Biology subjects

Lehner, P. J.

Publications and source records attributed to Lehner, P. J..

4 recordsLinked to original sources

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↗

bioRxiv2018

Functional proteomic atlas of HIV-infection inprimary human CD4+ T cells

Viruses manipulate host cells to enhance their replication, and the identification of host factors targeted by viruses has led to key insights in both viral pathogenesis and cellular physiology. We previously described global changes in cellular protein levels during human immunodeficiency virus (HIV) infection using transformed CEM-T4 T cells as a model. In this study, we develop an HIV reporter virus displaying a streptavidin-binding affinity tag at the surface of infected cells, allowing facile one-step selection with streptavidin-conjugated magnetic beads. We use this system to obtain pure populations of HIV-infected primary human CD4+ T cells for detailed proteomic analysis, including quantitation of >9,000 proteins across 4 different donors, and temporal profiling during T cell activation. Remarkably, amongst 650 cellular proteins significantly perturbed during HIV infection of primary T cells (q<0.05), almost 50% are regulated directly or indirectly by the viral accessory proteins Vpr, Vif, Nef and Vpu. The remainder have not been previously characterised, but include novel Vif-dependent targets FMR1 and DPH7, and 192 targets not identified and/or regulated in T cell lines, such as AIRD5A and PTPN22. We therefore provide a high-coverage functional proteomic atlas of HIV infection, and a mechanistic account of HIV-dependent changes in its natural target cell.

microbiology↗

bioRxiv2018

Vpr drives massive cellular proteome remodelling in HIV-1 infection

Viral infection causes global remodelling of the cellular proteome. We previously mapped the temporal changes in abundance of thousands of proteins in HIV-1 infected cells (Greenwood & Matheson, 2016). While a small proportion of these changes were attributable to specific HIV-1 accessory proteins, most were unexplained. Here, we use complementary unbiased mass spectrometry-based approaches to demonstrate that a single viral protein, Vpr, is both necessary and sufficient to cause the vast majority of these changes. This global protein regulation requires substrate binding and degradation via DCAF1, but is mostly independent of Vpr-mediated cell cycle arrest. Combined approaches of pulsed-Stable Isotope Labelling with Amino Acids in Cell Culture (pulsed-SILAC) and immunoprecipitation-mass spectrometry (IP-MS) identified at least 38 such cellular proteins directly targeted for degradation by Vpr. Thus, whilst other HIV-1 accessory proteins downregulate a small number of host factors, Vpr degrades multiple protein targets, causing systems-level remodelling of the cellular proteome.\n\nImpact statementHIV infection causes massive changes to the cellular proteome and a single HIV protein, Vpr, is necessary and sufficient to drive almost all these changes by degrading multiple host proteins.

microbiology↗

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↗