Expression of Kelch-like protein 11 (KLHL11) in N471D Washc5 knock-in mouse tissues

Abstract

Retrograde degeneration of nerve fibers in the spinal cord are critical characteristics for hereditary spastic paraplegia (HSP), a neurodegenerative disorder. Several subtypes of HSP are known, with SPG8 being one of the autosomal dominant forms that is known to develop from mutations in strumpelin/WASHC5, a core member of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. Previous studies generated N471D knock-in mice to investigate SPG8 (Clemen et al., 2021). Notably, KLHL11 was reported to be significantly downregulated in homo- and heterozygous knock-in mice brain tissue. Indeed, KLHL11 has priorly been described to be involved in neurological disorder, however, functions of KLHL11 in the context of SPG8 remained elusive. Nonetheless, KLHL11 is known to be part of Cullin 3 E3 ligase complexes, and to interact with LNX1, another E3 ligase complex. Even though the proteomic homeostasis is a highly regulated process and, therefore, extensively studied, little is known about KLHL11 expression and localization. Consequently, the objective of this study was to investigate KLHL11 in its context of cellular expression and localization, and to get first hints for its role in HSP/SPG8. Accordingly, protein levels were quantified to prove the hypothesis that KLHL11 is ubiquitously expressed in different tissues. In addition, immunofluorescence staining was performed for intercellular localization. Here, a striking punctuated cytoplasmic staining pattern around the nucleus is shown. Moreover, subcellular fractionation limited intercellular localization. The expression level in different genotypes of Washc5 knock-in mice tissue confirm previous studies by demonstrating significant downregulation of KLHL11. Furthermore, in a mass spectrometry approach, proteome analysis of different KLHL11-CFP transfected cells, provided insights to the identity of binding partners and co-localizations. Here, proteome analysis revealed enrichment of several proteasomal subunits, indicating the association of KLHL11 in the cellular degradation machinery. A key finding is that downregulation of KLHL11 in Washc5 knock-in mice might be related to the strumpellin binding partner VCP/p97, which has implications for future studies on the function, bindings partners, and expression pattern of KLHL11. In total, the present study paves the way towards a more exhaustive understanding of KLHL11, its localization, and its role in Washc5 knock-in mice.