNA-binding domain. We found that the intrahelical hydrogen bond formed by the T202 hydroxyl group in the C-terminal zinc finger of TIS11d is necessary to allow for π-π stacking between the side chains of a conserved phenylalanine and the zinc-coordinating histidine. We demonstrated that the lack of this hydrogen bond in TTP is responsible for the reduced zinc affinity of the C-terminal zinc finger.

Three Residues Make an Evolutionary Switch for Folding and RNA-Destabilizing Activity in the TTP Family of Proteins.

Tristetraprolin (TTP) binds to mRNA transcripts to promote their degradation. The TTP protein family in humans includes two other proteins, TIS11b and TIS11d. All three proteins contain a highly homologous RNA binding domain (RBD) that consists of two CCCH zinc fingers (ZFs). Both ZFs are folded in the absence of RNA in TIS11d and TIS11b. In TTP, however, only ZF1 adopts a stable fold. The focus of this study is to understand the origin and biological significance of the structural differences of the RBD. We identified three residues that affect the affinity for the structural Zn(2+) and determine the folding of ZF2 in the absence of RNA. We observed that the mRNA destabilizing activity of TTP was increased when the partially disordered RBD of TTP was replaced with the fully structured RBD of TIS11d, indicating that differences in the folded state of the RBD affect the activity of the proteins in the cell.

Probing the structural and dynamical effects of the charged residues of the TZF domain of TIS11d.

A member of the TTP family of proteins, TIS11d binds RNA with high specificity using a pair of CCCH-type tandem zinc fingers separated by a 18 residue long linker. Our previous work showed that the formation of hydrogen bonds between the C-terminal residue E220 and the residues of the linker region stabilized a compact structure of TIS11d in the absence of RNA. To investigate the role of the C-terminal residues in the structure of unbound TIS11d, the E220A mutant and the truncation mutant lacking the last two residues (D219/E220) were studied using molecular dynamics, NMR spectroscopy, and biochemical methods. This study confirmed the importance of the charged residues D219 and E220 in maintaining structural stability in unbound TIS11d and elucidated the underlying physical mechanisms. We observed a greater structural heterogeneity for the residues of the linker in the molecular dynamics trajectories of both mutant proteins relative to the wild-type. This heterogeneity was more pronounced in the D219/E220 deletion mutant than in the E220A mutant, indicating that a greater reduction of the charge of the C-terminus results in greater flexibility. In agreement with the increased flexibility and the reduced number of negatively charged residues of the D219/E220 deletion mutant, we measured more unfavorable entropic and a more favorable enthalpic contribution to the free energy of RNA binding in the mutant than in the wild-type protein. The relative orientation of the zinc fingers was stabilized by the electrostatic interaction between E220 and positively charged residues of the linker in TIS11d. In the E220A mutant, the relative orientation of the zinc fingers was less constrained, whereas in the D219/E220 deletion mutant, little orientational preference was observed. We posit that favorable electrostatic interactions provide a mechanism to promote preferential orientation of separate domains without imposing structural rigidity.

A conserved three-nucleotide core motif defines Musashi RNA binding specificity.

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila MSI is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins.

Insight into the allosteric mechanism of Scapharca dimeric hemoglobin.

Allosteric regulation is an essential function of many proteins that control a variety of different processes such as catalysis, signal transduction, and gene regulation. Structural rearrangements have historically been considered the main means of communication between different parts of a protein. Recent studies have highlighted the importance, however, of changes in protein flexibility as an effective way to mediate allosteric communication across a protein. Scapharca dimeric hemoglobin (HbI) is the simplest possible allosteric system, with cooperative ligand binding between two identical subunits. Thermodynamic equilibrium studies of the binding of oxygen to HbI have shown that cooperativity is an entropically driven effect. The change in entropy of the system observed upon ligand binding may arise from changes in the protein, the ligand, or the water of the system. The goal of this study is to determine the contribution of the change in entropy of the protein backbone to HbI cooperative binding. Molecular dynamics simulations and nuclear magnetic resonance relaxation techniques have revealed that the fast internal motions of HbI contribute to the cooperative binding to carbon monoxide in two ways: (1) by contributing favorably to the free energy of the system and (2) by participating in the cooperative mechanism at the HbI subunit interface. The internal dynamics of the weakly cooperative HbI mutant, F97Y, were also investigated with the same methods. The changes in backbone NH dynamics observed for F97Y HbI upon ligand binding are not as large as for the wild type, in agreement with the reduced cooperativity observed for this mutant. The results of this study indicate that interface flexibility and backbone conformational entropy of HbI participate in and are important for the cooperative mechanism of carbon monoxide binding.

Allosteric inhibition of a stem cell RNA-binding protein by an intermediary metabolite.

Gene expression and metabolism are coupled at numerous levels. Cells must sense and respond to nutrients in their environment, and specialized cells must synthesize metabolic products required for their function. Pluripotent stem cells have the ability to differentiate into a wide variety of specialized cells. How metabolic state contributes to stem cell differentiation is not understood. In this study, we show that RNA-binding by the stem cell translation regulator Musashi-1 (MSI1) is allosterically inhibited by 18-22 carbon ω-9 monounsaturated fatty acids. The fatty acid binds to the N-terminal RNA Recognition Motif (RRM) and induces a conformational change that prevents RNA association. Musashi proteins are critical for development of the brain, blood, and epithelium. We identify stearoyl-CoA desaturase-1 as a MSI1 target, revealing a feedback loop between ω-9 fatty acid biosynthesis and MSI1 activity. We propose that other RRM proteins could act as metabolite sensors to couple gene expression changes to physiological state.

Clusters of branched aliphatic side chains serve as cores of stability in the native state of the HisF TIM barrel protein.

Imidazole-3-glycerol phosphate synthase is a heterodimeric allosteric enzyme that catalyzes consecutive reactions in imidazole biosynthesis through its HisF and HisH subunits. The unusually slow unfolding reaction of the isolated HisF TIM barrel domain from the thermophilic bacteria, Thermotoga maritima, enabled an NMR-based site-specific analysis of the main-chain hydrogen bonds that stabilize its native conformation. Very strong protection against exchange with solvent deuterium in the native state was found in a subset of buried positions in α-helices and pervasively in the underlying ß-strands associated with a pair of large clusters of isoleucine, leucine and valine (ILV) side chains located in the α7(ßα)8(ßα)1-2 and α2(ßα)3-6ß7 segments of the (ßα)8 barrel. The most densely packed region of the large cluster, α3(ßα)4-6ß7, correlates closely with the core of stability previously observed in computational, protein engineering and NMR dynamics studies, demonstrating a key role for this cluster in determining the thermodynamic and structural properties of the native state of HisF. When considered with the results of previous studies where ILV clusters were found to stabilize the hydrogen-bonded networks in folding intermediates for other TIM barrel proteins, it appears that clusters of branched aliphatic side chains can serve as cores of stability across the entire folding reaction coordinate of one of the most common motifs in biology.

Molecular mechanisms of viral and host cell substrate recognition by hepatitis C virus NS3/4A protease.

Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.