Recent advances in glycosome biogenesis and its implications for drug discovery
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Lisa Hohnen
,
Chethan K. Krishna
,
Lewis Walker
Abstract
The phylum Euglenozoa, within the Eukaryote domain, includes diverse protists such as the medically significant kinetoplastids, characterized by their unique kinetoplast DNA. Both kinetoplastids and their sister class Diplonemea possess glycosomes – specialized microbodies that compartmentalize glycolysis and other metabolic pathways. Glycosomes likely evolved in a common ancestor of kinetoplastid and diplonemids, conferring metabolic flexibility and reducing cellular toxicity. These organelles are essential for parasite survival and thus, represent promising drug targets for treating kinetoplastid diseases. While the basic principles of peroxisome and glycosome biogenesis are conserved, distinct features in glycosome biogenesis machinery and a lower level of sequence conservation enables pathogen specific drug design for developing new therapies. This review summarizes our current knowledge on glycosome biogenesis, recent advances, and therapeutic potential for treating trypanosomatid infections.
1 Introduction
The phylum Euglenozoa belongs to protists of the Eukaryote domain (Cavalier-Smith 2016; d’Avila-Levy et al. 2015). It comprises a variety of free living and parasitic protists, which are medically relevant, particularly within the class Kinetoplastea (d’Avila-Levy et al. 2015). These include Trypanosoma and Leishmania, which cause vector-borne neglected tropical diseases: Human African Trypanosomiasis (HAT, also known as sleeping sickness) and Nagana in livestock animals caused by Trypanosoma brucei (Lejon et al. 2025; Steverding 2017), Chagas disease caused by Trypanosoma cruzi (Swett et al. 2024) and various forms of leishmaniasis caused by several species of Leishmania parasites (Mathison and Bradley 2023). Kinetoplastid protists are defined by the presence of a kinetoplast – a dense, DNA-rich structure within a single, branched mitochondrion located at the base of the flagellum. Kinetoplast DNA (kDNA), a complex network of mitochondrial interlocked circular DNA molecules, is essential for mitochondrial function (d’Avila-Levy et al. 2015). The further subdivision of the Kinetoplastea class into separate orders can be determined by the distribution pattern of kDNA within the kinetoplast. For example, when kDNA is compacted and positioned close to the flagellar pocket, the arrangement is termed eukinetoplastic, a characteristic feature of organisms in the order Trypanosomatida (d’Avila-Levy et al. 2015).
Kinetoplastic protists, along with their sister class Diplonemea, are part of the subphylum Glycomonada that exclusively possess specialized microbodies, which compartmentalize part of the glycolytic pathway, termed glycosomes (Cavalier-Smith 2016). It is believed that the glycosome evolved within the subphylum Glycomonada, in a common ancestor of Kinetoplastea and Diplonemea, since both contain this organelle (Andrade-Alviárez et al. 2022; Gualdrón-López et al. 2012). It is likely that glycosomes originated through a mislocalization of metabolic enzymes to peroxisomes, which provided a selective advantage by enhancing metabolic flexibility (Gualdrón-López et al. 2012).
Historically, the function of glycosomes was a subject of debate; one early hypothesis proposed that the high density of glycolytic enzymes facilitated higher glycolytic flux (Opperdoes 1987). However, this was considered unlikely when glycosomes were discovered in some kinetoplastids that did not have high glycolytic flux (Gualdrón-López et al. 2012). A prevailing view is that glycosomes enable rapid shifts in metabolic potential, as dynamic turnover allows kinetoplastids to efficiently adapt to changing extracellular environment (Andrade-Alviárez et al. 2022; Szöör et al. 2014). Furthermore, by compartmentalizing metabolic pathways, the glycosome helps to minimize the exposure of the cytosol and other organelles to potentially toxic intermediates, such as sugar-phosphates (glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate) or methylglyoxal (Bakker et al. 2000; Haanstra et al. 2008; Wyllie and Fairlamb 2011). For an extensive study on the evolution of glycosomes, refer to the recent report by Andrade-Alviárez et al. (2022).
Glycosomes are single membrane-bound organelles with a dense protein matrix, which compartmentalizes a variety of metabolic processes depending on the species and life cycle stage; pathways include glycolysis (with the first seven enzymes inside glycosomes), gluconeogenesis, pentose phosphate pathway, purine and pyrimidine biosynthesis, fatty acid beta-oxidation and sugar nucleotide synthesis (Allmann and Bringaud 2017). Due to the organelles’ critical role in metabolism, particularly in housing most of the glycolytic pathway, glycosomes are essential for parasite survival and disruption of their biogenesis is lethal (Furuya et al. 2002). Therefore, they represent an ideal drug target for kinetoplastid pathologies (Haanstra et al. 2016; Kalel et al. 2018).
As peroxisome-related organelles, glycosomes import their proteins post-translationally by the concerted action of various peroxin (PEX) proteins. In this review, we briefly summarize the current understanding of glycosome biogenesis, with a particular emphasis on the molecular machineries for glycosomal protein import and emerging therapeutic strategies targeting these glycosomal pathways in trypanosomatid infections.
2 Glycosomal membrane protein import
Glycosomal/peroxisomal membrane proteins (PMPs) are required for glycosome biogenesis but also for maintaining the organelle’s metabolic roles by enabling transport of solutes across the membrane and supporting redox shuttling (Theodoulou et al. 2013). Accordingly, the import of membrane proteins into glycosomes is a fundamental process. Glycosome biogenesis involves the targeting of PMPs and the import of matrix proteins (Figure 1), both processes mediated by PEX proteins. Of the 20 peroxins (and respective homologs) identified in trypanosomatids, which are summarized in Table 1, 13 are glycosomal membrane proteins, underscoring the central role of the glycosomal membrane in glycosome dynamics and maintenance (Krishna et al. 2025a).
Membrane protein and matrix protein import into glycosomes in kinetoplastids. Glycosomal proteins are synthesized in the cytosol and subsequently targeted to the glycosomal membrane or lumen. Proteins destined for the glycosomal membrane are recognized by the cytosolic receptor PEX19, bound to the novel euglenozoan-specific peroxin PEX38, via their membrane peroxisome targeting signal (mPTS). PEX19 interacts with its membrane anchor and docking factor PEX3 to target glycosomal membrane proteins to the membrane (left). In contrast, glycosomal matrix proteins are recognized by the cytosolic receptor PEX5 and co-receptor PEX7, depending on whether they carry a PTS1 or PTS2 targeting signal, respectively (middle, step 1). The cargo-loaded receptor binds to the membrane-bound docking complex consisting of PEX13.1, PEX13.2 and PEX14, which form an import pore (step 2). The cargo is released into the glycosomal lumen (step 3). While PEX7 is known to enter the glycosomal matrix, this is not resolved for PEX5. During or after cargo release, PEX5 is monoubiquitinated by the RING-finger complex PEX2–10–12, and PEX4–22 (step 4). Finally, the AAA+ ATPase PEX1–PEX6, which is recruited to the membrane by PEX15, extracts ubiquitinated PEX5 from the membrane for recycling and another round of matrix protein import (step 5). How PEX7 translocates back to the cytosol is not yet clarified. The novel peroxin PEX39 involved in PTS2 import has not been identified in kinetoplastids. PEX11 family proteins, absence of PEX37, and Ykt6 are shown on the right. Lower left inset: Three different import complexes have been identified in Trypanosoma brucei: a large complex (L) containing PEX13.1, PEX13.2 and PEX14, an intermediate complex (I) containing PEX13.1 and PEX13.2 and a small complex (S) with PEX14 proteins only. In other eukaryotes, it was shown that the intrinsically disordered regions including the YG-rich stretches form condensates. While it is very likely to also be the case in kinetoplastids, it remains to be experimentally verified for PEX13.1 and PEX13.2. Both termini of PEX13.1 and PEX13.2 were found to face the cytosol. In other organisms, PEX13 has dual topology leading to presence of the SH3 domain in both the matrix and the cytosol. Lower right inset: In the case of PEX5 monoubiquitination, PEX5 gets recycled and is made available for another round of matrix protein import with the aid of the PEX1–PEX6–PEX15 complex (left). In the case of polyubiquitination, PEX5 is degraded by the ubiquitin proteasome system (UPS) via the RADAR pathway (right). Msp1/ATAD1 and/or Cdc48/p97/VCP and its cofactor FAF2 might be involved in extraction of polyubiquitinated PEX5 as was shown for yeast and human. This remains to be clarified. The figure was created using BioRender.
Peroxins (PEX) and accessory proteins involved in peroxisome (human) and respective homologs involved in glycosome (Trypanosoma brucei, Trypanosoma cruzi, Leishmania spp., diplonemids) biogenesis. The Table summarizes identified (with bioinformatic tools) and characterized PEX proteins and its unique features in kinetoplastids and diplonemids. If a protein has only been identified but not yet experimentally characterized, it is shown in grey. If bioinformatic searches did not yield a certain homolog, it is denoted as „absent“. Homologs, which are still unkown, are indicated with a dash (−). Peroxins with unique characteristics may serve as potential drug targets to specifically disrupt glycosome biogenesis.
PMPs possess a specific membrane peroxisome targeting signal (mPTS), which is recognized by the cytosolic receptor PEX19. In addition to recognizing the mPTS, PEX19 functions as a chaperone, stabilizing newly synthesized PMPs in the cytosol (Jones et al. 2004). The resulting PEX19–PMP complex is directed to the glycosomal membrane, where it interacts with the docking proteins PEX3 and PEX16 to mediate membrane insertion of the PMP cargo (reviewed by Kalel et al. (2018) and Michels and Gualdrón-López (2022)). Following docking, PMPs are inserted into the membrane, and PEX19 is released for reuse in subsequent import cycles (Figure 1, left). Despite these insights, the detailed molecular mechanisms underlying PMP insertion and PEX19 release remain poorly understood.
2.1 Kinetoplastid-specific differences in PMP import
In various organisms, including humans and yeast, the flexible N-terminal region of PEX19 contains a PEX3 binding motif that is essential for docking at the glycosomal or peroxisomal membrane, while the structured C-terminal region acts as the binding domain for PMPs (PEX3 binding motif in dark green, PMP binding domain in deep salmon, Figure 2A) (Fransen et al. 2005; Kalel et al. 2019). A notable feature of PEX19 in humans, yeast, and plants is the presence of a C-terminal CaaX motif that undergoes farnesylation, which enhances its binding affinity for peroxisomal membrane proteins (yellow, Figure 2A) (Rucktäschel et al. 2009). Structural studies of farnesylated human PEX19 have shown that the farnesyl group is embedded within the C-terminal domain, triggering conformational changes, which result in the formation of two distinct hydrophobic pockets that are essential for recognizing mPTS-containing peptides (Figure 2A, inset) (Emmanouilidis et al. 2017; Giannopoulou et al. 2016). In addition, binding analyses have identified the first α-helix in the C-terminal region of PEX19 as a critical determinant for PMP recognition and binding in humans (blue, Figure 2A) (Schueller et al. 2010). Together, these findings offer insights into the structural basis of PEX19–PMP interaction. Bioinformatical searches revealed that PEX19 candidates are present in most kinetoplastids but are absent in diplonemids (Andrade-Alviárez et al. 2022; Banerjee et al. 2005). In kinetoplastids, PEX19 notably lacks the C-terminal CaaX motif found in other eukaryotes and thus does not undergo farnesylation. Consequently, the mechanism, by which PEX19 recognizes and stabilizes PMPs in these organisms remains poorly understood, raising the possibility of an as-yet unidentified co-factor or regulatory mechanism compensating for the missing CaaX-mediated function. Recently, a novel peroxin, PEX38, has been identified in T. brucei as a major cytosolic binding partner of PEX19 that is essential for glycosome biogenesis and parasite viability (Krishna et al. 2025b). Distinct domains of PEX38 interact with the co-chaperone Hip and the PEX3-binding motif of PEX19, suggesting a mechanism that facilitates PMP folding and prevents premature membrane association. These findings highlight how PEX19 engages the cytosolic chaperone machinery to safeguard PMP transport.
Comparison of PEX19, PEX3, PEX11, PEX26/PEX15 and PEX13 proteins from humans and Trypanosoma brucei. (A) Binding sites and domain architecture of PEX19 homologs (top) and structural model comparison of HsPEX19 (AF-P40855-F1-v6, pale cyan, left) and TbPEX19 (AF-Q38DH6-F1-v6, pale green, right) (bottom). The PEX3 binding site is depicted in dark green, the PEX14 binding site in light pink, the mPTS recognition site in navy, the PMP binding domain in deep salmon and the CaaX motif for farnesylation in yellow. Upon farnesylation, the C-terminus (yellow) is embedded within the C-terminal domain (deep salmon, PDB: 5LNF, inset). (B) Binding sites and domain architecture of PEX11 family proteins in humans (HsPEX11α, HsPEX11β, HsPEX11γ) and T. brucei (TbPEX11, TbGIM5A, TbGIM5B and the recently novel identified PEX11 family members, termed TbPEX11 HP1 (Tb927.10.8410) and TbPEX11 HP2 (Tb927.9.11640). For the TbPEX11 HP1 and HP2, no respective PEX19 binding sites (deep petrol) or transmembrane spans (grey) have been determined so far. (C) Comparison of binding sites, domain architecture (top) and predicted structures for PEX3 homologs. Structural models of HsPEX3 (AF-P56589-F1-v6, pale cyan) and TbPEX3 (AF-Q383Q3-F1-v6, pale green) were compared. The PEX19 binding site is colored in deep petrol and the transmembrane domain (TMD) in grey (bottom). Sequence insertions in TbPEX3 compared to HsPEX3 are depicted in orange. (D) Comparison of binding sites, domain architecture and predicted structures of human PEX26 (AF-Q7Z412-F1-v6, pale cyan) and trypanosomatid PEX15 (AlphaFold server structure prediction of TriTrypDB: Tb927.10.1860 (Abramson et al. 2024), pale green). Putative PEX6 binding sites are shown in petrol and transmembrane spans in grey. A specific PEX6 binding site on TbPEX15 was not determined but was shown to involve amino acids 1-320aa (Krishna et al. 2025a). (E) Domain architecture and binding sites of HsPEX13, TbPEX13.1 and TbPEX13.2. The KPWE motif, which allows binding to PEX7, is only present in HsPEX13 and shown in light blue, the YG-rich domain responsible for condensate formation is shown in purple, the TMDs in grey, the Src homology 3 (SH3) domain, which can bind PEX14 and PEX5, in navy and C-terminal motifs in burgundy for the FxxxF motif in HsPEX13 (binds to itself and PEX14 (Gaussmann et al. 2024)) or mint for the PTS1-like signal in TbPEX13.1 (role remains enigmatic (Verplaetse et al. 2009)). (F) Topology studies revealed that the N- and C-termini of both PEX13 proteins face the cytosol (Crowe et al. 2020). The topology of PEX14 is not resolved in Glycomonada. Amino acid boundaries for domains and binding site positions are assigned according to literature or approximates based on bioinformatical approaches. Panel F was created using BioRender.
The PEX3 ortholog in kinetoplastids remained unknown for long time. In 2019, two studies independently identified Trypanosoma PEX3 by different approaches (Banerjee et al. 2019; Kalel et al. 2019). Although the structure of PEX3 is highly conserved, the parasite PEX3 proteins contain two large insertions, which hindered their identification based on primary sequence similarity (orange, Figure 2C). Likewise, identification of other euglenozoan PEX3 homologs/orthologs is also complicated due to a low sequence conservation even within protists (Andrade-Alviárez et al. 2022).
PEX16 is the third component of the PMP import machinery found in human, plant and some yeasts. PEX16 homologs were also identified in trypanosomatids but shared only low sequence conservations (∼16 % identity and ∼30 % similarity) with the known PEX16 proteins in other organisms (Kalel et al. 2015). Interestingly, trypanosomatid PEX16 orthologs are larger proteins compared to those in other organisms due to the presence of additional internal amino acid stretches. Different to PEX16 in yeast (Yarrowia) and plant (Arabidopsis), where PEX16 is a peripheral membrane protein, trypanosomatid PEX16 is an integral membrane protein like mammalian PEX16 (Kalel et al. 2015).
2.2 New insights into the PEX19-PMP interaction
The mPTS sequences in PMPs generally consist of α-helical regions that often include segments of the transmembrane domains along with a short adjacent sequence. These neighboring sequences contain either a cluster of basic residues or a combination of basic and hydrophobic amino acids that facilitate membrane anchoring (reviewed by van Ael and Fransen (2006)). PEX19 binding sites tend to be relatively degenerate and can occur multiple times within a single PMP (Rottensteiner et al. 2004). Despite this variability, PEX19 binding sites have been identified across various organisms, including yeast, human, and parasite PMPs, highlighting their evolutionary conservation throughout eukaryotes (Saveria et al. 2007a). Based on systematic studies of PEX19–PMP interactions in various organisms, an in silico prediction matrix to identify PEX19 binding sites or mPTS motifs in other PMPs was developed (Rottensteiner et al. 2004; Saveria et al. 2007a). Building on this knowledge, a recent study dissected the PEX19 binding sites within the most abundant PMP, PEX11, in trypanosomes (Krishna et al. 2023). PEX11 family proteins are involved in peroxisome proliferation, regulation of their size and number, and metabolite transport (Deori and Nagotu 2022; Mindthoff et al. 2016). Similar functions are also performed by PEX11 family proteins in T. brucei (Figure 1, right). Depletion of PEX11 results in a reduced number of glycosomes and their enlargement, while overexpression leads to glycosome clustering or the formation of long tubules (Lorenz et al. 1998). PEX11 as well as its two homologs – GIM5A and GIM5B – have also been suggested to be involved in metabolite transport into the glycosome (Gualdrón-López et al. 2013a; Voncken et al. 2003). TbPEX11 contains two PEX19 binding sites, one adjacent to the first predicted transmembrane domain (deep petrol, Figure 2B) (Krishna et al. 2023). The second PEX19 binding site in TbPEX11 is located close to the N-terminus and is highly conserved across yeast, human, plants and kinetoplastids.
PEX19 functions as an import receptor for newly synthesized PMPs and also acts as a chaperone to stabilize them in the cytosol (Jones et al. 2004). In cells with defects in PMP import, PMPs undergo degradation or are mislocalized to alternative subcellular compartments such as mitochondria, the endoplasmic reticulum (ER), or membranes of undefined origin (Hettema et al. 2000; Nuebel et al. 2021). Accordingly, Krishna et al. (2023) found that deletion or mutation of the PEX19 binding site in parasite (T. brucei) or yeast PEX11 results in its mislocalization to mitochondria. Bioinformatic analysis suggested that TbPEX11 contains an amphipathic helix and several putative TOM20 recognition motifs in the N-terminus. This supports the observation that PEX11 tends to mislocalize to mitochondria when its glycosomal transport is blocked (Krishna et al. 2023).
2.3 Establishment of glycosomal membrane protein inventory
Like peroxisomes, glycosomes adapt their proteome in different life cycle stages where they encounter different metabolic niches. Accordingly, several studies have elucidated the glycosomal proteome of T. brucei, T. cruzi and Leishmania species (Acosta et al. 2019; Colasante et al. 2006; Colasante et al. 2013; Güther et al. 2014; Jamdhade et al. 2015; Jardim et al. 2018; Vertommen et al. 2008). These studies were mostly focused on total glycosomal proteomes, which is often dominated by abundant glycosomal enzymes. In a recent study, Krishna et al. (2025a) elucidated the glycosomal membrane-specific proteome by employing carbonate extraction enrichment of PMPs from purified glycosomes and protein correlation profiling across subcellular fractions of T. brucei cells. This high-confidence glycosomal membrane protein inventory revealed 28 new PMPs. Structural modelling uncovered two additional proteins of the PEX11 family as well as the long unknown PEX15 in trypanosomatids among the newly identified proteins (Krishna et al. 2025a).
3 Glycosomal matrix protein import
Glycosomes, as peroxisome-related organelles, do not contain their own DNA or protein translation machinery (Lazarow and Fujiki 1985). Both PMPs and matrix proteins encoded by nuclear genes are translated in the cytosol and then imported into the organelle post-translationally. Notably, these proteins are imported in their folded state. The import of matrix proteins into peroxisomes or glycosomes occurs via an import pore involving various PEX proteins (Figure 1, middle). The general matrix protein import can be divided in five steps. First, cargo proteins are recognized via their peroxisome targeting signal (PTS) (step 1). Most cargo proteins have a C-terminal PTS1 with a tripeptide consensus sequence of [SAGPYN]-[KHRNQ]-[LMAVY] as defined for kinetoplastids based on an in silico screen (Opperdoes and Szikora 2006). For peroxisomes, it was shown that the upstream 9-amino acid sequence also plays a role in cargo recognition (Brocard and Hartig 2006). However, specific physicochemical properties could not be determined for kinetoplastids (Opperdoes and Szikora 2006), and no such study is available for diplonemids. A glycosome-specific PTS1 prediction algorithm has been reported (Durrani et al. 2020). The PTS1-containing proteins are recognized by the cytosolic receptor PEX5. Less common are cargos with a PTS2 signal, which is a nonapeptide close to the N-terminus and has a consensus sequence of [RK]-[LVIQ]-X2-[LVIHQ]-[LSGAK]-X-[HQ]-[LAF] in Opisthokonta (Petriv et al. 2004), which is similar to the [RK]-[LVI]-X5-[HKQR]-[LAIVFY] sequence used for in silico prediction of glycosomal PTS2 containing proteins (Durrani et al. 2020; Opperdoes and Szikora 2006). These proteins are bound by the import receptor PEX7, which then binds to the co-receptor PEX5 for glycosomal import (Galland et al. 2007). Additionally, very few proteins are targeted to glycosomes by an internal peroxisomal targeting sequence (iPTS), such as triosephosphate isomerase in T. brucei or phosphoglucomutase in T. cruzi (Galland et al. 2010; Penha et al. 2009). However, no consensus sequence has yet been identified.
Upon binding of cargo-loaded PEX5 to the membrane-associated docking complex consisting of PEX14 and PEX13, an import pore forms (step 2). Kinetoplastids are unique in that they contain two essential and non-redundant PEX13 proteins – PEX13.1 and PEX13.2 (Brennand et al. 2012; Verplaetse et al. 2009; Verplaetse et al. 2012). Notably, blue native PAGE analyses in T. brucei revealed the existence of three different import pore complexes: a large complex (L) containing PEX13.1, PEX13.2 and PEX14, an intermediary complex (I) comprising PEX13.1 and PEX13.2 and a small complex (S) consisting only of PEX14 proteins (Crowe et al. 2020) (Figure 1, lower left inset). Whether these complexes resemble distinct import pores or intermediates in import pore assembly remains unclear.
Cargo proteins are thereafter translocated across the membrane into the glycosomal matrix in a still enigmatic manner (step 3). While it was shown that PEX7 enters the peroxisomal/glycosomal matrix during PTS2 cargo import in human, yeast, plant and trypanosomatids, specifically shown for Leishmania (Nair et al. 2004; Pilar et al. 2008; Rodrigues et al. 2014; Singh et al. 2009), the behavior of PEX5 remains subject of considerable debate. In this regard, two different models have been proposed for peroxisomes: (1) PEX5 is involved in pore formation and remains in the membrane during and after cargo translocation (transient import pore) (Erdmann and Schliebs 2005; Meinecke et al. 2010). (2) The PEX5-cargo complex is translocated across the membrane into the peroxisomal matrix (extended shuttle) (Dammai and Subramani 2001; Gao et al. 2022; Skowyra et al. 2024).
During or after cargo release, a complex of a ubiquitin conjugating enzyme (PEX4, and its peroxisomal anchor PEX22 in yeast/plants/T. brucei) and E3-ligases (RING-finger peroxins PEX2, PEX10, PEX12) ubiquitinate the receptor (step 4) (Gualdrón-López et al. 2013b). This marks PEX5 for recycling upon monoubiquitination or for proteasomal degradation in case of polyubiquitination.
Monoubiquitinated PEX5 is exported back to the cytosol by the heterohexameric AAA+ ATPase PEX1 and PEX6 in an ATP-dependent manner (step 5) (Miyata and Fujiki 2005; Platta et al. 2005). The ATPase complex is anchored to the peroxisomal or glycosomal membrane by PEX15 (yeast, Trypanosoma)/PEX26 (human)/APEM9 (plant) (Birschmann et al. 2003; Goto et al. 2011; Grimm et al. 2016; Krishna et al. 2025a; Matsumoto et al. 2003). The receptor ubiquitination machinery, together with the AAA+ ATPase complex, constitutes peroxisomal exportomer. Following receptor export, deubiquitination is mediated by Ubp15 in yeast (Debelyy et al. 2011) and by USP9x in humans (Grou et al. 2012), thereby preparing it for another round of matrix protein import. In kinetoplastids, however, the specific deubiquitinase involved in this process is not yet known.
3.1 PEX5-PTS1 interaction is almost identical across parasites and human
While the structural properties of the TbPEX5–PTS1 interaction have been known since 2008 (Sampathkumar et al. 2008), little has been grasped about its dynamic behavior. A recent study revealed the dynamics of the interaction between the TPR domains of PEX5 and the PTS1 in T. brucei and T. cruzi (Banasik et al. 2024). In the apo state of PEX5, when no cargo protein is bound, the protein takes on a “snail-like” open conformation. Upon PTS1 binding, the two structurally rigid domains consisting of TPR1-5, and TPR6-7 and the C-terminal three-helix bundle (3H), respectively, undergo a 17° hinging motion between TPR5 and TPR6, more specifically Arg525-Asp532. PEX5 is now in a closed, “ring-like” conformation. The hinging motion does not only occur upon ligand binding but appears to also be characteristic for the apo structure, while PTS1 binding locks it in the “ring-like” structure. The overall binding mode of human, T. brucei and T. cruzi PEX5 to PTS1 are almost identical with similar motion of TPR domains 2, 3, 5, 6 and 7 in Hs and Tb homologs (see Figure 3A and B, respectively). Significant differences include the role of the 7-C loop upon interaction and the mode of stabilization of the most C-terminal carboxylate group of the PTS1. In the closed conformation of HsPEX5 the 7-C loop interacts with TPR1 and thereby displacing the polypeptide chain N-terminal to TPR1 from the position in apo-form. This is not observed in TbPEX5, instead the 7-C loop becomes partially unstructured as concluded from the lack of defined electron density. There is also differences in the stabilization of the last carboxylate group of the PTS1 signal by human and T. cruzi PEX5.
Comparison of apo and PTS1-bound PEX5 structures of Trypanosoma brucei (A) and H. sapiens (PEX5L, B). Both PEX5 proteins undergo a hinging motion between the TPR1-5 domains and TPR6-7 domains plus the C-terminal 3-helix bundle (3H) upon PTS1-binding, thereby changing from an open conformation to a closed conformation. In T. brucei the hinging motion occurs along the Arg525-Arg532 axis, in humans along Thr512-Asn520 according to sequence alignment. The structures are colored as follows: TbPEX5 apo structure (PDB: 9F8W, chain A) – TPR1-5 in pale green, TPR6-7+3H in pale blue and the Arg525-Arg532 axis in magenta; TbPEX5 + 7-SKL (cargo protein not shown, PDB: 3CVQ) – TPR1-5 in green, TPR6-7+3H in wheat and the Arg525-Arg532 axis in orange; HsPEX5 apo structure (PDB: 2C0M, chain A) – TPR1-5 in pale cyan, TPR6-7+3H in pale yellow and the Thr512-Asn520 axis in sand; HsPEX5 + HsMSCP2 (cargo protein not shown, PDB: 2C0L) – TPR1-5 in cyan, TPR6-7+3H in light pink and the Thr512-Asn520 axis in red.
3.2 PEX13 contributes to protein import into peroxisomes through a nuclear pore-like phase
There have been recent advances in elucidating the formation of the peroxisomal import pore and the translocation of both PTS1 and PTS2 cargo proteins in several organisms. Novel findings suggest that PEX13 and PEX14 proteins in yeast and human undergo liquid-liquid phase separation (LLPS) and form transient condensates with PEX5-cargo complexes (Gaussmann et al. 2024; Ravindran et al. 2023; Wu et al. 2025). Here, PEX13, PEX14 and PEX5 might act as a transient conduit for cargo translocation across the peroxisomal membrane. In a different study, PEX13 proteins were shown to form hydrogels in vitro, suggesting the formation of a stable pore (Gao et al. 2022). According to this model, the pore consists of ten to twelve PEX13 molecules, which exhibit dual topology. Both LLPS and hydrogel formation were shown to depend on the intrinsically disordered N-terminal region in PEX13, which is rich in Tyrosine-Glycine (YG) repeats, closely resembling the Phenylalanine-Glycine (FG) repeats of the permeability filter of the nuclear pore complex (Gao et al. 2022).
The YG-rich domain and the following amphipathic helices (AH) are conserved in PEX13 proteins across organisms (Gao et al. 2022), which is also true for the two non-redundant and essential PEX13 proteins – PEX13.1 and PEX13.2 – in T. brucei, T. cruzi, Leishmania ssp. and Crithidia (Brennand et al. 2012; Verplaetse et al. 2009). While PEX13.1 lacks the typical N-terminal proline-rich region, it contains the YG-rich domain at its far N-terminal part, followed by an AH and an SRC homology 3 (SH3) domain, resembling PEX13 proteins from vertebrates and yeast (Figure 2E). A unique feature of the glycosomal PEX13.1 is the presence of conserved PTS1-like sequence (-TKL) at its C-terminus present in all kinetoplastids and diplonemids, with the exception of Angomonas deanai, Paratrypanosoma confusum and Bodo saltans (Andrade-Alviárez et al. 2022; Verplaetse et al. 2009). The function of this C-terminal motif is still not fully understood. Microscopy studies with procyclic form T. brucei parasites, however, showed that PEX13.1 localizes to the ER in a PTS1-dependent manner in low-glucose and hence, glycosome-independent growth conditions (Bauer et al. 2017). Interestingly, this protein is unique to kinetoplastids and could not be found in diplonemids (Andrade-Alviárez et al. 2022; Brennand et al. 2012). PEX13.2 on the other hand, which was found in kinetoplastids (no data available on diplonemids) (Andrade-Alviárez et al. 2022), contains an intrinsically disordered N-terminal region, including the proline-rich region, the YG-rich domain and an AH, but lacks the SH3 domain (Brennand et al. 2012). With this architecture, PEX13.2 is similar to plant PEX13 (Mano et al. 2006).
The presence of two PEX13 proteins in kinetoplastids and their role in glycosomal matrix protein import remains elusive. However, evidence from T. brucei studies suggests that PEX13.2 specifically functions in PTS2 import (Crowe et al. 2020). TbPEX13.1 on the other hand is involved in both PTS1 and PTS2 import pathways (Verplaetse et al. 2009). Since PEX13.1 is absent in diplonemids (Andrade-Alviárez et al. 2022), PEX13.2 might enable both PTS1 and PTS2 import in these organisms, or an unrelated protein might substitute for the function of PEX13.1. While there is no data on the specific composition of the import pore in Trypanosoma, complexes containing both PEX13.1 and PEX13.2 with and without PEX14 were identified in T. brucei (Crowe et al. 2020), indicating that both PEX13 proteins are (equally) involved in building the glycosomal translocon. A direct interaction between these proteins has also been shown by yeast two-hybrid (Y2H) analysis (Brennand et al. 2012). Whether PEX13.1 and PEX13.2 also form condensates remains to be experimentally proven. Furthermore, different to yeast and human, both PEX13.1 and PEX13.2 have one clear topology: N- and C-termini face the cytosol (Figure 2F). The topology for PEX14 on the other hand is still under investigation in several organisms (Gaussmann et al. 2024).
The PTS2-receptor PEX7 is also able to partition in the hydrogel phase formed by PEX13 proteins (Skowyra and Rapoport 2025), but interacts with PEX13 via a conserved [K/R]-PWE motif in various organisms (Chen et al. 2025). Interestingly, such PEX7 binding motifs are completely absent in all kinetoplastid PEX13.1 proteins, and only found in the PEX13.2 proteins of certain kinetoplastids (T. cruzi, B. saltans, P. confusum) and closely related euglenids (Euglena gracilis and Rhabdomonas costata), which harbor peroxisomes and not glycosomes (Andrade-Alviárez et al. 2022). The PEX7 binding motif in these few PEX13.2 proteins have a consensus [K/R]-P-[W/Y]-[E/D] sequence similar to the [K/R]-PW-[E/D] motif found in other organisms (Andrade-Alviárez et al. 2022; Chen et al. 2025). Noteworthy, for plant PEX13 (AtPEX13), which resembles PEX13.2, only an interaction with AtPEX7 but not AtPEX5 was detected by Y2H analysis (Mano et al. 2006). Due to autoactivation of TbPEX7 in Y2H, no such results could be obtained for either of the T. brucei PEX13 proteins (Verplaetse et al. 2009). After release of the PTS2 cargo in the peroxisomal matrix, PEX7 returns to the cytosol and is extracted from the YG-rich phase by the newly identified PEX39 via its own [K/R]-PW-[E/D] motif (Chen et al. 2025; Skowyra and Rapoport 2025). No homolog with a comparable size (<200 amino acids) could be identified in kinetoplastids and diplonemids through a PROSITE search using the generic [K/R]-P-[W/Y][-E/D/Q] motif. Hence, it remains unknown how PEX7 is removed from the glycosomal matrix and recycled in these species.
3.3 Missing components of the exportomer complex identified in kinetoplastids
Recently, two previously uncharacterized trypanosomatid PEX proteins involved in receptor export and recycling, PEX1 (Mahadevan et al. 2024) and PEX15 (Krishna et al. 2025a), were identified and characterized in T. brucei.
Although the identity of PEX1 had already been proposed through bioinformatic analysis alongside PEX6 (Krazy and Michels 2006; Opperdoes and Szikora 2006), its experimental confirmation and functional characterization were still outstanding. Mahadevan et al. (2024) demonstrated that the putative TbPEX1 protein localizes to glycosomes and interacts with TbPEX6 in Y2H assays, indicating that it is a true PEX1 ortholog.
PEX15 on the other hand remained unknown in kinetoplastids for long time. Similar to PEX1, most peroxins in trypanosomes were identified through primary sequence similarity searches such as BLAST. However, this strategy failed to uncover the trypanosomal orthologue of PEX15. Notably, yeast PEX15, human PEX26 and plant APEM9 also share minimal primary sequence conservation, yet they show high structural similarity, and all belong to the class of tail-anchored (TA) proteins (Jansen et al. 2021; Judy et al. 2022). A recent study by Krishna et al. (2025a) identified two TA-proteins within a high-confidence inventory of glycosomal membrane proteins from T. brucei. Structural modelling of the two candidates using AlphaFold2 revealed that one of the proteins possesses the characteristic N-terminal α-helical bundle, which is known to interact with the N-domain of PEX6 in Saccharomyces cerevisiae and other organisms (Figure 2D) (Ali et al. 2024; Birschmann et al. 2003; Gardner et al. 2018; Nashiro et al. 2011; Rüttermann et al. 2023; Tamura et al. 2006; Weller et al. 2005). It was further shown that the putative TbPEX15 interacts with the TbPEX6 protein in Y2H assay, and it localizes to glycosomes in T. brucei.
Knock-down of either TbPEX1 (Mahadevan et al. 2024) or TbPEX15 (Krishna et al. 2025a) by RNAi leads to cell death proving that these proteins are essential for the parasite survival of T. brucei, similar to all other peroxins (Crowe and Morris 2021). This along with the low sequence conservation for both PEX1 and PEX15 (below 33 % among T. brucei, T. cruzi and Leishmania donovani and below 17 % compared to homologs from yeast, plant and human), makes both proteins attractive candidates for drug targeting as it opens the possibility of identifying selective inhibitors, offering potential for the development of novel anti-trypanosomal therapies (Krishna et al. 2025a; Mahadevan et al. 2024).
3.4 Recent studies suggest that RADAR is also present in kinetoplastids
In the absence of any component of the receptor export machinery – PEX1, PEX6 or PEX15 – a functional AAA+ ATPase complex and thus exportomer cannot be assembled. As a result, monoubiquitinated PEX5 cannot be exported and recycled for subsequent rounds of matrix protein import (Nordgren et al. 2015). Instead, it accumulates at the membrane, where it gets polyubiquitinated (Brown et al. 2014; Kiel et al. 2005; Kragt et al. 2005; Law et al. 2017; Nuttall et al. 2014; Platta et al. 2004). This process was shown to either lead to extraction and degradation by the ubiquitin proteasome system – a process called receptor aggregation and degradation in the absence of recycling (RADAR) (Léon et al. 2006; Yusuf et al. 2025) – or induce pexophagy (Law et al. 2017; Nordgren et al. 2015; Nuttall et al. 2014). Consequently, peroxisomal proteins synthesized in the cytosol fail to be imported into peroxisomes.
Knockdown of either TbPEX1 or TbPEX15 by RNAi resulted in mislocalization of glycolytic enzymes to the cytosol (Krishna et al. 2025a; Mahadevan et al. 2024). Glycosomal glycolytic enzymes lack feedback regulation (Cronin and Tipton 1985; Nwagwu and Opperdoes 1982; Urbina and Crespo 1984) and therefore exhibit unregulated activity when mislocalized to the cytosol (Bakker et al. 2000). This unrestrained activity leads to rapid ATP depletion (Bakker et al. 2000), which was also observed upon PEX1 knockdown (Mahadevan et al. 2024). As mentioned above, a lack of PEX1, PEX6 or PEX15 is expected to result in the accumulation of import receptors in the glycosomal matrix or membrane as observed for yeast and human (Brown et al. 2014; Kiel et al. 2005; Kragt et al. 2005; Law et al. 2017; Nuttall et al. 2014; Platta et al. 2005). However, instead a specific degradation of PEX5 was observed upon knockdown of PEX1 and PEX15 (Krishna et al. 2025a; Mahadevan et al. 2024), similar to the observation in plants (Goto-Yamada et al. 2022). In the case of PEX1 depletion, a delayed degradation of PEX7 was also detected (Mahadevan et al. 2024). Inhibition of proteasome activity by MG-132 treatment led to a partial rescue of PEX5 and PEX7 levels, suggesting that, in the absence of the functional exportomer complex, import receptors are specifically removed from the glycosomal membrane by the RADAR pathway (Mahadevan et al. 2024). The conclusion that the reduction in steady-state PEX5 levels was caused by the RADAR pathway, rather than polyubiquitinated PEX5-induced pexophagy, was also supported by unchanged steady-state levels of PEX11 upon PEX1 knockdown, proving the intactness of glycosomal membranes.
Recently, other AAA+ ATPases, specifically Msp1/ATAD1 and Cdc48/VCP/p97, which are involved in quality control mechanisms of other organelles, such as mitochondria-associated degradation (MAD, (Ravanelli et al. 2020)) or endoplasmic reticulum-associated degradation (ERAD, (Krshnan et al. 2022)), respectively, were shown to play a role in PEX5 quality control and peroxisome homeostasis. In S. cerevisiae, Msp1 but mostly Cdc48 with its cofactors Npl4 and Ufd1 showed to be involved in clearance of PEX5 from the peroxisomal membrane in the absence of PEX1 via the RADAR pathway (Yusuf et al. 2025). Also in humans, PEX5 was degraded in an ATAD1-dependent manner when it could not be recycled (Ott et al. 2022). Furthermore, depletion of p97 or its cofactor UBXD8/FAF2 led to an accumulation of ubiquitinated PEX5 in the peroxisomal membrane, which ultimately led to pexophagy (Montes et al. 2025). While neither ATPase was shown to be involved in glycosome-related RADAR pathway yet, Msp1/ATAD1 was shown to dually localize to both the outer membrane of mitochondria and glycosomal membranes (Gerber et al. 2023; Krishna et al. 2025a), suggesting a role in removal of ubiquitinated integral membrane proteins. Besides, a recent study on the interaction network of Cdc48/p97/VCP in Leishmania major found that the putative FAF2 partially localizes to glycosomes. Proteomic studies in T. brucei also revealed presence of FAF2 in glycosomes (Güther et al. 2014; Krishna et al. 2025a). Whether these proteins are involved in the glycosomal RADAR pathway remains to be investigated (Figure 1, lower right inset).
4 Glycosome homeostasis
Peroxisomes and glycosomes proliferate primarily through two main mechanisms: (1) growth and division of existing organelles and (2) de novo biogenesis originating from the endoplasmic reticulum (ER) (Bauer and Morris 2017). The fission process of peroxisomes is initiated by the integral membrane protein PEX11 promoting elongation of the peroxisomal membrane, which is also observed for glycosomes (Lorenz et al. 1998). In mammals, further key adaptor proteins such as fission 1 (Fis1) and mitochondrial fission factor (MFF) recruit the dynamin-related protein DRP1, a GTPase that mediates constriction and scission, resulting in two daughter peroxisomes (Carmichael et al. 2022). The role of DRPs and their adaptors in glycosome biogenesis, however, has not yet been described in the literature. The de novo biogenesis of peroxisomes involves PEX3, PEX19 and PEX16 proteins and such pathway for glycosomes has only been implied indirectly (Bauer and Morris 2017; Bauer et al. 2017; Kalel et al. 2015). So far, only TbPEX13.1 has been shown to localize to ER in response to glucose levels (Bauer et al. 2017).
Recently, a novel peroxin Pex37, a member of the Pxmp2-family, has been identified as a fission factor in Hansenula polymorpha (Singh et al. 2020). In peroxisome-repressing conditions Pex37 causes a reduction in peroxisome number and a defect in segregation of peroxisomes. The defects can be partially restored upon introduction of the human homolog PXMP2. Kinetoplastids appear to not harbor a Pxmp2 homolog (Colasante et al. 2013).
Interestingly, the SNARE protein Ykt6 has also been implicated in glycosome biogenesis (Banerjee and Rachubinski 2017). Knockdown of Ykt6 by RNAi results in reduction of glycosome number, mislocalization of glycosomal matrix proteins and cell death in T. brucei, similar to the phenotypes observed for PEX proteins. GFP-tagged Ykt6 colocalized partially to glycosomes. Generally, SNARE proteins play important roles in vesicular trafficking and mediating a wide range of protein-protein interactions. How Ykt6 participates in glycosome biogenesis and whether it is involved in glycosome proliferation remains to be investigated.
The glycosomal protein composition varies according to the different metabolic environments that trypanosomatids encounter throughout their life cycle (Bauer et al. 2013; Colasante et al. 2006). Turnover of glycosomes could be responsible for this flexible metabolic adaptability, however, little is known about the degradation of glycosomes. There is strong evidence that glycosomes undergo a specialized form of autophagy, known as pexophagy, which facilitates the degradation of entire glycosomes and recycling of their contents as shown for T. brucei (Herman et al. 2008). Supporting this, glycosomes have been observed to localize to the lysosome during shifts in life cycle stages (Herman et al. 2008). Additionally, genome database searches reveal that trypanosomatids possess orthologues of roughly half of the core autophagy-related proteins identified in S. cerevisiae, the best-studied organism for autophagy (Brennand et al. 2011). Among these proteins was the classical autophagy marker ATG8. While there is only one ATG8 gene in yeast and fungi, higher eukaryotes and kinetoplastids contain several (Shpilka et al. 2011). ATG8 has undergone paralogue expansion by gene multiplication in Leishmania and T. brucei. For example, in L. major a total of 25 genes code for four families of ATG8-like proteins: the canonical ATG8 (one annotated gene), ATG8A (three gene copies), ATG8B (eight gene copies) and ATG8C (13 gene copies) (Williams et al. 2009). For a detailed review on trypanosomal autophagy and pexophagy, please refer to (Brennand et al. 2011; Romano et al. 2023). The observed genetic expansion potentially highlights the importance of autophagy in general and in glycosomal turnover in kinetoplastids (Brennand et al. 2011).
5 Glycosome biogenesis as a potential drug target
Glycosome biogenesis has been validated as a promising drug target for combating trypanosomatid parasite infections. RNAi-mediated knockdown studies have demonstrated that several peroxins are essential for glycosome biogenesis and parasite survival, underscoring the essentiality of the organelle in maintaining parasite viability (Barros-Alvarez et al. 2014; Kalel et al. 2018). Dawidowski et al. (2017) provided the first proof-of-concept study demonstrating the druggability of the glycosome biogenesis machinery by developing small-molecule inhibitors that disrupt glycosomal matrix protein import. The identified compounds specifically target the interaction between T. brucei PEX14 and PEX5, effectively impairing glycosomal protein import and function, and demonstrating therapeutic efficacy in mouse models of HAT (T. brucei rhodesiense). Since then, structure-activity relationship studies based on in silico 3D pharmacophore screening led to optimized drugs of the original pyrazolo[4,3-c]pyridine scaffold with trypanocidal effects in the submicromolar range against T. brucei gambiense and T. cruzi (Dawidowski et al. 2020). Further medicinal chemistry campaigns were based on the chemically advanced template search (CATS) algorithm (Fino et al. 2021), and high-throughput screening (HTS) of a compound library (Napolitano et al. 2022a), which revealed further PEX14–PEX5 inhibitor classes based on a 2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine and dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold, respectively. The binding site between PEX5 and PEX14 is a flat interface that is characterized by hydrophobic and aromatic (π-π) interactions between Trp and Phe/Tyr on Trypanosoma PEX14 and the aromatic residues WxxxF/Y motif on PEX5, with two further solvent-exposed cavities on PEX14. It was found that all drugs based on the above-mentioned scaffolds show similar binding characteristics with the central core shielding solvent-exposed Phe17 and Phe34 in Trypanosoma PEX14 and aromatic substituents interacting with the respective Trp and Phe hotspots (summarized in Figure 4A and B). Based on these results, novel peptidomimetic drugs based on an oxopiperazine scaffold were developed that follow spatial arrangements of PEX5 WxxxF/Y motif for interaction with the Trp and Phe pockets on PEX14 (Figure 4B) (Marciniak et al. 2023). These drugs were found to inhibit the interaction in the mid- to low-micromolar range.
Drug targets in glycosome biogenesis machinery and identified drug scaffolds/candidates in Trypanosoma brucei. (A) Schematic overview of the crystal structure of TbPEX14 (violet, PDB: 6S9Y) interacting with a PEX5-derived peptide (navy, PDB: 2W84), demonstrating the binding interface. TbPEX14 was aligned with HsPEX14 of the NMR structure for the HsPEX14–PEX5 complex (PDB: 2W84). (B) Several scaffolds were identified, which mimic the interaction of PEX5 to PEX14, specifically the interaction with the tryptophan and phenylalanine pockets. Based on these scaffolds, compounds had been identified that did not only inhibit the interaction in vitro but also showed anti-trypanocidal activity against T. brucei (and Trypanosoma cruzi). (C) X-ray crystal structure of TbPEX5 (navy) in complex with a PTS1 peptide (7-SKL, yellow, PDB: 3CVL). The PTS1 signal is embedded in a deep pocket between the TPR domains. (D) An initial screening was performed, which resulted in the identification of six hits (1-6) with selective inhibition in vitro and anti-trypanocidal activity against T. brucei (Napolitano et al. 2022b). (E) AlphaFold3 multimer structure prediction model of an N-terminal TbPEX19 peptide (9-21aa, deep petrol) with TbPEX3 (dark green). The interaction was predicted using AlphaFold server (Abramson et al. 2024). (F) Two groups independently identified several compounds that inhibited the TbPEX3–PEX19 interaction: Compound 7/DNMQ (Banerjee et al. 2021) and compounds 8 to 11 (Li et al. 2021) inhibited the interaction in vitro and showed anti-trypanocidal activity against T. brucei. Compound 10 was also toxic against human cells (Li et al. 2021).
Different to the hydrophobic interaction surface between PEX5 and PEX14, which is challenging to address by small molecules, the PEX5–cargo interaction offers a deep pocket that is better suited for drug design and development. Along this line, several scaffolds capable of disrupting T. cruzi PEX5–PTS1 cargo interaction in vitro and killing T. brucei parasites have been reported (Figure 4C and D) (Napolitano et al. 2022b). A similar approach was also reported for Leishmania, where L. donovani PEX5–PTS1 interaction inhibitors were identified through fluorescence polarization-based high throughput screening (Phan et al. 2024).
Glycosomal matrix protein import depends on several PEX proteins, many of which are glycosomal membrane proteins. Targeting the import of PMPs therefore offers another effective strategy, since disruption of membrane protein import not only compromises membrane assembly, but also indirectly blocks matrix protein import, ultimately impairing glycosome biogenesis. In absence of the peroxisomal membrane, some PMPs mislocalize to mitochondria resulting in mitochondrial dysfunction in humans (Nuebel et al. 2021). Such phenotype may additionally contribute to the anti-parasitic activities, particularly against the intracellular stages of T. cruzi and Leishmania, both of which exhibit active mitochondrial metabolism (Pedra-Rezende et al. 2022). In this context, two recent studies (Banerjee et al. 2021; Li et al. 2021) focused on the docking step of glycosomal membrane protein import in parasites, which is mediated by the PMP receptor PEX19 and the glycosomal membrane protein PEX3. Notably, both studies identified inhibitors, which disrupt the TbPEX19–PEX3 interaction and show in vitro trypanocidal activities against T. brucei (Figure 4E and F) (Banerjee et al. 2021; Li et al. 2021). Similarly, inhibitors of Leishmania PEX19–PEX3 were also reported in a recent preprint (Chou et al. 2024).
In summary, PEX protein interactions that are essential for cargo recognition (PEX5–PTS1, PEX19–PMP) and membrane docking (PEX14–PEX5, PEX3–PEX19) have been chemically validated as anti-trypanosomatid drug targets. The peculiar PEX13.1 and PEX13.2 proteins or the recently identified components of receptor export machinery such as PEX15 may serve as promising molecular targets in glycosome biogenesis for developing new anti-trypanosomatid infection therapies.
Funding source: Deutsche Forschungsgemeinschaft
Award Identifier / Grant number: ER178/17-1
Funding source: Ruhr University Bochum, InnovationsFoRUM – Host Microbe Interactions
Award Identifier / Grant number: IF-009N-22
Award Identifier / Grant number: IF-018N-22
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Research ethics: Not applicable.
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Informed consent: Not applicable.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: The authors state no conflict of interest.
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Research funding: The work was funded by DFG grant ER178/17-1 (to R.E) and supported by Ruhr University Bochum, InnovationsFoRUM – Host Microbe Interactions: IF-009N-22 and IF-018N-22 (to R.E.).
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Data availability: Not applicable.
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