Structure of the reduced microsporidian proteasome bound by PI31-like peptides in dormant spores

Abstract

Proteasomes play an essential role in the life cycle of intracellular pathogens with extracellular stages by ensuring proteostasis in environments with limited resources. In microsporidia, divergent parasites with extraordinarily streamlined genomes, the proteasome complexity and structure are unknown, which limits our understanding of how these unique pathogens adapt and compact essential eukaryotic complexes. We present cryo-electron microscopy structures of the microsporidian 20S and 26S proteasome isolated from dormant or germinated Vairimorpha necatrix spores. The discovery of PI31-like peptides, known to inhibit proteasome activity, bound simultaneously to all six active sites within the central cavity of the dormant spore proteasome, suggests reduced activity in the environmental stage. In contrast, the absence of the PI31-like peptides and the existence of 26S particles post-germination in the presence of ATP indicates that proteasomes are reactivated in nutrient-rich conditions. Structural and phylogenetic analyses reveal that microsporidian proteasomes have undergone extensive reductive evolution, lost at least two regulatory proteins, and compacted nearly every subunit. The highly derived structure of the microsporidian proteasome, and the minimized version of PI31 presented here, reinforce the feasibility of the development of specific inhibitors and provide insight into the unique evolution and biology of these medically and economically important pathogens.

High-resolution structure of mammalian PI31–20S proteasome complex reveals mechanism of proteasome inhibition

“…The relationship between PI31-mediated proteasome inhibition and such functions is unclear and would appear to require release of inhibition upon delivery. Although we have not identified in vitro conditions that reverse PI31-proteasome binding and inhibition, such a process has been proposed as a mechanism for termination of PI31-like mediated proteasome inhibition that allows assembly of active 20S-19S/PA700 holoenzyme complexes after germination of dormant Microporidia spores [23]. The generality of the latter process is unknown and appears to be inconsistent with previous evidence that PI31 blocks the in vitro assembly and activation of proteasome holoenzymes with 19S/PA700 and PA28 regulators [16,43].…”

“…More recently, the structure of a complex between 20S proteasome and a truncated PI31-like protein has been described from dormant spores of Microsporidia [23]. Although these structures are in agreement regarding a general mechanism by which PI31 interacts with and inhibits the proteasome, they have a number of specific significant differences.…”

Functional annotation of a divergent genome using sequence and structure-based homology

“…We manually curated each protein from V. necatrix and updated or complemented the functional annotation. In addition, we used two previous structural studies ( 56, 57 ) to annotate the ribosomal and proteasomal genes. The high-quality annotation of these proteins can help to improve and correct the functional annotation of other microsporidian organisms.…”

“…When comparing our annotations with the assignments of ProtNLM, 42% of the gene function predictions had the same name or were homologous, and 33% were different at first glance and were further looked at in more detail below. Both approaches failed to annotate 22% of all gene functions, while 3% could be assigned using solved protein structures ( 56, 57 ). The 42% identical annotations, included cases where one approach predicted a gene function while the other identified a protein domain typically fulfilling this gene function, or vice versa.…”

A bacterial ribosome hibernation factor with evolutionary connections to eukaryotic protein synthesis

“…The phenomenon of molecular hibernation has been observed for many cellular enzymes, including RNA polymerases [3][4][5] , proteasomes 6 , and catalases 7 . Most extensively, this process has been studied in ribosomes -essential ribonucleoprotein complexes that catalyse protein synthesis, also known as translation.…”