Biology subjects

Williams, J. K.

Publications and source records attributed to Williams, J. K..

15 recordsLinked to original sources

bioRxiv2026

Sorcin couples Annexin A11 recruitment and ESCRT-III assembly during plasma membrane repair

The absence of a cell wall affords animal cells diverse functionality at the cost of acute sensitization to plasma membrane (PM) damage. Thus, animal cells tightly monitor and maintain PM integrity to prevent cell death. Genetic loss of PM repair factors is associated with human diseases such as muscular dystrophy. Despite evidence that annexin and endosomal sorting complex required for transport (ESCRT) proteins are required for PM repair, the extent to which their recruitment is coordinated at sites of membrane damage remains unclear. Here, leveraging quantitative organellar proteomics and genome-wide CRISPR interference screens, we identify sorcin as a new PM repair factor that couples annexin A11 (ANXA11)-mediated sensing of PM damage to ESCRT-III assembly. We show that sorcin directly binds ANXA11 and ALIX in the presence of Ca2+ via its penta-EF-hand domain and flexible N-terminus, respectively, and is required for ESCRT-III recruitment to PM lesions and membrane resealing. Our data support a model in which ANXA11, recruited to the PM upon damage-induced Ca2+ influx, serves as an anchor that facilitates the sequential recruitment of sorcin and ESCRT-III at PM lesions. Together, these findings establish a Ca2+-dependent scaffolding mechanism that couples PM damage sensing to ESCRT-III assembly for PM repair.

cell biology↗

bioRxiv2025

Is synuclein aggregation a derived or ancestral trait? Ancestral sequence reconstruction uncovers stepwise evolution of synuclein aggregation

Protein aggregation drives many neurodegenerative diseases, including Parkinsons disease, where misfolded -synuclein (Syn) forms fibrillar assemblies that accumulate as Lewy bodies. Although Syn aggregation has been extensively characterized, its evolutionary origins and sequence determinants remain unresolved. Here, we use ancestral sequence reconstruction (ASR) to trace the emergence of fibril-forming ability in the synuclein family. We inferred synuclein phylogeny and experimentally resurrected common ancestors, including ROOT synuclein, the last common ancestor of all synucleins, and key intermediates along the Syn lineage. Strikingly, ROOT synuclein is non-aggregating, demonstrating that fibril formation is an evolved, rather than ancestral property. Aggregation first emerges at the ancestral {beta} node, is retained in Syn, and suppressed in {beta}-synuclein. Biophysical analyses including mass spectrometry and NMR reveal that aggregation aligns with greater complexity and heterogeneity in the monomer conformational ensemble, suggesting that evolutionary sequence changes progressively remodel monomer landscapes to favor fibril formation. Complementing these insights, comparative sequence analysis reveals that the transition from ROOT to -WT is marked by the stepwise acquisition of residues critical for stabilizing the fibril core. Early mutations stabilized the {beta}-arch core, enabling the onset of fibril formation, followed by substitutions that reinforce protofilament-protofilament interactions. Together, ASR defines an evolutionary framework for synuclein aggregation linking progressive sequence evolution and conformational complexity to the molecular origins of Syn fibril formation.

biophysics↗

bioRxiv2025

Endosome transcriptomics reveal trafficking of Cajal bodies into multivesicular bodies

All eukaryotic cells secrete exosomes, a type of extracellular vesicles (EVs) derived from the endocytic compartments known as multivesicular bodies (MVBs), or late endosomes (LEs). Exosomes contain a diverse range of cargo such as nucleic acids, proteins, lipids and small molecules but whether these contents have a biological function remains an area of intense investigation. Over the last decade, numerous studies have described the transcriptome of exosomes but very little is known about the RNA content of the MVBs, the source compartment for exosome biogenesis. Here we determine the small-RNA transcriptome of highly purified MVBs and report that various classes of nuclear small regulatory RNAs such as small-Cajal body associated RNAs (scaRNAs), small-nucleolar RNAs (snoRNAs) and small-nuclear RNAs (snRNAs) traffic to MVBs. We show that this RNA-trafficking requires the function of ESCRT machinery but is independent of canonical LC3 lipidation mediated selective autophagy. Furthermore, blocking the activity of a PI3K Class 3 enzyme, VPS34, required for recruitment of the ESCRT machinery to the endosome, prevents the turnover of these nuclear RNAs in MVBs. Our results provide a mechanism for targeting nuclear ribonucleoprotein complexes (RNPs), such as Cajal bodies, for degradation and turnover by the cytoplasmic endo-lysosomal pathway. Significance StatementEndosomes are cytoplasmic, membrane-bound subcellular organelles that are sites for biogenesis of exosomes, a class of extracellular vesicles, thought to mediate intercellular communication via their packaged cargo such as RNA. Previous studies have focused on the transcriptome of exosomes however very little is known about the identity of RNAs and mechanisms by which they are sorted into endosomes. Here we report a comprehensive endosome transcriptome and provide evidence that several nuclear RNA-protein complexes (RNPs) sort into endosomes, a previously unappreciated phenomenon. We show that this process requires the activity of endosomal sorting complexes and phospholipids characteristic of cellular endocytic compartments. Our study provides a mechanism for recycling and disposal of unwanted nuclear RNPs by the cytoplasmic endolysosomal pathway.

biochemistry↗

bioRxiv2025

p62 sorts Lupus La and selected microRNAs into breast cancer-derived exosomes

Exosomes are multivesicular body-derived extracellular vesicles that are secreted by metazoan cells. Exosomes have utility as disease biomarkers, and exosome-mediated miRNA secretion has been proposed to facilitate tumor growth and metastasis. Previously, we demonstrated that the Lupus La protein (La) mediates the selective incorporation of miR-122 into metastatic breast cancer-derived exosomes; however, the mechanism by which La itself is sorted into exosomes remains unknown. Using unbiased proximity labeling proteomics, biochemical fractionation, superresolution microscopy and genetic tools, we establish that the selective autophagy receptor p62 sorts La and miR-122 into exosomes. We then performed small RNA sequencing and found that p62 depletion reduces the exosomal secretion of tumor suppressor miRNAs and results in their accumulation within cells. Our data indicate that p62 is a quality control factor that modulates the miRNA composition of exosomes. Cancer cells may exploit p62-dependent exosome cargo sorting to eliminate tumor suppressor miRNAs and thus to promote cell proliferation.

cell biology↗

bioRxiv2025

Void spot assay visualization optimization for use of Void Whizzard in rats (Rattus norvegicus)

Void spot assay (VSA) noninvasively evaluates urination. VSA is often not performed in rats due to difficulty analyzing larger papers compared with mouse. This study optimizes VSA for rats by comparing post-assay visualization techniques: bright field light (BF), ultraviolet light (UV), and ninhydrin spray (N). Male rats were placed in cages lined by filter paper for 4 hours. Water and food was provided ad lib. After the assay all papers were dried overnight. BF images were photographed (digital camera). UV images were captured after cutting papers in half using a Darkroom ultraviolet imaging cabinet. Papers were sprayed with ninhydrin and photographed (digital camera). All images were converted to black and white for analysis with Void Whizzard. UV vs. BF showed significant differences in area. All three groups had significant differences in overall spot count and in the smallest sized spots (0-0.1 cm2). UV vs. N and UV vs. BF showed significant differences in 0.1-0.25 cm2 spots, UV vs. N in 0.25-0.5 cm2, and N vs. BF in spots 0.5-1 cm2. Overall BF visualization proved difficult. N provided an ideal way to highlight urine and image with a digital camera. Human fingerprints from pre-assay handling of paper interfered with analysis of the smallest sized spots however there were no differences in detection of larger spots, spot distribution, or overall spot area. This study contributes to the development of a standardized VSA protocol for assessing bladder function in both mouse and rat models.

physiology↗

bioRxiv2024

Calpains Orchestrate Secretion of Annexin-containing Microvesicles during Membrane Repair

Microvesicles (MVs) are membrane-enclosed, plasma membrane-derived particles released by cells from all branches of life. MVs have utility as disease biomarkers and may participate in intercellular communication; however, physiological processes that induce their secretion are not known. Here, we isolate and characterize annexin-containing MVs and show that these vesicles are secreted in response to the calcium influx caused by membrane damage. The annexins in these vesicles are cleaved by calpains. After plasma membrane injury, cytoplasmic calcium-bound annexins are rapidly recruited to the plasma membrane and form a scab-like structure at the lesion. In a second phase, recruited annexins are cleaved by calpains-1/2, disabling membrane scabbing. Cleavage promotes annexin secretion within MVs. Our data supports a new model of plasma membrane repair, where calpains relax annexin-membrane aggregates in the lesion repair scab, allowing secretion of damaged membrane and annexins as MVs. We anticipate that cells experiencing plasma membrane damage, including muscle and metastatic cancer cells, secrete these MVs at elevated levels.

cell biology↗

bioRxiv2023

Evolving Membrane-Associated Accessory Protein Variants for Improved Adeno-Associated Virus Production

Manufacturing sufficient Adeno-Associated Virus (AAV) to meet current and projected clinical needs is a significant hurdle to the growing gene therapy industry. The recently discovered membrane-associated accessory protein (MAAP) is encoded by an alternative open reading frame in the AAV cap gene that is found in all presently reported natural serotypes. Recent evidence has emerged supporting a functional role of MAAP in AAV egress, though the underlying mechanisms of MAAP function remain unknown. Here, we show that inactivation of MAAP from AAV2 by a single point mutation that is silent in the VP1 ORF (AAV2-{Delta}MAAP) decreased exosome-associated and secreted vector genome production. We hypothesized that novel MAAP variants could be evolved to increase AAV production and thus subjected a library encoding over 1E6 MAAP protein variants to five rounds of packaging selection into the AAV2-{Delta}MAAP capsid. Between each successive packaging round, we observed a progressive increase in both overall titer and ratio of secreted vector genomes conferred by the bulk selected MAAP library population. Next-generation sequencing uncovered enriched mutational features, and a resulting selected MAAP variant containing missense mutations and a novel C-terminal domain increased overall GFP transgene packaging in AAV2, AAV6, and AAV9 capsids.

bioengineering↗

bioRxiv2022

Annexin A6 mediates calcium-dependent secretion of exosomes

Exosomes are a subtype of extracellular vesicles (EV) that are secreted upon fusion of multivesicular bodies (MVBs) with the plasma membrane. In order to identify proteins responsible for this fusion event, we developed sensitive cellular and cell-free assays to measure exosome secretion. Our results suggest that exosome secretion is coupled to Ca2+-dependent plasma membrane repair. Using a targeted proteomics approach, we identify annexin A6 (ANXA6), a well-known plasma membrane repair protein, as a MVB binding protein and demonstrate that it is required for Ca2+-dependent exosome secretion both in intact and in permeabilized cells. Our results suggest that cells employ MVBs as a membrane source for plasma membrane lesion repair during physiological mechanical stress or treatment with a bacterial pore-forming toxin, and that this repair process results in the coincident secretion of exosomes. We propose that this unconventional secretion process may contribute significantly to the heterogeneity of EVs in biological fluids.

molecular biology↗

bioRxiv2022

Broadly neutralizing antibodies target the coronavirus fusion peptide

The potential for future coronavirus outbreaks highlights the need to develop strategies and tools to broadly target this group of pathogens. Here, using an epitope-agnostic approach, we identified six monoclonal antibodies that bound to spike proteins from all seven human-infecting coronaviruses. Epitope mapping revealed that all six antibodies target the conserved fusion peptide region adjacent to the S2 cleavage site. Two antibodies, COV44-62 and COV44-79, broadly neutralize a range of alpha and beta coronaviruses, including SARS-CoV-2 Omicron subvariants BA.1 and BA.2, albeit with lower potency than RBD-specific antibodies. In crystal structures of Fabs COV44-62 and COV44-79 with the SARS-CoV-2 fusion peptide, the fusion peptide epitope adopts a helical structure and includes the arginine at the S2 cleavage site. Importantly, COV44-79 limited disease caused by SARS-CoV-2 in a Syrian hamster model. These findings identify the fusion peptide as the target of the broadest neutralizing antibodies in an epitope-agnostic screen, highlighting this site as a candidate for next-generation coronavirus vaccine development. One-Sentence SummaryRare monoclonal antibodies from COVID-19 convalescent individuals broadly neutralize coronaviruses by targeting the fusion peptide.

immunology↗

bioRxiv2021

Tannic Acid Inhibits α-Synuclein Amyloid Fibril Formation via Binding to the Monomer N-terminal Domain

-Synuclein (S) is an intrinsically disordered protein (IDP) that aggregates into amyloid fibrils during the progression of Parkinsons Disease and other synucleinopathies. The N-terminal domain (residues 1-60) is now understood to play a critical role in the initial nucleation of aggregation, as well as a pivotal role in the monomer-fibril interaction underlying amyloid seeding. Here we report on the interaction between S and the polyphenol tannic acid (TA), where a combination of solution NMR, atomic force microscopy (AFM), and ThT assays have identified that TA targets the S N-terminal domain to inhibit amyloid fibril formation in a pH dependent manner. This work highlights the importance of targeting the N-terminus of S to arrest fibril formation, and suggests the importance of including polyphenolic moieties in future amyloid inhibitors.

biochemistry↗

bioRxiv2021

Potent neutralization of SARS-CoV-2 variants of concern by an antibody with a unique genetic signature and structural mode of spike recognition

The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.

immunology↗

bioRxiv2021

Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo), and others recognizing non-RBD epitopes that bound the heptad repeat 1 region of the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.

immunology↗

bioRxiv2021

Molecular Dynamics Analysis of a Flexible Loop at the Binding Interface of the SARS-CoV-2 Spike Protein Receptor-Binding Domain

Since the identification of the SARS-CoV-2 virus as the causative agent of the current COVID-19 pandemic, considerable effort has been spent characterizing the interaction between the Spike protein receptor-binding domain (RBD) and the human angiotensin converting enzyme 2 (ACE2) receptor. This has provided a detailed picture of the end point structure of the RBD-ACE2 binding event, but what remains to be elucidated is the conformation and dynamics of the RBD prior to its interaction with ACE2. In this work we utilize molecular dynamics simulations to probe the flexibility and conformational ensemble of the unbound state of the receptor-binding domain from SARS-CoV-2 and SARS-CoV. We have found that the unbound RBD has a localized region of dynamic flexibility in Loop 3 and that mutations identified during the COVID-19 pandemic in Loop 3 do not affect this flexibility. We use a loop-modeling protocol to generate and simulate novel conformations of the CoV2-RBD Loop 3 region that sample conformational space beyond the ACE2 bound crystal structure. This has allowed for the identification of interesting substates of the unbound RBD that are lower energy than the ACE2-bound conformation, and that block key residues along the ACE2 binding interface. These novel unbound substates may represent new targets for therapeutic design.

biophysics↗

bioRxiv2020

Evolution of the SARS-CoV-2 proteome in three dimensions (3D) during the first six months of the COVID-19 pandemic

Three-dimensional structures of SARS-CoV-2 and other coronaviral proteins archived in the Protein Data Bank were used to analyze viral proteome evolution during the first six months of the COVID-19 pandemic. Analyses of spatial locations, chemical properties, and structural and energetic impacts of the observed amino acid changes in >48,000 viral proteome sequences showed how each one of the 29 viral study proteins have undergone amino acid changes. Structural models computed for every unique sequence variant revealed that most substitutions map to protein surfaces and boundary layers with a minority affecting hydrophobic cores. Conservative changes were observed more frequently in cores versus boundary layers/surfaces. Active sites and protein-protein interfaces showed modest numbers of substitutions. Energetics calculations showed that the impact of substitutions on the thermodynamic stability of the proteome follows a universal bi-Gaussian distribution. Detailed results are presented for six drug discovery targets and four structural proteins comprising the virion, highlighting substitutions with the potential to impact protein structure, enzyme activity, and functional interfaces. Characterizing the evolution of the virus in three dimensions provides testable insights into viral protein function and should aid in structure-based drug discovery efforts as well as the prospective identification of amino acid substitutions with potential for drug resistance.

molecular biology↗

bioRxiv2020

Probing acetylated-α-synuclein monomer-aggregate complexes by NMR elucidates mechanism of fibril seeding

Amyloid fibril formation of -synuclein (S) is associated with multiple neurodegenerative diseases, including Parkinsons Disease (PD). Growing evidence suggests that progression of PD is linked to cell-to-cell propagation of S fibrils, which leads to seeding of endogenous intrinsically disordered monomer via templated elongation and secondary nucleation. A molecular understanding of the seeding mechanism and driving interactions is crucial to inhibit progression of amyloid formation. Here, using relaxation-based solution NMR experiments designed to probe large complexes, we probe weak interactions of intrinsically disordered acetylated-S (Ac-S) monomers with seeding-competent Ac-S fibrils and seeding-incompetent off-pathway oligomers to identify Ac-S monomer residues at the binding interface. Under conditions that favor fibril elongation, we determine that the first 11 N-terminal residues on the monomer form a common binding site for both fibrils and off-pathway oligomers. Additionally, the presence of off-pathway oligomers within a fibril seeding environment suppresses seeded amyloid formation, as observed through thioflavin-T fluorescence experiments. This highlights that off-pathway S oligomers can act as an auto-inhibitor against S fibril elongation. Based on these data taken together with previous results, we propose a model in which Ac-S monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N-terminus and the intrinsically disordered flanking regions (IDR) on the fibril surface. We suggest that this monomer recruitment may play a role in the elongation of amyloid fibrils and highlight the potential of the IDRs of the fibril as important therapeutic targets against seeded amyloid formation. SignificanceCell-to-cell spreading of S fibrils leads to amyloid seeding of endogenous monomer. Detailed atomic-level mechanistic understanding of the fibril seeding process of S is essential for design of therapeutic approaches against Parkinsons disease. In light of its complexity, this process remains ill-defined at the molecular level. Using relaxation-based solution NMR experiments, we mapped a common N-terminal binding interface of the Ac-S intrinsically disordered monomer with Ac-S fibrils and off-pathway oligomers to elucidate critical monomer-aggregate interactions during seeded aggregation and in equilibrium with mature aggregates. From this work, we propose a new paradigm, in which Ac-S monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N-terminus and the flanking IDRs on the fibril surface.

biophysics↗