Substrate Trapping To Discover The Role Of Histone Deacetylase Proteins Beyond Epigenetics

Gene expression is regulated by chromatin remodeling factors and histone modifications, such as phosphorylation, methylation and acetylation. Acetylation is regulated by histone acetyltransferases and histone deacetylases. Histone deacetylases remove acetyl groups from ε-N-acetyl lysine amino acids, which allows DNA to wrap around the histones more tightly. The discovery of a wide variety of acetylated proteins suggests that HDAC proteins likely deacetylate other substrates in addition to histones. By identifying non-histone substrates, HDAC proteins have been linked to multiple cellular processes in addition to gene expression. However, with only few known substrates, the full role of HDAC proteins in cellular events was unclear. In prior HDAC-related research, substrate identification has been largely serendipitous. This thesis work focused on the development of substrate trapping mutants of HDAC proteins, namely HDAC1 and HDAC6, to discover new substrates. Discovery of novel substrates for HDAC1 and HDAC6 using trapping mutants may lead to a better understanding of the role of the proteins in diseases. Given the high sequence similarity between HDAC family members, substrate trapping was applied to identify novel substrates of HDAC6. Due to the presence of two catalytic domains in HDAC6, four mutants (two from each domain) were screened to assess the relative trapping ability towards a known substrate, tubulin. H611A, a second catalytic site mutant, was identified as the optimal substrate trapping mutant for HDAC6. Proteomics-based substrate trapping using H611A identified a profile of potential HDAC6 substrates. Many of the previously known HDAC6 substrates were identified, validating the method. Protein arginine methyltransferase 5 (PRMT5) was identified as a new substrate of HDAC6, which further revealed a crosstalk between acetylation and methyltransferase activity. Another HDAC6 substrate, Serine/threonine kinase 38 like protein (STK38L), was shown to have a novel role in ciliogenesis through HDAC6-mediated deacetylation. To optimize substrate trapping for HDAC1, the relative trapping abilities of 17 inactive HDAC1 mutants were assessed using two known substrates of HDAC1. HDAC1 H141A, F150A, and C151A showed strong binding to the protein substrates LSD1 and p53. Interestingly, each mutant preferentially trapped a different substrate. By combining several inactive mutants, the trapping strategy facilitates discovery of new HDAC1 substrates and shed light on the variety of HDAC1-related functions in cell biology. In summary, this thesis work established a systematic method to identify novel substrates of HDAC proteins to uncover new HDAC-related functions

Preventable hand injuries: A national audit

Summary: Little is known of the scale of avoidable injuries presenting to medical services on a national level in the UK. This study aimed to assess the type and incidence of preventable wrist and hand injuries (as defined by the core research team) at a national level in the UK. 28 UK hospitals undertook a service evaluation of all hand trauma cases presenting to their units over a 2 week period in early 2021 identifying demographical and aetiological information about injuries sustained. 1909 patients were included (184 children) with a median age of 40 (IQR 25-59) years. The commonest five types of injury were fractures of the wrist; single phalangeal or metacarpal fractures; fingertip injuries; and infection, with the most common mechanisms being mechanical falls and manual labour. This is the first extensive survey of preventable hand injuries in the UK, identifying a need for further work into prevention to reduce healthcare burden and cost. 50% of injuries presenting to hand surgeons are preventable, with the most common injuries being single fractures of the wrist, phalanx and metacarpal. Few preventable injuries were related to alcohol or narcotic intoxication. Further research is needed to identify how to initiate injury prevention measures for hand injuries, particularly focussed towards hand fracture prevention