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Published on: February 25, 2011
Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors
Jan Silhan1, Pavla Fajtova2,3, Jitka Bartosova1
1Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Prague, Czech Republic.
Abstract:
The proteasome is a proteolytic enzyme complex essential for protein homeostasis in mammalian cells and protozoan parasites like Trichomonas vaginalis (Tv), the cause of the most common, non-viral sexually transmitted disease. Tv and other protozoan 20S proteasomes have been validated as druggable targets for antimicrobials. However, low yields and purity of the native proteasome have hindered studies of the Tv 20S proteasome (Tv20S). We address this challenge by creating a recombinant protozoan proteasome by expressing all seven α and seven β subunits of Tv20S alongside the Ump-1 chaperone in insect cells. The recombinant Tv20S displays biochemical equivalence to its native counterpart, confirmed by various assays. Notably, the marizomib (MZB) inhibits all catalytic subunits of Tv20S, while the peptide inhibitor carmaphycin-17 (CP-17) specifically targets β2 and β5. Cryo-electron microscopy (cryo-EM) unveils the structures of Tv20S bound to MZB and CP-17 at 2.8 Å. These findings explain MZB's low specificity for Tv20S compared to the human proteasome and demonstrate CP-17's higher specificity. Overall, these data provide a structure-based strategy for the development of specific Tv20S inhibitors to treat trichomoniasis.
Insights
Researchers developed a recombinant Trichomonas vaginalis proteasome (Tv20S) for drug development. Structural studies revealed specific inhibitors for treating trichomoniasis, a common sexually transmitted disease.
Area of Science:
- Biochemistry
- Structural Biology
- Parasitology
Background:
- The proteasome is crucial for protein homeostasis in cells.
- Trichomonas vaginalis (Tv) proteasomes are potential antimicrobial targets.
- Native Tv proteasome isolation challenges hinder research.
Purpose of the Study:
- To create a recombinant Tv 20S proteasome (Tv20S) for structural and biochemical studies.
- To characterize Tv20S inhibition by marizomib (MZB) and carmaphycin-17 (CP-17).
- To provide a basis for developing specific inhibitors against Tv.
Main Methods:
- Recombinant expression of Tv20S subunits and Ump-1 chaperone in insect cells.
- Biochemical assays to confirm recombinant Tv20S activity.
- Cryo-electron microscopy (cryo-EM) to determine Tv20S-inhibitor complex structures.
Main Results:
- Recombinant Tv20S is biochemically equivalent to the native proteasome.
- Marizomib (MZB) inhibits all catalytic subunits, while CP-17 specifically targets β2 and β5.
- Cryo-EM structures reveal inhibitor binding modes and explain differential specificities.
Conclusions:
- A recombinant Tv20S system facilitates structural and biochemical investigations.
- CP-17 exhibits higher specificity for Tv20S than MZB.
- Structural insights enable the design of targeted inhibitors for trichomoniasis treatment.
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