CB-5339 – Her2 Signal

p97 Inhibitor CB-5083 Blocks ERAD in Trypanosoma brucei

Paige Garrison, James D. Bangs*
Department of Microbiology and Immunology, Jacobs School of Medicine and Biomedical Sciences, University at Buﬀalo (SUNY), Buﬀalo, NY, 14203, USA

Abstract

Misfolded proteins trapped in the endoplasmic reticulum (ER) are speciﬁcally recognized and retrotranslocated to the cytosol by the ER-Associated Degradation (ERAD) system and delivered to the proteasome for destruction. This process was recently described in Trypanosoma brucei (T. brucei) using the misfolded epitope tagged Transferrin Receptor subunits ESAG7:Ty and HA:ESAG6 (HA:E6). Critical to this work was the proteasomal inhibitor MG132. However, MG132 has oﬀ-target inhibitory eﬀects on lysosomal Cathepsin L that could cause misinterpretation of turnover results. Here, we evaluate an orally bioavailable p97 inhibitor, CB-5083, for use in
T. brucei. p97 is a ubiquitous protein involved in many cellular events including the membrane extraction step of ERAD. CB-5083 strongly inhibits turnover of HA:E6, with comparable protein recovery to MG132 treatment. Interestingly, little deglycosylated cytoplasmic species accumulates, though it normally emerges with MG132 treatment. This suggests that CB-5083 blocks ERAD upstream of the proteasome, as expected for inhibition of the trypanosomal p97 orthologue TbVCP. Under CB-5083 treatment, HA:E6 is also strongly membrane-associated, suggesting ER localization. Finally, we provide an experimental example where CB-5083 treatment oﬀers clarity to the oﬀ-target eﬀects of MG132 treatment.

African trypanosomes (T. brucei ssp.) are extracellular parasites that cause the human and veterinary diseases Human African Trypanosomiasis (HAT) and nagana, respectively. Following a tsetse ﬂy bite, parasites populate the local dermis [1,2] and bloodstream, where they are confronted with the host immune system. However, certain trypanosomes stochastically switch their immunogenic coat protein, the Variant Surface Glycoprotein (VSG), and so evade the immune system, causing characteristic waves of parasitemia [3,4]. Though the annual incidence of HAT is on the decline [5], the capacity for diagnosing and treating HAT patients remains very limited, as it requires traveling screening teams, trained clinical professionals, and often long courses of in-patient treatment at a health center. Similarly, nagana poses a huge economic burden to sub-Saharan Africa, where livestock are a vital resource.

As in all eukaryotes, T. brucei utilizes two major pathways for degradation of secretory and endocytic proteins. First, proteins are de- graded in the lysosome during secretory traﬃcking or endocytosis through the ﬂagellar pocket. This is the case for the transmembrane Invariant Surface Glycoprotein 65 (ISG65) and GPI-anchored Transferrin Receptor (TfR) [6,7]. Both ISG65 and TfR are endocytosed and recycled through the ﬂagellar pocket and recycling endosomes but ultimately are turned over in the lysosome. Second, newly translated secretory proteins translocated into the ER lumen undergo quality control (QC), which mitigates cellular stress caused by the aggregation and accumulation of terminally misfolded and toXic proteins within the ER [8]. During this process, proteins are folded in the calnexin/calre- ticulin cycle and packaged in COPII-coated vesicles for anterograde transport to the Golgi. However, proteins that have repeatedly under- gone the folding cycle without success are recognized as terminally misfolded and turned over by the ER-Associated Degradation (ERAD) machinery [9,10]. This involves N-glycan trimming by ER α-mannosi- dase 1 and multiple related EDEMs (ER degradation-enhancing α- mannosidase I–like proteins) before transfer by the OS-9 lectin to a membrane complex containing the ubiquitin ligase Hrd1.

The Hrd1 complex aids in ubiquitination and retrotranslocation of the misfolded protein. The ubiquitinated protein is recognized by cytoplasmic p97 (aka VCP or Cdc48) and extracted from the ER membrane using energy from ATP hydrolysis [11]. Note that we will refer throughout to the trypanosomal orthologue as TbVCP in keeping with our prior publica- tions on this essential protein [12,13]. To ensure eﬃcient turnover, the N-glycans are then trimmed by a cytoplasmic N-glycanase prior to degradation by the 26S proteasome [14–16] (for an excellent review on ERAD, see [17]). p97, the subject of this report, is a highly abundant AAA (ATPases Associated with a variety of cellular Activities) protein containing distinct D1 and D2 ATPase domains [18] and participating in diverse cellular functions including ERAD [19–21], mitochondria- associated degradation [22,23], chromatin-associated degradation [24–26], ER and Golgi membrane dynamics [27–29], endosomal sorting [30–32], and many more. All these critical ERAD components, except ER α-mannosidase 1 and cytoplasmic N-glycanase, have obvious orthologues in trypanosomes (TbEDEM1-3, Tb927.8.2910-2930; TbOS-9, Tb927.11.10700; TbHrd1, Tb927.9.5260; TbVCP, Tb927.10.5770).

Fig. 1. CB-5083 Treatment. A. Cultured bloodstream form (BSF) TfR RNAi trypanosomes containing the HA:E6 reporter in situ in the ESAG6 locus (33) were seeded (5 × 104/mL) in the presence of increasing concentrations of CB-5083 and counted after 24 hrs (mean ± std. dev., n = 3). B. Cells were treated (24 hr) with tetracycline to induce knockdown of endogenous TfR. Cells were then treated with cycloheximide (100 μg/mL) in the absence (-) or presence of MG132 (MG, 25 μM) and/or CB-5083 (CB, 10 μM) for 4 hrs. Steady state levels of misfolded HA:E6 reporter were analyzed by immunoblotting (107 cell equivalents/lane) with (αHA) and anti-Hsp70 (αHsp70, loading control) antibodies.

The mobilities of Hsp70 (H), intact HA:E6 (E), and deglycosylated HA:E6 (E*) are indicated on left. Molecular mass markers (kDa) are indicated on right. C. HA:E6 was normalized to Hsp70 and quantiﬁed relative to initial HA:E6 (mean ± std. dev., n = 3). MG132 (MG), and CB- 5083 (CB) treatment as indicated.

The roles of ER α-mannosidase 1 and N-glycanase in T. brucei are likely carried out by the EDEMs and an orthologue of cytoplasmic endo-β-N- acetylglucosaminidase (TbENG, Tb927.9.3400), respectively. Of these components only TbVCP has been characterized in any detail [12,13]. We have veriﬁed that ERAD indeed functions in T. brucei [33,34] using misfolded soluble and GPI-anchored subunits of TfR, soluble ESAG7 (E7:Ty) and GPI-anchored ESAG6 (HA:E6). These misfolded reporters accumulate in the ER, even though GPI anchors are known forward traﬃcking signals in T. brucei [35,36]. Both reporters are ra- pidly degraded and their turnover blocked by MG132, a widely used embrane-permeable proteasomal inhibitor. In each case this treat- ment also produces a smaller protected species. Further analysis of misfolded HA:E6 indicates that this species represents the deglycosy- lated, GPI-hydrolyzed form of HA:E6 that has been extracted from the ER membrane, but blocked from proteasomal degradation [33]. Overall, these data conﬁrm that ERAD is active in T. brucei.

The only inhibitors available for ERAD studies in trypanosomes are proteasomal inhibitors such as MG132. However, this inhibitor can also cross-inhibit lysosomal thiol proteases (e.g., cathepsin L [34] and re- ferences therein), and so can cause unclear or misinterpreted results. For this reason, another ERAD inhibitor could be of much use. To this end, we tested an orally bioavailable inhibitor of p97 that causes re- tention of ERAD substrates in the ER and accumulation of ubiquitinated proteins in mammalian cells, and has been previously explored in clinical trials for its broad anti-tumor activity [37–40]. This inhibitor, CB-5083, is extremely selective and potent (IC50 ∼11 nM) in an in vitro assay against the p97 D2 ATPase site in a competitive manner with ATP [38,41]. Notably, previous reports suggest that in T. brucei, the D2 domain of TbVCP is essential for in vivo activity but relies on aug- mentation by the D1 domain [12,13]. For these reasons, we evaluated the potential laboratory use of CB-5083 for speciﬁc inhibition of TbVCP in regard to ERAD in trypanosomes.

To test the eﬃcacy of CB-5083 against TbVCP, drug was added in increasing concentrations to cultured 427 Lister strain bloodstream trypanosomes. Over 24 hrs, treatment with CB-5083 caused strong growth inhibition with an in vivo IC50 of ∼3-4 μM (Fig. 1A), suggesting inhibition of TbVCP. This also indicates that TbVCP is essential for growth, consistent with previous reports [12]. To test the activity of CB- 5083 against a recognized ERAD substrate, we utilized the misfolded reporter HA:E6 in a cycloheximide chase protocol [42]. As seen pre- viously, untreated cells displayed rapid turnover of the substrate, while MG132 treatment (25 μM) signiﬁcantly slowed turnover and produced
the deglycosylated protected species HA:E6* (Fig. 1B &C). CB-5083 (10 μM) blocked HA:E6 turnover, displaying a similar turnover rate to MG132 treatment, with ∼80% recovery of reporter protein after 4 hours. Interestingly, CB-5083 treatment did not produce the protected
species. This suggests that CB-5083 inhibits the ERAD process upstream of the deglycosylation step. Dual MG132 and CB-5083 treatment si- milarly inhibited turnover, with a modest increase in recovery of re- porter protein (∼90%) compared to either treatment alone. Notably, dual treatment resulted in reduced production of the protected species HA:E6*, suggesting again that CB-5083 inhibits the ERAD pathway upstream of the MG132. Overall, these data indicate that CB-5083 treatment is as eﬀective as MG132 at blocking ERAD turnover of mis- folded proteins in T. brucei.

We next evaluated the membrane association of HA:E6 after 4 hours of MG132 or CB-5083 treatment. We subjected control and treated cells to hypotonic lysis on ice and then separated membrane and cytosolic fractions [33]. Without treatment, HA:E6 was membrane-associated, colocalizing with ER reporter BiP (Fig. 2, lanes 1-2). Under MG132 treatment, HA:E6 appeared as full-length and protected species HA:E6*. Full-length HA:E6 colocalized exclusively with BiP in the membrane fraction, while HA:E6* associated with both membrane and cytosolic fractions (Fig. 2, lanes 5-6), as we have observed previously [33]. CB- 5083 treatment similarly produced a strongly membrane-associated HA:E6 (Fig. 2, lanes 3-4), with comparably much less HA:E6*. From this, we conclude that CB-5083 inhibition occurs upstream of ER membrane extraction.

We lastly compared the speciﬁcity of CB-5083 or MG132 inhibition in the case of endogenous Cathepsin L (TbCatL) processing. Normally, TbCatL is synthesized as immature precursors (P and X) that are rapidly converted to the mature form (M) upon arrival in the lysosome [43].

Fig. 2. HA:E6 Reporter Localization. HA:E6-expressing cells were treated with tetracycline (24 hr) to induce knockdown of endogenous TfR and then incubated (2 hr) with CB-5083 (CB, 10μM) or MG132 (MG, 25μM). Cells were lysed hypotonically and membrane (M) and cytoplasmic (C) fractions were prepared by centrifugation (4°C, 10 min at 17,000 X g). Fractions (107 cell equivalents/lane) were analyzed by immunoblotting with anti-HA (E6 and E6*), anti-BiP (B, ER marker), and anti-Hsp70 (H, cytoplasmic marker).

Fig. 3. Turnover of TbCatL. HA:E6 cells (without tetracycline induction) were pulse-chase radiolabeled with [35S]Met/Cys in the presence of CB-5083 (CB, 10 μM) or MG132 (25 μM) and endogenous TbCatL was immunoprecipitated from cell lysates at the indicated times.
Immunoprecipitates were fractionated by 12% SDS-PAGE (107 cell equivalents per lane) and visualized using a Molecular Dynamics Typhoon FLA 9000 system. A representative phosphorimage is pre- sented. Processing of TbCatL to mature (M) form was quantiﬁed relative to initial precursors (P + X) (mean ± std. dev., n = 3).

This processing is mediated by mature TbCatL itself and is blocked by the selective lysosomal thiol protease inhibitor FMK024. However, treatment with MG132 also blocked the processing of TbCatL to the mature form, indicating cross inhibition [34]. To evaluate the speciﬁ- city of the inhibitors during TbCatL turnover, we treated cells with ei- ther MG132 or CB-5083 for 4 hours during a standard pulse-chase protocol [44]. Typical processing of TbCatL was observed without treatment, and addition of MG132 blocked processing to mature form as previously described. However, treatment with CB-5083 did not alter processing of TbCatL (Fig. 3). This indicates that the processing block observed with MG132 is caused by an oﬀ-target eﬀect on lysosomal TbCatL. This oﬀers an excellent example of the importance of utilizing CB-5083 when conﬁrming speciﬁc turnover by ERAD, where MG132 provides ambiguous results.

ERAD should be further explored to better understand under what circumstances, and to what extent, misfolded proteins are degraded by this process in T. brucei. In this regard, we suggest that the p97-speciﬁc inhibitor CB-5083 is superior to MG132 for laboratory experimentation, as it circumvents the cross-inhibition of lysosomal TbCatL evident during MG132 treatment. In addition, our results indicate that TbVCP activity occurs upstream of many ERAD events, including ER membrane extraction and de-glycosylation. Furthermore, TbVCP may facilitate retrotranslocation itself, although we have not formally tested this. It is also possible that TbVCP interacts in a complex with TbENG and other proteins to enable these coordinated events. This would be consistent with other systems, where Rad23 and cytosolic N-glycanase (PNG/Png1) interact with Cdc48/p97 and may deliver misfolded proteins to the proteasome as a complex [45–48].

CRediT authorship contribution statement

Paige Garrison: Conceptualization, Investigation, Formal analysis, Writing – original draft, Writing – review & editing. James D. Bangs: Supervision, Funding acquisition, Writing – review & editing.

Declaration of Competing Interest

None.

Acknowledgements

The authors are grateful to Professor Mark Carrington (Cambridge University, UK) for anti-ISG65 antibody. This work was supported by United States Public Health Service Grant R01 AI035739, and funds from the Jacobs School of Medicine and Biomedical Sciences to JDB.

References

[1] S. Trindade, F. Rijo-Ferreira, T. Carvalho, D. Pinto-Neves, F. Guegan, F. Aresta- Branco, F. Bento, S.A. Young, A. Pinto, J. Van Den Abbeele, R.M. Ribeiro, S. Dias,
T.K. Smith, L.M. Figueiredo, Trypanosoma brucei Parasites Occupy and Functionally Adapt to the Adipose Tissue in Mice, Cell Host Microbe 19 (6) (2016) 837–848.
[2] G. Caljon, N. Van Reet, C. De Trez, M. Vermeersch, D. Perez-Morga, J. Van Den
Abbeele, The Dermis as a Delivery Site of Trypanosoma brucei for Tsetse Flies, PLoS Pathog 12 (7) (2016) e1005744.
[3] R. Ross, D. Thomson, A Case of Sleeping Sickness showing Regular Periodical Increase of the Parasites Disclosed, Br Med J 1 (2582) (1910) 1544–1545.
[4] J. Pinger, S. Chowdhury, F.N. Papavasiliou, Variant surface glycoprotein density deﬁnes an immune evasion threshold for African trypanosomes undergoing anti- genic variation, Nat Commun 8 (1) (2017) 828.
[5] Trypanosomiasis, human African (sleeping sickness), W.H. Organization, 2019, http://www.who.int/mediacentre/factsheets/fs259/en/.2017.
[6] K.J. Schwartz, R.F. Peck, N.N. Tazeh, J.D. Bangs, GPI valence and the fate of se- cretory membrane proteins in African trypanosomes, J Cell Sci 118 (Pt 23) (2005) 5499–5511.
[7] J.S. Silverman, K.A. Muratore, J.D. Bangs, Characterization of the late endosomal
ESCRT machinery in Trypanosoma brucei, Traﬃc 14 (10) (2013) 1078–1090.
[8] D. Ron, P. Walter, Signal integration in the endoplasmic reticulum unfolded protein response, Nat Rev Mol Cell Biol 8 (7) (2007) 519–529.
[9] J.L. Brodsky, Cleaning up: ER-associated degradation to the rescue, Cell 151 (6) (2012) 1163–1167.
[10] R.Y. Hampton, T. Sommer, Finding the will and the way of ERAD substrate retro-
translocation, Curr Opin Cell Biol 24 (4) (2012) 460–466.
[11] X. Zhang, A. Shaw, P.A. Bates, R.H. Newman, B. Gowen, E. Orlova, M.A. Gorman,
H. Kondo, P. Dokurno, J. Lally, G. Leonard, H. Meyer, M. van Heel, P.S. Freemont, Structure of the AAA ATPase p97, Mol Cell 6 (6) (2000) 1473–1484.
[12] J.R. Lamb, V. Fu, E. Wirtz, J.D. Bangs, Functional analysis of the trypanosomal AAA protein TbVCP with trans-dominant ATP hydrolysis mutants, J Biol Chem 276 (24) (2001) 21512–21520.
[13] J.L. Roggy, J.D. Bangs, Molecular cloning and biochemical characterization of a
VCP homolog in African trypanosomes, Mol Biochem Parasitol 98 (1) (1999) 1–15.
[14] S. Misaghi, M.E. Pacold, D. Blom, H.L. Ploegh, G.A. Korbel, Using a small molecule inhibitor of peptide: N-glycanase to probe its role in glycoprotein turnover, Chem Biol 11 (12) (2004) 1677–1687.
[15] D. Blom, C. Hirsch, P. Stern, D. Tortorella, H.L. Ploegh, A glycosylated type I
membrane protein becomes cytosolic when peptide: N-glycanase is compromised, EMBO J 23 (3) (2004) 650–658.
[16] T. Suzuki, Catabolism of N-glycoproteins in mammalian cells: Molecular mechan- isms and genetic disorders related to the processes, Mol Aspects Med 51 (2016) 89–103.
[17] S.S. Vembar, J.L. Brodsky, One step at a time: endoplasmic reticulum-associated
degradation, Nat Rev Mol Cell Biol 9 (12) (2008) 944–957.
[18] T.F. Chou, S.L. Bulfer, C.C. Weihl, K. Li, L.G. Lis, M.A. Walters, F.J. Schoenen,
H.J. Lin, R.J. Deshaies, M.R. Arkin, Speciﬁc inhibition of p97/VCP ATPase and kinetic analysis demonstrate interaction between D1 and D2 ATPase domains, J Mol Biol 426 (15) (2014) 2886–2899.
[19] Y. Ye, H.H. Meyer, T.A. Rapoport, The AAA ATPase Cdc48/p97 and its partners
transport proteins from the ER into the cytosol, Nature 414 (6864) (2001) 652–656.
[20] Y. Ye, Y. Shibata, C. Yun, D. Ron, T.A. Rapoport, A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol, Nature 429 (6994) (2004) 841–847.
[21] E. Jarosch, C. Taxis, C. Volkwein, J. Bordallo, D. Finley, D.H. Wolf, T. Sommer,
Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48, Nat Cell Biol 4 (2) (2002) 134–139.
[22] S. Xu, G. Peng, Y. Wang, S. Fang, M. Karbowski, The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover, Mol Biol Cell 22 (3) (2011) 291–300.
[23] A. Tanaka, M.M. Cleland, S. Xu, D.P. Narendra, D.F. Suen, M. Karbowski, R.J. Youle, Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin, J Cell Biol 191 (7) (2010) 1367–1380.
[24] K. Ramadan, p97/VCP- and Lys48-linked polyubiquitination form a new signaling pathway in DNA damage response, Cell Cycle 11 (6) (2012) 1062–1069.
[25] M. Meerang, D. Ritz, S. Paliwal, Z. Garajova, M. Bosshard, N. Mailand, P. Janscak,
U. Hubscher, H. Meyer, K. Ramadan, The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks, Nat Cell Biol 13 (11) (2011) 1376–1382.
[26] K. Ramadan, R. Bruderer, F.M. Spiga, O. Popp, T. Baur, M. Gotta, H.H. Meyer,
Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin, Nature 450 (7173) (2007) 1258–1262.
[27] H. Kondo, C. Rabouille, R. Newman, T.P. Levine, D. Pappin, P. Freemont,
G. Warren, p47 is a cofactor for p97-mediated membrane fusion, Nature 388 (6637) (1997) 75–78.
[28] M. Latterich, K.U. Frohlich, R. Schekman, Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes, Cell 82 (6) (1995) 885–893.
[29] C. Rabouille, T.P. Levine, J.M. Peters, G. Warren, An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments, Cell 82 (6) (1995) 905–914.
[30] D. Ritz, M. Vuk, P. Kirchner, M. Bug, S. Schutz, A. Hayer, S. Bremer, C. Lusk,
R.H. Baloh, H. Lee, T. Glatter, M. Gstaiger, R. Aebersold, C.C. Weihl, H. Meyer, Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations, Nat Cell Biol 13 (9) (2011) 1116–1123.
[31] I.T. Pleasure, M.M. Black, J.H. Keen, Valosin-containing protein, VCP, is a ubiqui-
tous clathrin-binding protein, Nature 365 (6445) (1993) 459–462.
[32] H.N. Ramanathan, Y. Ye, The p97 ATPase associates with EEA1 to regulate the size of early endosomes, Cell Res 22 (2) (2012) 346–359.
[33] C. Tiengwe, C.M. Koeller, J.D. Bangs, Endoplasmic reticulum-associated degrada-
tion and disposal of misfolded GPI-anchored proteins in Trypanosoma brucei, Mol Biol Cell 29 (20) (2018) 2397–2409.
[34] C. Tiengwe, K.A. Muratore, J.D. Bangs, Surface proteins, ERAD and antigenic var- iation in Trypanosoma brucei, Cell Microbiol (2016).
[35] M.A. McDowell, D.M. Ransom, J.D. Bangs, Glycosylphosphatidylinositol-dependent secretory transport in Trypanosoma brucei, Biochem J 335 (Pt 3) (1998) 681–689.
[36] V.P. Triggs, J.D. Bangs, Glycosylphosphatidylinositol-dependent protein traﬃcking
in bloodstream stage Trypanosoma brucei, Eukaryot Cell 2 (1) (2003) 76–83.
[37] H.J. Zhou, J. Wang, B. Yao, S. Wong, S. Djakovic, B. Kumar, J. Rice, E. Valle,
F. Soriano, M.K. Menon, A. Madriaga, S. Kiss von Soly, A. Kumar, F. Parlati,
F.M. Yakes, L. Shawver, R. Le Moigne, D.J. Anderson, M. Rolfe, D. Wustrow, Discovery of a First-in-Class, Potent, Selective, and Orally Bioavailable Inhibitor of the p97 AAA ATPase (CB-5083), J Med Chem 58 (24) (2015) 9480–9497.
[38] D.J. Anderson, R. Le Moigne, S. Djakovic, B. Kumar, J. Rice, S. Wong, J. Wang,
B. Yao, E. Valle, S. Kiss von Soly, A. Madriaga, F. Soriano, M.K. Menon, Z.Y. Wu,
M. Kampmann, Y. Chen, J.S. Weissman, B.T. Aftab, F.M. Yakes, L. Shawver,
H.J. Zhou, D. Wustrow, M. Rolfe, Targeting the AAA ATPase p97 as an Approach to Treat Cancer through Disruption of Protein Homeostasis, Cancer Cell 28 (5) (2015) 653–665.
[39] R. Le Moigne, B.T. Aftab, S. Djakovic, E. Dhimolea, E. Valle, M. Murnane, E.M. King,
F. Soriano, M.K. Menon, Z.Y. Wu, S.T. Wong, G.J. Lee, B. Yao, A.P. Wiita, C. Lam,
J. Rice, J. Wang, M. Chesi, P.L. Bergsagel, M. Kraus, C. Driessen, S. Kiss von Soly,
F.M. Yakes, D. Wustrow, L. Shawver, H.J. Zhou, T.G. Martin, J.L. Wolf 3rd,
C.S. Mitsiades, D.J. Anderson, M. Rolfe, The p97 Inhibitor CB-5083 Is a Unique Disrupter of Protein Homeostasis in Models of Multiple Myeloma, Mol Cancer Ther 16 (11) (2017) 2375–2386.
[40] A. Gareau, C. Rico, D. Boerboom, M.E. Nadeau, In vitro eﬃcacy of a ﬁrst-generation
valosin-containing protein inhibitor (CB-5083) against canine lymphoma, Vet Comp Oncol 16 (3) (2018) 311–317.
[41] W.K. Tang, T. Odzorig, W. Jin, D. Xia, Structural Basis of p97 Inhibition by the Site- Selective Anticancer Compound CB-5083, Mol Pharmacol 95 (3) (2019) 286–293.
[42] J.S. Silverman, K.J. Schwartz, S.L. Hajduk, J.D. Bangs, Late endosomal Rab7 reg- ulates lysosomal traﬃcking of endocytic but not biosynthetic cargo in Trypanosoma brucei, Mol. Microbiol. 82 (2011) 664–678.
[43] C. Koeller, J.D. Bangs, Processing and targeting of Cathepsin L (TbCatL) to the
Lysosome in Trypanosoma brucei, Cell Microbiol (2018) e12980.
[44] R.F. Peck, A.M. Shiﬂett, K.J. Schwartz, A. McCann, S.L. Hajduk, J.D. Bangs, The LAMP-like protein p67 plays an essential role in the lysosome of African trypano- somes, Mol. Microbiol. 68 (2008) 933–946.
[45] I. Kim, J. Ahn, C. Liu, K. Tanabe, J. Apodaca, T. Suzuki, H. Rao, The Png1-Rad23
complex regulates glycoprotein turnover, J Cell Biol 172 (2) (2006) 211–219.
[46] T. Suzuki, H. Park, M.A. Kwoﬁe, W.J. Lennarz, Rad23 provides a link between the Png1 deglycosylating enzyme and the 26 S proteasome in yeast, J Biol Chem 276 (24) (2001) 21601–21607.
[47] G. Li, G. Zhao, X. Zhou, H. Schindelin, W.J. Lennarz, The AAA ATPase p97 links
peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptor, Proc Natl Acad Sci U S A 103 (22) (2006) 8348–8353.
[48] H. Richly, M. Rape, S. Braun, S. Rumpf, C. Hoege, S. Jentsch, A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting, CB-5339 Cell 120 (1) (2005) 73–84.