Biology subjects

Timms, R. T.

Publications and source records attributed to Timms, R. T..

9 recordsLinked to original sources

bioRxiv2026

Proteasomal surveillance of exposed cysteines

Cysteine residues are critical for both the catalytic activity and structural stability of many proteins, yet their high chemical reactivity threatens proteome integrity. Whether cells have specialized mechanisms to recognize and eliminate proteins with aberrant or exposed cysteines is unknown. By systematically analyzing the stability of thousands of cysteine-containing peptides, here we identify two complementary surveillance mechanisms targeting proteins with exposed cysteine residues. First, a set of E3 ubiquitin ligases, including AMBRA1, TRPC4AP, FBXO3, and FBXO21, detects double cysteines and targets proteins for ubiquitin-dependent proteasomal degradation. Second, the PSMD14/Rpn11 subunit of the 26S proteasome recognizes exposed cysteines in a ubiquitin-independent manner. Both pathways function in quality control to clear misfolded proteins exposing cysteine residues, including zinc finger-containing proteins. Mutational escape from the ubiquitin-independent pathway through dysregulation of ornithine decarboxylase or FOS levels is responsible for two human diseases, Bachmann-Bupp Syndrome and osteoblastoma, respectively. Our findings reveal conserved mechanisms for degrading proteins with exposed cysteines and uncover an alternative mode of substrate recognition by the 26S proteasome.

cell biology↗

bioRxiv2026

Regulation of proteasome activation by a ubiquitin-independent feedback mechanism

The ubiquitin-proteasome system is the major pathway for selective protein degradation in eukaryotic cells. Some proteins are degraded by the proteasome without ubiquitination, but the prevalence and underlying mechanisms remain poorly understood. Here, we use pulsed-SILAC proteomics to systematically identify proteins undergoing ubiquitin-independent proteasomal degradation (UbInPD). We identify the paralogous proteasome activation factors ZFAND5 and ZFAND6 (ZFAND5/6) and show that ZFAND5 contains a ubiquitin-independent degron that can both promote its rapid turnover and allosterically activate the proteasome. These findings support a model in which proteasome activation and activator degradation are coupled through a ubiquitin-independent feedback mechanism. We further show that ZFAND5/6, together with the scaffold protein p62, restrain NF-{kappa}B activation in the TLR4 pathway. We link this regulation to the degradation of UBCH5c, an abundant E2 ubiquitin-conjugating enzyme that can initiate or prime ubiquitin chain synthesis. Our findings expand the known landscape of UbInPD and reveal unexpected links between proteasome activation, E2 enzyme regulation, and inflammatory signalling.

biochemistry↗

bioRxiv2026

Proteome-wide C-degron activity profiling connects conditional regulation of the CTLH E3 ligase complex to ribosome biogenesis

Many E3 ubiquitin ligases recognize cognate degron motifs located at protein termini, but the paucity of bona fide substrates of N-degron and C-degron pathways hampers our understanding of their physiological significance. Here, by devising an expression screening approach to assess the effect of C-terminal "capping" on the stability of thousands of human proteins, we systematically identify a suite of full-length substrates harboring C-terminal degrons. Interrogating one leading candidate, ZMYND19, we characterize a C-degron pathway governed by the Muskelin substrate adaptor of the CTLH E3 ligase complex. Cell-to-cell variability in ZMYND19 stability uncovered conditional regulation, with CTLH-mediated degradation impaired by TNF- stimulation but enhanced by mTOR inhibition. Parallel genetic and proteomic screens identified two poorly characterized proteins, AAMP and AEN, as additional substrates of the CTLHMuskelin C-degron pathway, leading us to define an essential role for AAMP in ribosome maturation through chaperone activity towards ribosomal protein uL16. Altogether, these data define a C-degron pathway through which the Muskelin substrate adaptor connects conditional regulation of the CTLH E3 ligase complex to control of ribosome biogenesis.

molecular biology↗

bioRxiv2025

LRRC58 defines an E3 ubiquitin ligase complex sensitive to cysteine abundance

Adaptation to fluctuating nutrient supply is essential for organismal survival, but how cells sense and respond to changes in the abundance of many critical nutrients remains undefined. One such example is the amino acid cysteine, whose reactive thiol group is exploited for diverse cellular functions. Here, by characterizing the machinery required for the conditional degradation of cysteine dioxygenase type I (CDO1), the critical enzyme responsible for cysteine catabolism, we identify a Cul2 E3 ligase complex containing the uncharacterized substrate adaptor LRRC58 that is sensitive to cysteine abundance. When cysteine is replete, LRRC58 activity is restrained through auto-ubiquitination and proteasomal degradation; upon cysteine deprivation, LRRC58 is stabilized to permit CDO1 degradation. Through saturation mutagenesis stability profiling, we systematically validate a structural model of the CDO1-LRRC58 interaction and identify residues at the LRRC58 C-terminus required for cysteine-dependent instability. The LRRC58-mediated degradation of CDO1 is essential to prevent ferroptotic cell death under conditions of cysteine scarcity, and mutations in CDO1 which cause neurodevelopmental defects in humans encode dominant-active proteins refractory to LRRC58 recognition. Altogether, these data reveal the CDO1-LRRC58 axis as a critical regulator of cysteine homeostasis that safeguards neural development.

molecular biology↗

bioRxiv2024

Loss-of-function mutations in the dystonia gene THAP1 impair proteasome function by inhibiting PSMB5 expression

The 26S proteasome is a multi-catalytic protease that serves as the endpoint for protein degradation via the ubiquitin-proteasome system. Proteasome function requires the concerted activity of 33 distinct gene products, but how the expression of proteasome subunits is regulated in mammalian cells remains poorly understood. Leveraging coessentiality data from the DepMap project, here we characterize an essential role for the dystonia gene THAP1 in maintaining the basal expression of PSMB5. PSMB5 insufficiency resulting from loss of THAP1 leads to defects in proteasome assembly, impaired proteostasis and cell death. Exploiting the fact that the toxicity associated with loss of THAP1 can be rescued upon exogenous expression of PSMB5, we define the transcriptional targets of THAP1 through RNA-seq analysis and perform a deep mutational scan to systematically assess the function of thousands of single amino acid THAP1 variants. Altogether, these data identify THAP1 as a critical regulator of proteasome function and suggest that aberrant proteostasis may contribute to the pathogenesis of THAP1 dystonia.

genetics↗

bioRxiv2020

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

SummaryThe Human Silencing Hub (HUSH) complex epigenetically represses retroviruses, transposons and genes in vertebrates. HUSH therefore maintains genome integrity and is central in the interplay between intrinsic immunity, transposable elements and transcriptional regulation. Comprising three subunits - TASOR, MPP8 and Periphilin - HUSH regulates SETDB1-dependent deposition of the transcriptionally repressive epigenetic mark H3K9me3 and recruits MORC2 to modify local chromatin structure. However the mechanistic roles of each HUSH subunit remain undetermined. Here we show that TASOR lies at the heart of HUSH, providing a platform for assembling the other subunits. Targeted epigenomic profiling supports the model that TASOR binds and regulates H3K9me3 specifically over LINE-1 repeats and other repetitive exons in transcribed genes. We find TASOR associates with several components of the nuclear RNA processing machinery and its modular domain architecture bears striking similarities to that of Chp1, the central component of the yeast RNA-induced transcriptional silencing (RITS) complex. Together these observations suggest that an RNA intermediate may be important for HUSH activity. We identify the TASOR domains necessary for HUSH assembly and transgene repression. Structural and genomic analyses reveal that TASOR contains a poly-ADP ribose polymerase (PARP) domain dispensable for assembly and chromatin localization, but critical for epigenetic regulation of target elements. This domain contains a degenerated and obstructed active site and has hence lost catalytic activity. Together our data demonstrate that TASOR is a pseudo-PARP critical for HUSH complex assembly and H3K9me3 deposition over its genomic targets.

molecular biology↗

bioRxiv2019

Periphilin self-association underpins epigenetic silencing by the HUSH complex

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin forms -helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts.

biochemistry↗

bioRxiv2018

The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1

HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78, independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR and Insig-1, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent ligase partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation.

cell biology↗

bioRxiv2017

Neuropathic mutations in MORC2 perturb GHKL ATPase dimerization dynamics and epigenetic silencing by multiple structural mechanisms

Missense mutations in MORC2 cause neuropathies including spinal muscular atrophy and Charcot-Marie-Tooth disease. We recently identified MORC2 as an effector of epigenetic silencing by the HUSH complex. Here we report the biochemical and cellular activities of MORC2 variants, alongside crystal structures of wild-type and neuropathic forms of a human MORC2 fragment comprising the GHKL-type ATPase module and CW-type zinc finger. This fragment dimerizes upon binding ATP and contains a hinged, functionally critical coiled coil insertion absent in other GHKL ATPases. We find that dimerization and DNA binding of the MORC2 ATPase module transduce HUSH-dependent silencing. Disease mutations change the dynamics of dimerization by distinct structural mechanisms: destabilizing the ATPase-CW module, trapping the ATP lid or perturbing the dimer interface. These defects lead to modulation of HUSH function, thus providing a molecular basis for understanding MORC2-associated neuropathies.

biochemistry↗