Biology subjects

Lynch, E. M.

Publications and source records attributed to Lynch, E. M..

13 recordsLinked to original sources

bioRxiv2026

VCP is a critical component for TDP-43 disaggregase activity in skeletal muscle

TDP-43 aggregates are a unifying pathological feature between several neurodegenerative diseases and myopathies. Mutations in the AAA ATPase protein VCP have been linked to TDP-43 proteinopathies in both brain and skeletal muscle including frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and inclusion body myopathy (IBM). In the case of multisystem proteinopathy (MSP), patients with VCP mutations may present with combinations of these phenotypes. Of these MSP phenotypes IBM is the most common, affecting around 90% of patients. However, it is not well understood how mutations in VCP contribute to the accumulation of insoluble TDP-43 aggregates in the context of skeletal muscle. To study this further, we used in vitro and in vivo models of TDP-43 aggregation in skeletal muscle in the presence of VCP mutation or inhibition to study its effects on the ability to clear TDP-43 aggregates. Across multiple model systems we found that VCP disease mutations or inhibition causes a reduced capacity to clear insoluble TDP-43, suggesting a VCP loss of function in patients with VCP-related MSP.

pathology↗

bioRxiv2025

A Covalent PFKL Activator Suppresses Tumor Growth

Glycolysis fuels vital cellular functions and its dysregulation is implicated in cancer, neurodegeneration, antibiotic resistance and diabetes. The glycolytic dependency of cancer, known as the Warburg effect, presents a key vulnerability for developing targeted anticancer agents but remains challenging due to metabolic heterogeneity and resistance. Here, we developed a first-in-class covalent phosphofructokinase-1 liver type (PFKL) activator that induces metabolic imbalance coupled to delivery of a cytotoxic payload to cancer cells in vitro and in vivo. The electrophile-drug conjugate (EDC) site-specifically and proteome-wide selectively modifies K677 in the allosteric effector site to stabilize the R-state tetramer of PFKL and destabilize cell metabolism. We introduce EDCs as a new delivery mechanism analogous to antibody-drug conjugates but differentiated by selective covalent targeting of intracellular proteins.

biochemistry↗

bioRxiv2025

De-novo design of a random protein walker

Molecular machines hold great potential. Design of static monomeric and oligomeric protein structures has advanced tremendously, but few dynamic protein systems have been designed. Here we present the design and characterization of a random protein walker that diffuses along a designed protein track. For the track, we designed micro-meter long fibres and developed a method to rigidly decorate them with arbitrary proteins. The walkers consist of homo-oligomers that reversibly bind the track using heterodimeric feet; we tested multiple heterodimer interfaces for reversibility and designed six walkers with different numbers of feet. Cryo-EM experiments confirmed the structure of the track and walkers. We performed detailed single molecule tracking and kinetics characterisation and found that the walkers with more feet diffuse along the track faster. The system represents a tuneable starting point for future powered protein molecular robots.

synthetic biology↗

bioRxiv2025

De novo design of inducibly assembling multi-component filaments

The design of multi-component nanomaterials is an outstanding challenge. Here, we describe the computational design of protein filaments with two or three distinct structural components that assemble into micron-scale, well-ordered fibers when mixed. CryoEM structure determination of four fiber designs was close to the computational design models. Filament assembly can be initiated by mixing the components, and modulated by addition and/or phosphorylation of designed regulatory subunits. This work demonstrates that regulatable multi-component protein filament systems can now be designed, opening the door to a wide range of engineered materials.

bioengineering↗

bioRxiv2024

Computational redesign of TALE proteins for DNA-templated assembly of protein fibers

Many viral proteins self-assemble into capsid structures, often using their genetic material as a template for assembly. To date, de novo designed capsid-like proteins do not require genetic material as a template for assembly, which can be both an advantage and a disadvantage depending on the use case. Templates are indispensable, for example, in the assembly of linear structures with well-defined lengths. As a first step towards fully de novo designed templated assembly, here we redesign proteins from the Transcription activator-like effector (TALE) family of transcriptional regulators to polymerize on double-stranded DNA (dsDNA) templates. Starting from natural TALE protein sequences, we created idealized repeat proteins with sequence-independent DNA binding properties that cooperatively self-assemble to form linear protein-DNA complexes with template-controlled lengths. We used high-resolution atomic force microscopy (AFM) and cryo electron microscopy (cryo-EM) to characterize the three-dimensional structures of the DNA-protein hybrid complexes. In these structures, a protein filament helically wraps around the dsDNA using a binding mode similar to that of natural TALE proteins. As an example application of these materials, we show the system can be used for repetitive peptide antigen display at precisely controlled repeat distances, and that such immunogens elicit robust antigen-specific antibodies in mice.

biochemistry↗

bioRxiv2024

Nucleation limited assembly and polarized growth of a de novo-designed allosterically modulatable protein filament

The design of inducibly assembling protein nanomaterials is an outstanding challenge. Here, we describe the computational design of a protein filament formed from a monomeric subunit that binds a peptide ligand. The cryoEM structure of the micron-scale fibers is very close to the computational design model. The ligand acts as a tunable allosteric modulator: while not part of the fiber subunit-subunit interfaces, the assembly of the filament is dependent on ligand addition, with longer peptides having more extensive interaction surfaces with the monomer, promoting more rapid growth. Seeded growth and capping experiments reveal that the filaments grow primarily from one end. We show that designed nucleators that present 12 copies of the peptide ligand promote fiber assembly at concentrations where otherwise assembly occurs very slowly, likely by generating critical local concentrations of monomer in the assembly competent conformation. Following filament assembly, the peptide ligand can be exchanged with free peptide in solution fused to any functional protein of interest, opening the door to a wide variety of tunable engineered materials.

bioengineering↗

bioRxiv2024

Evolutionarily divergent Mycobacterium tuberculosis CTP synthase filaments are under selective pressure

The final and rate-limiting enzyme in pyrimidine biosynthesis, CTP synthase (CTPS), is essential for the viability of Mycobacterium tuberculosis and other mycobacteria. Its product, CTP, is critical for RNA, DNA, lipid and cell wall synthesis, and is involved in chromosome segregation. In various organisms across the tree of life, CTPS assembles into higher-order filaments, leading us to hypothesize that M. tuberculosis CTPS (mtCTPS) also forms higher-order structures. Here, we show that mtCTPS does assemble into filaments but with an unusual architecture not seen in other organisms. Through a combination of structural, biochemical, and cellular techniques, we show that polymerization stabilizes the active conformation of the enzyme and resists product inhibition, potentially allowing for the highly localized production of CTP within the cell. Indeed, CTPS filaments localize near the CTP-dependent complex needed for chromosome segregation, and cells expressing mutant enzymes unable to polymerize are altered in their ability to robustly form this complex. Intriguingly, mutants that alter filament formation are under positive selection in clinical isolates of M. tuberculosis, pointing to a critical role needed to withstand pressures imposed by the host and/or antibiotics. Taken together, our data reveal an unexpected mechanism for the spatially organized production of a critical nucleotide in M. tuberculosis, which may represent a vulnerability of the pathogen that can be exploited with chemotherapy.

biochemistry↗

bioRxiv2024

Seeding competent TDP-43 persists in human patient and mouse muscle

TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous system of some neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy (IBM). TDP-43 aggregates from ALS and FTD brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in IBM patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with IBM, IMNM and ALS we found that TDP-43 seeding capacity was specific to IBM. Surprisingly, TDP-43 seeding capacity anti-correlated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease. SummaryTDP-43 aggregate seeds persist in mouse and human skeletal muscle independent of large TDP-43 inclusions.

pathology↗

bioRxiv2024

Structural basis for allosteric regulation of human phosphofructokinase-1

Phosphofructokinase-1 (PFK1) catalyzes the rate-limiting step of glycolysis, committing glucose to conversion into cellular energy. PFK1 is highly regulated to respond to the changing energy needs of the cell. In bacteria, the structural basis of PFK1 regulation is a textbook example of allostery; molecular signals of low and high cellular energy promote transition between an active R-state and inactive T-state conformation, respectively. Little is known, however, about the structural basis for regulation of eukaryotic PFK1. Here, we determine structures of the human liver isoform of PFK1 (PFKL) in the R- and T-state by cryoEM, providing insight into eukaryotic PFK1 allosteric regulatory mechanisms. The T-state structure reveals conformational differences between the bacterial and eukaryotic enzyme, the mechanisms of allosteric inhibition by ATP binding at multiple sites, and an autoinhibitory role of the C-terminus in stabilizing the T-state. We also determine structures of PFKL filaments that define the mechanism of higher-order assembly and demonstrate that these structures are necessary for higher-order assembly of PFKL in cells.

biochemistry↗

bioRxiv2021

Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or hyper-assembly mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

biochemistry↗

bioRxiv2019

Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

Many enzymes assemble into defined oligomers, providing a mechanism for cooperatively regulating enzyme activity. Recent studies in tissues, cells, and in vitro have described a mode of regulation in which enzyme activity is modulated by polymerization into large-scale filaments1-5. Enzyme polymerization is often driven by binding to substrates, products, or allosteric regulators, and tunes enzyme activity by locking the enzyme in high or low activity states1-5. Here, we describe a unique, ultrasensitive form of polymerization-based regulation employed by human CTP synthase 2 (CTPS2). High-resolution cryoEM structures of active and inhibited CTPS2 filaments reveal the molecular basis of this regulation. Rather than selectively stabilizing a single conformational state, CTPS2 filaments dynamically switch between active and inactive filament forms in response to changes in substrate and product levels. Linking the conformational state of many CTPS2 subunits in a filament results in highly cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 tetramer alone. The structures also reveal a link between conformational state and control of ammonia channeling between the enzymes two active sites. This filament-based mechanism of enhanced cooperativity demonstrates how the widespread phenomenon of enzyme polymerization can be adapted to achieve different regulatory outcomes.

biochemistry↗

bioRxiv2019

Architecture of the Mto1/2 microtubule nucleation complex

Proteins that contain a Centrosomin Motif 1 (CM1) domain are key regulators of{gamma} -tubulin complex-dependent microtubule nucleation, but how they are organized in higher-order structures is largely unknown. Mto1[bonsai], a truncated functional version of the Schizosaccharomyces pombe CM1 protein Mto1, interacts with Mto2 to form an Mto1/2[bonsai] complex in vivo. Here we show that recombinant Mto1/2[bonsai] forms higher-order multimers in vitro and that Mto2 alone can also multimerize. We demonstrate that Mto2 multimerization involves two separate homodimerization domains, the near N-terminal domain (NND) and the twin-cysteine domain (TCD). The TCD crystal structure reveals a stable homodimer with a novel dimerization interface. While the NND homodimer is intrinsically less stable, using crosslinking mass spectrometry we show that within Mto1/2[bonsai] complexes, it can be reinforced by additional cooperative interactions involving both Mto2 and Mto1[bonsai]. We propose a model for Mto1/2[bonsai] complex architecture that is supported by functional analysis of mutants in vivo.

cell biology↗

bioRxiv2019

Independent evolution of polymerization in the Actin ATPase clan regulates hexokinase activity

The actin protein fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, like the carbohydrate kinases, do not polymerize. We find that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with unique ultrastructure, distinct from that of cytoskeletal polymers. In cells, Glk1 polymerizes upon sugar addition and depolymerizes upon sugar withdrawal. Glk1 polymerization inhibits its enzymatic activity, thus the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation eliminating Glk1 polymerization alleviates concentration-dependent enzyme inhibition, causing glucokinase activity to become unconstrained. Polymerization-based regulation of Glk1 activity serves an important function in vivo: yeast containing non-polymerizing Glk1 are less fit when growing on sugars and more likely to die when refed glucose. Glucokinase polymerization arose within the ascomycete fungi and is conserved across a group of divergent (150-200 mya) yeast. We show that Glk1 polymerization arose independently from other actin-related filaments and allows yeast to rapidly modulate glucokinase activity as nutrient availability changes.\n\nOne-sentence summaryYeast glucokinase activity is limited by its polymerization, which is critical for cell viability during glucose refeeding.

biochemistry↗