The Eps15-Kelch13 protein complex – a potential regulator of endocytosis and a possible

The protein-complex, of which Eps15 is part of, is well described for CME in model organisms but seems to be different in P. falciparum according to the results of the DiQ-BioID with this protein.

Identification of potential interaction partners using the BioID technique, which allows in vivo tagging of proximal proteins, revealed a protein complex that is likely specific for Plasmodium. The proteins identified in the Eps15 DiQ-BioID do not suggest an obvious endocytosis-complex, but with a closer look based on amino acid homologies roles in endocytosis can be hypothesized for some of the identified proteins and this is discussed in the following paragraphs.

The results of the DiQ-BioID were confirmed with CoIP for Eps15 and Kelch13. This newly identified interactor Kelch13 is not just temporarily interacting with Eps15 but rather accompanying during the cycling of Eps15 movement between the PPM and the FV. Kelch-domain containing proteins are usually present in high numbers in eukaryotic genomes and have versatile functions (Adams et al.,

128 2000; Stogios et al., 2005). Specific roles are often defined by molecular architecture of the Kelch domains, additional functional domains or different binding partners of each Kelch-domain containing protein (Adams et al., 2000). The function of Kelch13 of P. falciparum was extensively discussed but remains unclear (Dogovski et al., 2015; Mbengue et al., 2015). It was already linked to the here also identified de-ubiquitinating enzyme (DUB) called UBP1 which is the critical hint toward a potential function in ubiquitination-pathways (Leznicki and Kulathu, 2017). This is in agreement with the identification of several RING-domain (Really Interesting New Gene-domain) containing proteins in the DiQ-BioID for Eps15. Proteins with such domains play a key role in ubiquitin-protein ligase interaction, often due to binding to UIM-containing proteins (Genschik et al., 2013; van Bergen En Henegouwen, 2009). Along with other multiple functions, ubiquitination has been shown to regulate endocytic and trafficking processes by controlling protein-protein interaction of E3 ubiquitin ligase, scaffolding proteins and Eps15 in Cullin3-based E3 ligase complexes (MacGurn et al., 2012; Yuan et al., 2014). This complex assembly relies on the RING-domain, the UIM of Eps15 and the BTB domain of a Kelch-protein (also termed BTB-Kelch) (Stogios et al., 2005). This BTB-Kelch domain is also present in the Eps15-interacting Kelch13 but a function in endocytosis has been excluded due to the lack of a BACK domain which is usually located between the BTB-Domain and the Kelch-Repeats (Tilley et al., 2016). Even though that many BTB-Kelch proteins do contain the BACK-domain, its importance and precise function has not been shown yet (Stogios et al., 2005). Crucial protein interaction with proteins of the Cullin3-based E3 ligase complexes is rather dependent on a short amino acid sequence at the C-terminus of the BTB-domain termed the 3-Box (structural similarity to F-Box or SOCS-Box) which is the binding site for Cullin3 in the BTB-Cul3 Ubiquitin Ligases (Canning et al., 2013; Stogios et al., 2005; Zhuang et al., 2009). Kelch13 indeed contains such a 3-Box sequence in its BTB domain (Figure 40). It is therefore possible that Kelch13 has a function in an endocytosis regulating Ubiquitin-Ligase dependent complex via ubiquitination of PfEps15 itself or interaction of ubiquitinated enzymes with the UIM of PfEps1.

129 Figure 40: The Kelch13 protein of P. falciparum contains a 3-Box domain for potential binding of ubiquitin ligases. A) The consensus sequence of the conserved 3-box of the BTB-domain in five eukaryotic proteins is shown. Structure based sequence alignment of 32 amino acids corresponding to 3-boxes from five Cul3-interacting BTB domain containing proteins. Consensus sequence of amino acids conserved in more than 50 % of the aligned proteins. Amino acids with red background are present in all aligned proteins. Blue boxes show conserved amino acid residues (highlighted with red font) (modified from Zhuang et al., 2009). B) Alignment (ClustalO) of the Eps15 interacting protein Kelch13 of P. falciparum with the 5 proteins from A. The 32 amino acids corresponding to the putative 3-box of PfKelch13 were found by alignment of the whole Kelch13 amino acid sequence to the consensus sequence from A. Amino acids present in Kelch13 that are conserved in more than 50 % of the aligned eukaryotic proteins are shown as consensus sequence. Amino acids conserved in all aligned proteins are shown in red bold letters and are highlighted by red frames in the alignment.

Proteins conserved in 50 % to 80 % are highlights in blue letters and blue frames.

Due to the fact that ubiquitinylating enzymes were concurrently linked with Artemisinin resistance, it was hypothesized that it is based on oxidative stress response and the proteasomal degradation pathways reacting to Artemisinin (Adams et al., 2018; Dogovski et al., 2015). Based on the indication from this thesis that Kelch13 and UBP1 interact with the possible endocytic protein Eps15 in P.

falciparum, different explanation of which role these de- and ubiquityniting enzymes play in this context is offered. The formation of endocytic vesicles is a highly orchestrated process which requires multiple regulation-sites. Besides membrane identity by different species of phosphatidyl inositides and protein-phosphorylation, ubiquitynliation is a modification that does not just destine proteins for degradation but also is used for regulation of protein-protein-interactions (Andersen et al., 2005). This has been shown especially for Eps15 (van Bergen En Henegouwen, 2009; Weinberg and Drubin, 2014). Since PfEps15 contains several putative ubiquitinylation-sites and a possible ubiquitin interaction motif (UIM) at its C-terminus, which has been shown to be of importance for regulation of endocytosis in neurons (Fallon et al., 2006; van Delft et al., 1997a), UBP1, which is one of the most significantly enriched proteins in the DiQ-BioID experiment for Eps15, could be the enzyme being

130 responsible for modification of Eps15 at its UIM. Overexpression of Eps15 was not possible before but since a codon-adjusted, intron-free eps15 gene of P. falciparum is available (unpublished), overexpression of Eps15 proteins with mutated or deleted ubiqitynilation-sites could be used to check for an altered localization or movement of these mutants. In case an efficient knock down or knock out of PfEps15 is available in the future, complementation with functional mutants after ablation of the endogenous protein to analyze the phenotype of the induced mutations would be highly informative in this respect.

Seen in a bigger context the hypothesized interactions of Eps15, Kelch13 and UBPs or DUBs are a matter of particular interest since mutations in Kelch13 are associated with resistance to the first-line anti-malaria drug Artemisinin (Ariey et al., 2014). It has already been shown, that Artemisinin function is dependent on Hemoglobin uptake (hence HCCU), hemoglobin digestion (Dogovski et al., 2015; Klonis et al., 2011; Mbengue et al., 2015; Tilley et al., 2016) and radical activation of Artemisinin by heme (Klonis et al., 2013; Meshnick et al., 1993) but no convincing mode of function of Artemisinin resistance could be drawn from these results (O'Neill et al., 2010). Data from this and previous work (Flemming, 2015) indicate a role for Eps15 in HCCU. Hence, the interacting artemisinin-resistance marker Kelch13 might also function in endocytosis. If HCCU is reduced because of Kelch13 variants, less hemoglobin is present and can be digested in the FV. This directly leads to less activated Artemisinin, possibly lowering it below the effective threshold inside the cell and therefore may allow survival of the parasites. A role of endocytosis in Artemisinin-resistance is supported by the identification of AP-2 in the DiQ-BioID with Eps15, which was recently shown to confer Artemisinin resistance (Adams et al., 2018; Henriques et al., 2014; Henriques et al., 2013;

Henriques et al., 2015; Sutherland et al., 2017). Taken together this offers an alternative hypothesis for Artemisinin resistance based on reduced HCCU.

Several also high confidence hits in the Eps15 DiQ-BioID are not obvious candidates of a potential enodcytosis complex. This includes a metacaspase, functioning as a possible cysteine-protease, or the 6-phsphofructokinase, which is important for glycolysis. The proteins identified with DiQ-BioID with Eps15 were confirmed with the vice-versa experiment using Kelch13 as target for DiQ-BioID (Birnbaum et al., 2017), supporting the identified interacting proteins and compartment neighbors for Eps15.