Project Overview: Introduction to WDR12 and WDR55 Proteins

I am currently completing my fourth-year thesis in Dr. Dalia Barsyte-Lovejoy’s lab. I have a strong interest in both pharmacology and biochemistry. I am interested in learning about biochemical pathways in the body, with the goal of finding compounds that can target those pathways and for them to potentially develop into therapeutics. My project focuses on investigating the localization and protein-protein interactions of 2 WD40 repeat proteins (WDR12 and WDR55) to understand the biological networks and functions of these proteins and facilitate the design of pharmacological targeting.

WD repeat proteins are protein-protein interaction modules that play roles in numerous biological processes and are emerging as attractive drug targets. WD40 repeat proteins have tryptophan-aspartic acid residue repeats and are defined by the doughnut shape-like structure: predominantly 7 blade β-propeller domain with a central pore¹ (Fig.1). The central pore, an important part of the protein that mediates PPIs, has a great potential to be druggable due to its size and shape that creates a suitable binding pocket for chemical probes¹. Chemical probes are potent, selective, and cell-active small molecules that are used as tools to investigate protein function². They can be used to enhance our understanding of proteins and can also be further developed into potential treatments for diseases². The two proteins in this study, WDR12, and WDR55, are associated with ribogenesis and are druggable targets with potential in oncology.

Figure 1. WDR12 protein structure from PDB (6N31), which shows the 7 blades β-propeller domain with a central pore.

WDR12 and Bop1 are WD40 repeat domain proteins that exhibit predominant nuclear localization, and they play an important role in the ribosomal biogenesis pathway³. They form a complex with Pes1 known as the PeBoW, and this complex regulates cell proliferation and cell cycle arrest. Hölzel et al. found that c-Myc upregulates the expression of all three proteins in cancer cells, and thus this upregulation of these proteins may drive cell proliferation³. PeBoW can also affect the cell cycle by regulating the activity of p53 through the amount of unprocessed rRNA³. Furthermore, mutations in WDR12 protein lacking the NH2-terminal Notchless-like domain still exhibits predominant nucleolar localization but leads to the accumulation of 32S rRNA and the reduction of 28S rRNA compared to wild type WDR12 (Fig. 2), which induces the p53-dependent cell cycle arrest. Additionally, Pes1, Bop1, and WDR12 proteins are a set of evolutionarily conserved proteins that are also found in yeast. The homologous proteins of Pes1, Bop1, and WDR12 in yeast are Nop7, Erb1, and Ytm1 respectively, and those proteins are also important for the ribosomal biogenesis in yeast. Erb1 has the defined doughnut shape structure like other WD40 repeat domain proteins, and it also has an extended N-terminus tail that mediates many protein-protein interactions in specific states of the pre-60S ribosome assembly pathway⁴. Both Nop7 and Ytm1 have specific interaction sites on Erb1. Erb1 interacts with Nop7 on the extended N-terminus tail that is closer to the C-terminus β-propeller domain and interacts with Ytm1 directly on the β-propeller domain⁴ (Fig. 3)⁴.

Figure 2. Ribosomal biogenesis pathway. PeBoW has been shown to play a crucial role in the synthesis of 32S to 28S rRNA; thus, the defect in this complex leads to the accumulation of 28S rRNA. The role of WDR55 in the synthesis of 32S to 5.8S rRNA is still unclear, but previous studies have reported that the defect in WDR55 leads to the accumulation of 5.8S rRNA.

 

Figure 3. Schematic of Erb1 protein-protein interacts during ribosomal biogenesis. Bop1, Pes1, and WDR12 are the human homologs of yeast Erb1, Nop7, and Ytm1, respectively.

WDR55 is another WD40 repeat domain protein that exhibits nucleolar localization, and there are very few studies that have been done on this protein. Iwanami et al. showed that WDR55 is localized in the dense fibrillar component of the nucleolus because of partial colocalization with fibrillarin (Fig. 4), which is where the early processes of the ribosomal biogenesis take place^5,6, including the synthesis, processing, and modification of rRNA⁷. Defective WDR55 has no effects on the overall ribosome production, but it causes the accumulation of partially processed 5.8S (Fig. 2) and p53 dependent cell cycle arrest in phase G1⁵. It also suggested that WDR55 does not associate with the PeBoW through co-immunoprecipitation. However, a recent study suggested otherwise. Huttlin et al. used WDR55 as bait in affinity-capture mass spectroscopy, and the results showed that WDR55 interacted with Bop1 and Pes1 with high confidence score⁸.

Figure 4. Different components of the nucleolus include fibrillar centre, dense fibrillar component, and granular component.

References:

1. Schapira M, Tyers M, Torrent M, Arrowsmith CH. WD40 repeat domain proteins: A novel target class? Nat Rev Drug Discov. 2017;16(11):773-786. doi:10.1038/nrd.2017.179
2. Arrowsmith CH, Audia JE, Austin C, et al. The promise and peril of chemical probes. Nat Chem Biol. 2015;11(8):536-541. doi:10.1038/nchembio.1867
3. Hölzel M, Rohrmoser M, Schlee M, et al. Mammalian WDR12 is a novel member of the Pes1-Bop1 complex and is required for ribosome biogenesis and cell proliferation. J Cell Biol. 2005;170(3):367-378. doi:10.1083/jcb.200501141
4. Kater L, Thoms M, Barrio-Garcia C, et al. Visualizing the Assembly Pathway of Nucleolar Pre-60S Ribosomes. Cell. 2017;171(7):1599-1610.e14. doi:10.1016/j.cell.2017.11.039
5. Iwanami N, Higuchi T, Sasano Y, et al. Correction: WDR55 Is a Nucleolar Modulator of Ribosomal RNA Synthesis, Cell Cycle Progression, and Teleost Organ Development. PLoS Genet. 2008;4(9). doi:10.1371/annotation/6683988c-4647-4550-88e3-e8ba052f7916
6. Cooper GM. The Nucleolus. 2000. https://www.ncbi.nlm.nih.gov/books/NBK9939/. Accessed November 25, 2020.
7. J Lafontaine DL, Riback JA, Bascetin meyza, Brangwynne CP. The nucleolus as a multiphase liquid condensate. Nat Rev Mol Cell Biol. doi:10.1038/s41580-020-0272-6
8. Huttlin EL, Bruckner RJ, Paulo JA, et al. Architecture of the human interactome defines protein communities and disease networks. Nature. 2017;545(7655):505-509. doi:10.1038/nature22366