Biology subjects

Liddle, J. C.

Publications and source records attributed to Liddle, J. C..

4 recordsLinked to original sources

bioRxiv2023

Proximity labeling reveals new insights into the relationships between meiotic recombination proteins in S. cerevisiae

Several protein ensembles facilitate MutS{gamma} crossover recombination and the associated process of synaptonemal complex (SC) assembly during meiosis, but the physical and functional relationships between the components involved remain obscure. We have employed proximity labeling as a phenotypic tool to discern functional relationships between meiotic recombination and SC proteins in S. cerevisiae, and to gain deeper insight into molecular deficits of crossover-defective mutants. We find that recombination initiation (Spo11) and the Mer3 helicase are dispensable for proximity labeling of the Zip3 E3 ligase by components of the ZZS ensemble (Zip2, Zip4 and Spo16) but are required for proximity labeling of Zip3 by Msh4, consistent with the possibility that MutS{gamma} joins Zip3 only after a specific recombination intermediate has been generated. Proximity labeling analysis of crossover-defective zip1 mutants suggests a key shared defect is a failure to assemble an early recombination ensemble where ZZS can properly engage Zip3. We furthermore discovered that Zip3s abundance within the meiotic cell is uniquely dependent on the presence of Zip1, and that the post-translational modification of Zip3 is promoted by most MutS{gamma} pathway proteins but countered by Zip1. Based on this and additional data, we propose a model whereby Zip1 stabilizes a functional, unmodified form of Zip3 until intermediate steps in recombination are complete. We also find that SC structural protein Ecm11 is proximity labeled by ZZS complex proteins in a Zip4-dependent manner, but by Zip3 and Msh4, at least in part, via a distinct pathway. Finally, streptavidin pulldowns followed by mass spectrometry on eleven different proximity labeling strains uncovers shared proximity targets of MutS{gamma}-associated proteins, some with known meiotic functions and others not yet implicated in a meiotic activity, highlighting the potential power of proximity labeling as a discovery tool.

genetics↗

bioRxiv2021

The Okur-Chung Neurodevelopmental Syndrome (OCNDS) mutation CK2K198R leads to a rewiring of kinase specificity

Okur-Chung Neurodevelopmental Syndrome (OCNDS) is caused by heterozygous mutations to the CSNK2A1 gene, which encodes the alpha subunit of casein kinase II (CK2). The most frequently occurring mutation is lysine 198 to arginine (K198R). To investigate the impact of this mutation, we first generated a high-resolution phosphorylation motif of CK2WT, including the first characterization of specificity for tyrosine phosphorylation activity. A second high resolution motif representing CK2K198R substrate specificity was also generated. Here we report for the first time the impact of the OCNDS associated CK2K198R mutation. Contrary to prior speculation, the mutation does not result in a loss of function, but rather shifts the substrate specificity of the kinase. Broadly speaking the mutation leads to 1) a decreased preference for acidic residues in the +1 position, 2) a decreased preference for threonine phosphorylation, 3) an increased preference for tyrosine phosphorylation, and 4) an alteration of the tyrosine phosphorylation specificity motif. To further investigate the result of this mutation we have developed a probability-based scoring method, allowing us to predict shifts in phosphorylation in the K198R mutant relative to the wild type kinase. As an initial step we have applied the methodology to the set of axonally localized ion channels in an effort to uncover potential alterations of the phosphoproteome associated with the OCNDS disease condition.

neuroscience↗

bioRxiv2020

Viral-mediated ubiquitination impacts interactions of host proteins with viral RNA and promotes viral RNA processing

Viruses promote infection by hijacking host ubiquitin machinery to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defenses and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K/E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins (RBPs) as high-confidence substrates which are not decreased in overall abundance. We focused on two RBPs, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance, and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with virus deleted for E1B55K resulted in increased interaction of hnRNP-C with viral RNA, and attenuation of viral RNA processing. These data suggest viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing, revealing an unexpected role for non-degradative ubiquitination in manipulation of cellular processes during virus infection.

microbiology↗

bioRxiv2019

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Modulating the dynamics of protein kinases expands the inhibitory mechanisms for small molecules. NMR measurements of the MAP kinase, ERK2, have shown that activation by dual-phosphorylation induces global motions involving exchange between two states, \"L\" and \"R\". We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions, while inhibitor GDC-0994 and ATP analogue AMP-PNP retain L{rightleftharpoons}R exchange. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analogue. Thus, the L[->]R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding modes for Vertex-11e, GDC-0994 and AMP-PNP to active vs inactive ERK2, where the extent of HX protection matches their degree of R-state formation. In addition, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop, suggesting that L{rightleftharpoons}R exchange involves coupling between the activation loop and the active site. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity towards 2P-ERK2. We conclude that global motions in ERK2 promote productive nucleotide binding, and couple with the activation loop to allow control of dephosphorylation by conformation-selective inhibitors.\n\nSIGNIFICANCE STATEMENTProtein kinases in the RAF/MKK/ERK signaling pathway are dysregulated in cancer and are important targets for inhibitor development. Catalytic activation of the MAP kinase, ERK2, induces global motions involving exchange between two conformational states. Using nuclear magnetic resonance (NMR) and hydrogen-exchange mass spectrometry, we show that inhibitors exploit these motions to trap ERK2 in distinct states. Our findings reveal motions of the activation loop coupled to the active site. Inhibitor binding can control these activation loop dynamics to alter its rate of dephosphorylation by MAP kinase phosphatase.

biochemistry↗