Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Reiter, Katherine H.; Mukhopadhyay, Debaditya; Zhang, Hong; Boucher, Lauren; Kumar, Nirbhay; Bosch, Jürgen; Matunis, Michael J.

doi:10.1074/jbc.m113.498410

Cited by 27 publications

(50 citation statements)

References 55 publications

(67 reference statements)

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“…Complete malaria culture media consisted of RPMI 1640 (Invitrogen) supplemented with 25 mM HEPES, 0.2% sodium bicarbonate (Invitrogen), 12.5 g/ml hypoxanthine (Sigma), and 5 mg/ml AlbuMAX II (Life Technologies). Dd2 attB strain parasites were grown as described above; synchronized with one cycle of sorbitol treatment at the ring stage, as described; and allowed to mature to schizonts (12). A further round of synchronization was performed using a MACS column (16).…”

Section: Methodsmentioning

confidence: 99%

“…For Western blot analysis, treated parasites were harvested via saponin lysis, as previously described (12). Parasite pellets were resuspended in PBS lysis buffer containing 200 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.05% sodium deoxycholate, 100 M leupeptin, 1 mM PMSF, 5 mM N-ethylmaleimide, 20 g/ml aprotinin, and 1 g/ml pepstatin and lysed using a Biorupter water bath (Diagenode) for five rounds of 30 s on, 30 s with ice.…”

Section: Methodsmentioning

confidence: 99%

“…Membranes were probed with 1:1000 mAb Pf SUMO 7E11 and 1:15,000 HRPconjugated G␣M (Jackson) (12). Signals were detected by ECL Prime chemiluminescent substrate (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

“…The SUMO conjugation machinery is conserved in P. falciparum; however, we have previously determined that the surface residues important for interactions between the E1-activating and E2-conjugating enzymes have diverged significantly from humans (12). Analysis of human and P. falciparum Ubc9 chimeric proteins revealed that residues comprising the ␣1 helix and ␤1-␤2 loop are critical for functional and also species-specific interactions with cognate E1-activating enzymes.…”

mentioning

confidence: 99%

“…We previously showed that SUMOylation levels peak during the trophozoite stage of the intraerythrocyte developmental cycle, which is characterized by enhanced oxidative stress caused by parasite digestion of host hemoglobin (12). Notably, anti-malarials, such as chloroquine and artemisinin, are thought to kill parasites at least in part by overloading oxidative stress response pathways (13,14).…”

mentioning

confidence: 99%

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Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems

Reiter¹,

Ramachandran²,

Xia³

et al. 2016

Journal of Biological Chemistry

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Protein modification by small ubiquitin-related modifiers (SUMOs) is essential and conserved in the malaria parasite, Plasmodium falciparum. We have previously shown that interactions between the SUMO E1-activating and E2-conjugating enzyme in P. falciparum are distinct compared with human, suggesting a potential target for development of parasite-specific inhibitors of SUMOylation. The parasite asexual trophozoite stage is susceptible to iron-induced oxidative stress and is subsequently a target for many of the current anti-malarial drugs. Here, we provide evidence that SUMOylation plays a role in the parasite response to oxidative stress during red blood cell stages, indicative of a protective role seen in other organisms. Using x-ray crystallography, we solved the structure of the human SUMO E1 ubiquitin fold domain in complex with the E2, Ubc9. The interface defined in this structure guided in silico modeling, mutagenesis, and in vitro biochemical studies of the P. falciparum SUMO E1 and E2 enzymes, resulting in the identification of surface residues that explain species-specific interactions. Our findings suggest that parasite-specific inhibitors of SUMOylation could be developed and used in combination therapies with drugs that induce oxidative stress.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Membranes were probed with 1:1000 mAb Pf SUMO 7E11 and 1:15,000 HRPconjugated G␣M (Jackson) (12). Signals were detected by ECL Prime chemiluminescent substrate (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems

Reiter¹,

Ramachandran²,

Xia³

et al. 2016

Journal of Biological Chemistry

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Concepts and Methodologies to Study Protein SUMOylation: An Overview

Matunis

Rodríguez²

2016

Methods in Molecular Biology

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Protein modification by the small ubiquitin-related modifier (SUMO) was simultaneously discovered by several groups at the middle of the 1990s. Although distinct names were proposed including Sentrin, GMP1, PIC1, or SMT3, SUMO became the most popular. Early studies on the functions of SUMOylation focused on activities in the nucleus, including transcription activation, chromatin structure, and DNA repair. However, it is now recognized that SUMOylation affects a large diversity of cellular processes both in the nucleus and the cytoplasm and functions of SUMOylation appear to have undefined limits. SUMO-conjugating enzymes and specific proteases actively regulate the modification status of target proteins. The recent discoveries of ubiquitin-SUMO hybrid chains, multiple SUMO-interacting motifs, and macromolecular complexes regulated by SUMOylation underscore the high complexity of this dynamic reversible system. New conceptual frameworks suggested by these findings have motivated the development of new methodologies to study pre- and post-SUMOylation events in vitro and in vivo, using distinct model organisms. Here we summarize some of the new developments and methodologies in the field, particularly those that will be further elaborated on in the chapters integrating this book.

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Detection of SUMOylation in Plasmodium falciparum

Reiter

Matunis

2016

Methods in Molecular Biology

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Reversible protein modification by small ubiquitin-related modifiers (SUMOs) regulates many cellular processes, including transcription, protein quality control, cell division, and oxidative stress. SUMOylation is therefore essential for normal cell function and represents a potentially valuable target for the development of inhibitors of pathogenic eukaryotic organisms, including the malaria parasite, Plasmodium falciparum (Pf). The specific and essential functions of SUMOylation in Pf, however, remain largely uncharacterized. The further development of antimalarial drugs targeting SUMOylation would benefit significantly from a more detailed understanding of its functions and regulation during the parasite life cycle. The recent development of antibodies specific for Pf SUMO provides a valuable tool to study the functions and regulation of SUMOylation. In preliminary studies, we have used immunoblot analysis to demonstrate that SUMOylation levels vary significantly in parasites during different stages of the red blood cell cycle and also in response to oxidative stress. Owing to the dynamic nature of SUMOylation and to the robust activity of SUMO isopeptidases, analysis of SUMOylation in cultured Pf parasites requires a number of precautions during parasite purification and lysis. Here, we outline methods for preserving SUMO conjugates during isolation of Pf parasites from human red blood cell cultures, and for their detection by immunoblot analysis using PfSUMO-specific antibodies.

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Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Cited by 27 publications

References 55 publications

Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems

Characterization and Structural Insights into Selective E1-E2 Interactions in the Human and Plasmodium falciparum SUMO Conjugation Systems

Concepts and Methodologies to Study Protein SUMOylation: An Overview

Detection of SUMOylation in Plasmodium falciparum

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