Biology subjects

Shao, G.-C.

Publications and source records attributed to Shao, G.-C..

3 recordsLinked to original sources

bioRxiv2026

Enhancing the Identification of NHS Ester-Mediated Lysine-Cysteine Cross-Linking via Reduced Trypsin Digestion Time

Abstract Chemical cross-linking mass spectrometry (XL-MS) is a powerful technique for elucidating protein structures and interactions, with NHS ester-based cross-linkers being the most widely used. Traditionally, NHS esters are considered highly specific for primary amines; although their reactivity toward cysteine thiols has been reported, it has not been systematically characterized in XL-MS due to the extreme lability of the resulting thioester bonds. Here, we demonstrate that NHS esters efficiently label cysteine residues and form stable lysine-cysteine (K-C) cross-links--species previously deemed labile in XL-MS. To preserve these thioester-based cross-linked sites , we optimized the standard XL-MS workflow and applied it to four model proteins. K-C cross-links constitute 23% to 59% of total detected cross-links, with C-C distances predominantly ranging from 12 to 40 [A], and 60% satisfying theoretical linker constraints -- confirming that K-C cross-links are as reliable as canonical lysine-lysine (K-K) cross-link sites. Cross-software validation and benchmarking against the heterobifunctional K-C cross-linker GMBS further demonstrated that our optimized workflow captures ~ 40% of GMBS-identified K-C cross-linked sites. Collectively, these findings expand the practical utility of NHS esters and enhance protein structural characterization by enabling simultaneous acquisition of K-K and K-C distance constraints.

biochemistry↗

bioRxiv2025

Nucleophagy is promoted by two autophagy receptors and inhibited by chromatin-nuclear envelope tethering in fission yeast

Selective autophagy of the nucleus, known as nucleophagy, targets nuclear components for degradation. The molecular mechanisms underlying nucleophagy remain inadequately understood. In this study, we identify a nucleophagy receptor, Npr1, in the fission yeast Schizosaccharomyces pombe. Npr1 is an Atg8-binding multi-transmembrane protein localized to the outer nuclear membrane. It functions redundantly with another autophagy receptor Epr1 to promote nitrogen starvation-induced nucleophagy. In the absence of both Npr1 and Epr1, starved cells exhibit abnormal nuclear morphology and reduced survival. During nucleophagy, the nuclear envelope (NE) forms outward protrusions where Atg8 co-localizes with Npr1 and/or Epr1. These protrusions subsequently detach from the NE, resulting in the formation of autophagosomes that contain nucleophagy cargo. Notably, artificially enhancing chromatin association with the inner nuclear membrane leads to NE protrusions that fail to detach, thereby aborting nucleophagy. Our findings provide mechanistic insights into nucleophagy and suggest that abortive nucleophagy protects chromatin from degradation.

cell biology↗

bioRxiv2023

Ubiquitination-mediated Golgi-to-endosome sorting determines the poison-antidote duality of wtf meiotic drivers

Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. Here we investigate how the toxin and antidote products of a fission yeast wtf-family KMD gene can act antagonistically. Both the toxin and the antidote are multi-transmembrane proteins, differing only in their N-terminal cytosolic tails. We find that the antidote employs N-terminal PY motifs (Leu/Pro-Pro-X-Tyr) to bind Rsp5/NEDD4 family ubiquitin ligases, which ubiquitinate the antidote. Mutating PY motifs or attaching a deubiquitinating enzyme transforms the antidote into a toxic protein. Ubiquitination promotes the transport of the antidote from the trans-Golgi network to the endosome, thereby preventing it from causing toxicity. A physical interaction between the antidote and the toxin enables the ubiquitinated antidote to translocate the toxin to the endosome and neutralize its toxicity. We propose that post-translational modification-mediated protein localization and/or activity changes may be a common mechanism governing the antagonistic duality of single-gene KMDs.

cell biology↗