Biology subjects

Sitron, C. S.

Publications and source records attributed to Sitron, C. S..

7 recordsLinked to original sources

bioRxiv2026

Aggregate Deep Visual Proteomics reveals selective organization of α-synuclein inclusions

-Synuclein inclusions, the lesions typical of Parkinson's disease, have been interpreted as protective deposits, toxic species or failed degradation products, each predicting a different composition and correlating that enrichment with neuron dysfunction and eventual degeneration. Distinguishing them requires measuring what an inclusion concentrates relative to the cell that built it. Here we developed Aggregate Deep Visual Proteomics, which uses ultrasensitive mass spectrometry to profile laser-microdissected inclusions against the remainder of the same cell or a matched host. In HEK293 cells, iPSC-derived dopaminergic neurons and post-mortem patient brain, each inclusion concentrated a selective subset of its cell's proteome, independent of abundance and dominated by membrane, in neurons largely presynaptic. Inclusions took up whole assemblies, with subunits moving together in 421 of 438 complexes, and the V-ATPase alone was enriched in all three systems. Degradation cargo stayed with the receptors that route it, consistent with sequestration and failed degradation acting together. Neuritic and somatic inclusions differed, the former rich in UBQLN2 and the latter in intermediate filaments. In the cell line, phosphorylated -synuclein was tenfold higher in the inclusion than in the rest of the cell, with about one third of the rise already present before an inclusion was visible. Measured against its host, the composition of an inclusion becomes a readout of how it formed and a source of candidate markers and therapeutic targets.

biochemistry↗

bioRxiv2026

Q-DOAS: A Proximity Quenching Assay for Real-Time Detection of Early Protein Aggregation Events

Accurate measurement of protein aggregation is essential for studying neurodegenerative diseases. The standard ThT assay reports on amyloid formation but is blind to early oligomers and is prone to interference. We describe Q-DOAS, a plate-reader assay that quantifies protein self-assembly in real time via proximity-quenching of a single, site-specifically conjugated dye (BODIPY-TMR). Using mutant Huntingtin-exon 1 (mHTT-Ex1) and -Synuclein A53T, we show Q-DOAS detects pre-amyloid oligomers, yielding quantitative kinetic data compatible with mechanistic analysis. We demonstrate its utility to dissect mutational effects, screen for protein and small-molecule inhibitors, and quantify amyloid seeding activity in cellular and mouse models of Huntingtons disease. Q-DOAS also detects seeds in cerebrospinal fluid from Parkinsons disease patients without amplification. Q-DOAS provides a sensitive, robust, and scalable tool for studying the earliest events in amyloid pathologies and for advancing therapeutic development.

biochemistry↗

bioRxiv2026

Scanning DIA on the ZenoTOF 8600 system enables ultra-sensitive and quantitative proteomics from single cells to post-translational modifications in a compact platform

Mass spectrometry-based proteomics increasingly demands platforms that combine quantitative rigor with the discovery capabilities of accurate mass systems. Here we present the ZenoTOF 8600 system, a compact mass spectrometry system that integrates enhanced ion capture and transmission optics with an optical detection system, Zeno trap-enhanced MS/MS, electron-activated dissociation, and scanning quadrupole data-independent acquisition (ZT Scan DIA). We show that ZT Scan DIA outperforms conventional variable-window DIA (Zeno SWATH DIA) in both identifications and quantitative reproducibility, and demonstrate the platforms versatility across proteomics applications: thousands of protein groups from bulk samples at up to 500 samples per day, single-cell proteomics yielding up to 4,700 proteins, accurate ratio recovery in mixed-species quantitative benchmarks, low-attomole targeted quantitation, and detection of disease-relevant phosphorylation in a Parkinsons disease cellular model using complementary CID and EAD fragmentation. The instruments compact footprint makes it attractive for settings where both analytical breadth and operational robustness are required.

biophysics↗

bioRxiv2025

Alpha-Synuclein aggregates inhibit ESCRT-III through sequestration and collateral degradation

-Synuclein aggregation is a hallmark of Parkinsons disease and related synucleinopathies. Extracellular -synuclein fibrils enter naive cells via endocytosis, followed by transit into the cytoplasm to seed endogenous -synuclein aggregation. Intracellular aggregates sequester numerous proteins, including subunits of the ESCRT-III system for endolysosome membrane repair, but the toxic effects of these events remain poorly understood. Using cellular models and in vitro reconstitution, we found that -synuclein fibrils interact with an -helix common to ESCRT-III proteins. This interaction results in sequestration of ESCRT-III subunits and triggers their proteasomal destruction in a process of "collateral degradation." These twin mechanisms deplete the available ESCRT-III pool, initiating a toxic feedback loop. The ensuing loss of ESCRT function compromises endolysosome membranes, thereby facilitating escape of aggregate seeds into the cytoplasm, which in turn increases aggregation and ESCRT-III sequestration. We suggest that collateral degradation and triggering of self-perpetuating systems could be general mechanisms of sequestration-induced proteotoxicity. HIGHLIGHTSO_LI-Synuclein fibrils bind and sequester ESCRT-III endolysosome repair proteins C_LIO_LIAn -helical segment common to ESCRT-III mediates fibril-selective interaction C_LIO_LIFibril-bound ESCRT-III subunits undergo "collateral degradation" via the proteasome C_LIO_LIESCRT-III depletion damages endolysosomes and worsens -synuclein aggregation C_LI

biochemistry↗

bioRxiv2024

Mechanochemical forces regulate the composition and function of CAT tails

The ribosome-associated quality control (RQC) pathway resolves stalled ribosomes. As part of RQC, stalled nascent polypeptide chains (NCs) are appended with CArboxy-Terminal amino acids (CAT tails) in an mRNA-free, non-canonical elongation process. CAT tail composition includes Ala, Thr, and potentially other residues. The relationship between CAT tail composition and function has remained unknown. Using biochemical approaches in yeast, we discovered that mechanochemical forces on the NC regulate CAT tailing. We propose CAT tailing initially operates in an "extrusion mode" that increases NC lysine accessibility for on-ribosome ubiquitination. Thr in CAT tails enhances NC extrusion by preventing formation of polyalanine, which can form -helices that lower extrusion efficiency and disrupt termination of CAT tailing. After NC ubiquitylation, pulling forces on the NC switch CAT tailing to an Ala-only "release mode" which facilitates nascent chain release from large ribosomal subunits and NC degradation. Failure to switch from extrusion to release mode leads to accumulation of NCs on large ribosomal subunits and proteotoxic aggregation of Thr-rich CAT tails. One sentence summaryMechanochemical forces regulate the composition of CAT tails to extrude or release stalled nascent chains and recycle ribosomes.

biochemistry↗

bioRxiv2019

Rqc2 levels alter CAT tail composition, function, and toxicity in S. cerevisiae

The Ribosome-associated Quality Control (RQC) pathway co-translationally marks incomplete polypeptides from stalled translation with two signals that trigger their proteasome-mediated degradation. The E3 ligase Ltn1 adds ubiquitin and Rqc2 directs the large ribosomal subunit to append carboxy-terminal alanine and threonine residues (CAT tails). When excessive amounts of incomplete polypeptides evade Ltn1, CAT-tailed proteins accumulate and can self-associate into aggregates. CAT tail aggregation has been hypothesized to either protect cells by sequestering potentially toxic incomplete polypeptides or harm cells by disrupting protein homeostasis. To distinguish between these possibilities, we modulated CAT tail aggregation in Saccharomyces cerevisiae with genetic and chemical tools to analyze CAT tails in aggregated and un-aggregated states. We found that enhancing CAT tail aggregation induces proteotoxic stress and antagonizes degradation of CAT-tailed proteins, while inhibiting aggregation reverses these effects. Our findings suggest that CAT tail aggregation harms RQC-compromised cells and that preventing aggregation can mitigate this toxicity.

cell biology↗

bioRxiv2018

CAT tails drive on- and off-ribosome degradation of stalled polypeptides

Stalled translation produces incomplete, ribosome-associated polypeptides that Ribosome-associated Quality Control (RQC) targets for degradation via the ubiquitin ligase Ltn1. During this process, the Rqc2 protein and large ribosomal subunit elongate stalled polypeptides with carboxy-terminal alanine and threonine residues (CAT tails). Failure to degrade CAT-tailed proteins disrupts global protein homeostasis, as CAT-tailed proteins aggregate and sequester chaperones. Why cells employ such a potentially toxic process during RQC is unclear. Here, we developed quantitative techniques to assess how CAT tails affect stalled polypeptide degradation in Saccharomyces cerevisiae. We found that CAT tails improve Ltn1s efficiency in targeting structured polypeptides, which are otherwise poor Ltn1 substrates. If Ltn1 fails, CAT tails undergo a backup route of ubiquitylation off the ribosome, mediated by the ubiquitin ligase Hul5. Thus, CAT tails functionalize the carboxy-termini of stalled polypeptides to drive their degradation on and off the ribosome.

cell biology↗