Biology subjects

Tse, E.

Publications and source records attributed to Tse, E..

28 records · Page 2Linked to original sources

bioRxiv2022

Stacked binding of a small molecule PET tracer to Alzheimer's tau paired helical filaments

Neurodegenerative diseases (NDs) are characterized by the formation of amyloid filaments that adopt disease-specific conformations in the brain. Recently developed small molecules hold promise as diagnostics and possible therapeutics for NDs, but their binding mechanisms to amyloid filaments remain unknown. Here, we used cryo-electron microscopy (cryo-EM) to determine a 2.7 [A] structure of Alzheimers disease patient-derived tau paired-helical filaments incubated with the GTP-1 PET probe. GTP-1 is bound stoichiometrically along an exposed cleft of each protofilament in a stacked arrangement that matches the fibrils symmetry. Multiscale modeling revealed favorable pi-pi aromatic stacking interactions between GTP-1 molecules that, together with small molecule-protein contacts, result in high affinity binding. This binding mode offers new insight into designing compounds for diagnosis and treatment of specific NDs. One Sentence SummaryCryo-EM structure reveals a novel stacked arrangement of the GTP-1 PET ligand bound to Alzheimers disease tau filaments.

biophysics↗

bioRxiv2022

Unique structural features govern the activity of a human mitochondrial AAA+ disaggregase, Skd3.

The AAA+ protein, Skd3 (human CLPB), solubilizes proteins in the mitochondrial intermembrane space, which is critical for human health. Skd3 variants with impaired protein-disaggregase activity cause severe congenital neutropenia (SCN) and 3-methylglutaconic aciduria type 7 (MGCA7). Yet how Skd3 disaggregates proteins remains poorly understood. Here, we report a high-resolution structure of a Skd3-substrate complex. Skd3 adopts a spiral hexameric arrangement that engages substrate via pore-loop interactions in the nucleotide-binding domain (NBD). Unexpectedly, substrate-bound Skd3 hexamers stack head-to-head via unique, adaptable ankyrin-repeat domain (ANK)-mediated interactions to form dodecamers. Deleting the ANK-linker region reduces dodecamerization and disaggregase activity. We elucidate apomorphic features of the Skd3 NBD and C-terminal domain that regulate disaggregase activity. We also define how Skd3 subunits collaborate to disaggregate proteins. Importantly, SCN-linked subunits sharply inhibit disaggregase activity, whereas MGCA7-linked subunits do not. Our findings illuminate Skd3 structure and mechanism, explain SCN and MGCA7 inheritance patterns, and suggest therapeutic strategies.

biochemistry↗

bioRxiv2021

Structure of an Hsp90-immunophilin complex reveals cochaperone recognition of the client-maturation state

The Hsp90 chaperone promotes the folding and activation of hundreds of client proteins in the cell through an ATP-dependent conformational cycle guided by distinct cochaperone regulators. The FKBP51 immunophilin binds Hsp90 with its tetratricopeptide repeat (TPR) domain and catalyzes peptidyl-prolyl isomerase (PPIase) activity during the folding of kinases, nuclear receptors and tau. Here we have determined the cryo-EM structure of the human Hsp90:FKBP51:p23 complex to 3.3 [A] that, together with mutagenesis and crosslinking analysis, reveals the basis for cochaperone binding to Hsp90 during client maturation. A helix extension in the TPR functions as a key recognition element, interacting across the Hsp90 C-terminal dimer interface presented in the closed, ATP conformation. The PPIase domain is positioned along the middle domain, adjacent Hsp90 client binding sites, while a single p23 makes stabilizing interactions with the N-terminal dimer. With this architecture, FKBP51 could thereby act on specific client residues presented during Hsp90-catalyzed remodeling.

biophysics↗

bioRxiv2021

PIKfyve inhibition blocks endolysosomal escape of α-synuclein fibrils and spread of α-synuclein aggregation

The inter-cellular prion-like propagation of -synuclein aggregation is emerging as an important mechanism driving the progression of neurodegenerative diseases including Parkinsons disease and multiple system atrophy (MSA). To discover therapeutic strategies reducing the spread of -synuclein aggregation, we performed a genome-wide CRISPR interference screen in a human cell-based model. We discovered that inhibiting PIKfyve dramatically reduced -synuclein aggregation induced with both recombinant -synuclein fibrils and fibrils isolated from MSA patient brain. While PIKfyve inhibition did not affect fibril uptake or -synuclein clearance or secretion, it reduced -synuclein trafficking from the early endosome to the lysosome, thereby limiting fibril escape from the lysosome and reducing the amount of fibrils that reach cytosolic -synuclein to induce aggregation. These findings point to the endolysosomal transport of fibrils as a critical step in the propagation of -synuclein aggregation and a potential therapeutic target.

cell biology↗

bioRxiv2020

Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM

Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed nonCDS mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 [A] with a 1.346 [A] physical pixel). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834[A] physical pixel size) we achieved a resolution of 1.65 [A] within 9 hours of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 [A] resolution with 5 minutes of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.

biophysics↗

bioRxiv2019

Conformational Plasticity of the ClpAP AAA+ Protease Couples Protein Unfolding and Proteolysis

The ClpAP complex functions as a \"bacterial proteasome\" that simultaneously unfolds and degrades proteins targeted for destruction. ClpA utilizes two AAA+ domains per protomer to power substrate unfolding and translocation into the ClpP proteolytic chamber. To understand this mechanism, we determined high-resolution structures of wildtype E. coli ClpAP in distinct substrate-bound states. ClpA forms a spiral with substrate contacts across both AAA+ domains, while protomers at the seam undergo nucleotide-specific rearrangements indicating a conserved rotary mechanism. ClpA IGL loops extend flexibly to bind the planar, heptameric ClpP surface and support a large ClpA-P rotation that re-orients the translocation channel. The symmetry mismatch is maintained at the spiral seam through bind and release states of the IGL loops, which appear precisely coupled to substrate translocation. Thus, ClpA rotates around the apical surface of ClpP to processively translocate substrate into the protease.

biophysics↗

bioRxiv2019

Mechanistic Insights into the Protective Roles of Polyphosphate Against Amyloid Cytotoxicity

The universally abundant polyphosphate (polyP) accelerates fibril formation of disease-related amyloids and protects against amyloid cytotoxicity. To gain insights into the mechanism(s) by which polyP exerts these effects, we focused on -synuclein, a well-studied amyloid protein, which constitutes the major component of Lewy bodies found in Parkinsons Disease. Here we demonstrate that polyP is unable to accelerate the rate-limiting step of -synuclein fibril formation but effectively nucleates fibril assembly once -synuclein oligomers are formed. Binding of polyP to -synuclein either during fibril formation or upon fibril maturation substantially alters fibril morphology, and effectively reduces the ability of -synuclein fibrils to interact with cell membranes. The effect of polyP appears to be -synuclein fibril specific, and successfully prevents the uptake of fibrils into neuronal cells. These results suggest that altering the polyP levels in the extracellular space might be a potential therapeutic strategy to prevent the spreading of the disease.

cell biology↗

bioRxiv2019

Compromised function of an ESCRT complex promotes endolysosomal escape of tau seeds and propagation of tau aggregation

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimers Disease and frontotemporal dementia. However, we lack a systematic understanding of the cellular path-ways controlling prion-like propagation. To uncover such pathways, we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation. Our screens uncovered that knockdown of several components of the ESCRT machinery, including CHMP6, or CHMP2A in combination with CHMP2B (a gene linked to familial fronto-temporal dementia), promote propagation of tau aggregation. We found that knockdown of these genes caused damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation.

cell biology↗

bioRxiv2018

Structural basis for substrate gripping and translocation by the ClpB AAA+ disaggregase

Bacterial ClpB and yeast Hsp104 are homologous Hsp100 protein disaggregases that serve critical functions in proteostasis by solubilizing protein aggregates. Two AAA+ nucleotide binding domains (NBDs) power polypeptide translocation through a central channel comprised of a hexameric spiral of protomers that contact substrate via conserved pore-loop interactions. To elucidate the translocation mechanism, we determined the cryo-EM structure of a hyperactive ClpB variant to 2.9 [A] resolution bound to the model substrate, casein in the presence of slowly hydrolysable ATP{gamma}S. Distinct substrate-gripping mechanisms are identified for NBD1 and NBD2 pore loops. A trimer of N-terminal domains define a channel entrance that binds the polypeptide substrate adjacent the topmost NBD1 contact. NBD conformations at the spiral seam reveal how ATP hydrolysis and substrate engagement or disengagement are precisely tuned to drive a stepwise translocation cycle.

biophysics↗

bioRxiv2018

Whole-Genome Genomics Correlates of Response To Anti-PD1 Therapy in Relapsed/Refractory Natural Killer/T Cell Lymphoma

AbstractThis study aims to identify recurrent genetic alterations in relapsed or refractory (RR) natural-killer/T-cell lymphoma (NKTL) patients who have achieved complete response (CR) with programmed cell death 1 (PD-1) blockade therapy. Seven of the eleven patients treated with pembrolizumab achieved CR while the remaining four had progressive disease (PD). Using whole genome sequencing (WGS), we found recurrent clonal structural rearrangements (SR) of the PD-L1 gene in four of the seven (57%) CR patients pretreated tumors. These PD-L1 SRs consist of inter-chromosomal translocations, tandem duplication and micro-inversion that disrupted the suppressive function of PD-L1 3UTR. Interestingly, recurrent JAK3-activating (p.A573V) mutations were also validated in two CR patients tumors that did not harbor the PD-L1 SR. Importantly, these mutations were absent in the four PD cases. With immunohistochemistry (IHC), PD-L1 positivity could not discriminate patients who archived CR (range: 6%-100%) from patients who had PD (range: 35%-90%). PD-1 blockade with pembrolizumab is a potent strategy for RR NKTL patients and genomic screening could potentially accompany PD-L1 IHC positivity to better select patients for anti-PD-1 therapy.

genomics↗