Biology subjects

Kleiger, G.

Publications and source records attributed to Kleiger, G..

3 recordsLinked to original sources

bioRxiv2026

Global Data Fitting Enables Kinetic Dissection of Cullin-RING Ligase-Catalyzed Ubiquitylation: From Substrate Priming to Poly-ubiquitin Chain Formation

The discovery and characterization of the ubiquitin system was founded on classical enzymology approaches that established fundamental principles of protein ubiquitylation. Cullin-RING ligases (CRLs) represent the largest class of human ubiquitin ligases, yet detailed enzymology of these multi-subunit assemblies has lagged behind other systems such as nucleic acid polymerases. Here, we employed pre-steady state kinetics and global data fitting using KinTek Explorer to systematically investigate the kinetic parameters controlling CRL-dependent ubiquitin transfer. Using a well-characterized in vitro reconstituted system exploring CUL2-dependent CRLs, we modeled substrate priming, showing that the system is highly amenable to global fitting of kinetic data. The results show that product dissociation from the CRL is significantly slower than substrate dissociation, explaining previously reported slow steady-state ubiquitylation rates. Extending this framework to poly-ubiquitin chain formation revealed catalytic efficiencies 15-42-fold greater than substrate priming. Comparative analysis of CUL1-dependent and CUL2-dependent systems showed remarkably similar kinetic parameters, including the Km of ubiquitin-carrying enzyme for CRLs, despite differences in substrate receptor and enzyme architecture. We further examined drug-induced neo-substrate ubiquitylation using the pomalidomide-CRBN-SALL4 system, revealing atypical kinetics characterized by rapid ubiquitin transfer paired with an elevated Km, ultimately yielding catalytic efficiencies comparable to natural substrates. The findings establish pre-steady state kinetics and global data fitting as powerful enzymological tools for dissecting CRL-dependent ubiquitylation across different CRL families and substrate contexts, with important implications for understanding both physiological ubiquitylation and therapeutic targeted protein degradation strategies.

biochemistry↗

bioRxiv2025

Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1

Cellular responses to amino acid fluctuations often hinge on ubiquitin-mediated control of metabolic enzymes, yet the underlying E3 ligase pathways remain incompletely defined. Using quantitative proteomics and active cullin-RING ligase (CRL) profiling, we identify LRRC58 as a cysteine-responsive substrate receptor whose stability increases sharply under cysteine starvation. Proteomics reveals an inverse relationship between LRRC58 and the metabolic enzyme cysteine dioxygenase 1 (CDO1), suggesting a cysteine-linked regulatory axis. Biochemical reconstitution and cryo-EM structures show that LRRC58 forms an active CUL2-or CUL5-based CRL that selectively positions CDO1 for ubiquitylation at Lys8. By contrast, targeted protein degradation via the VHL-based CRL is achieved by recruitment of a distinct surface of CDO1, and more promiscuous ubiquitylation independent of Lys8. Together, our proteomics-guided discovery pipeline, cellular stability studies, and structural analyses uncover a metabolically-tuned LRRC58-CDO1 pathway that links cysteine availability to selective proteasomal turnover, reveals principles of metabolite-regulated CRL activity, and showcases mechanisms distinguishing endogenous and targeted protein degradation.

biochemistry↗

bioRxiv2024

Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination

Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of [~]20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2Ds central role in proteome management.

biochemistry↗