Syndicate content

Malaria Parasites Secrete EVs Containing Proteasome 20S Complex and Kinases to Target Specific Cytoskeleton Proteins and Weaken RBC Membrane to Greatly Enhance Infectivity; Possible Treatment Approaches Suggested; Wider Applications Envisioned

Red blood cells (RBCs) are the body's lifeline, but they also serve as the perfect hosts for one of the deadliest infectious organisms: the malaria parasite. About two weeks after infecting the body, the deadliest malaria parasite, Plasmodium falciparum (Pf), launches its invasion, rapidly taking over masses of red blood cells so as to grow within them. That's when the disease can turn life-threatening, ultimately killing approximately a thousand children around the world every day. Two research teams at the Weizmann Institute of Science in Israel have joined forces to reveal what enables the malaria parasite to mount such an effective takeover. Neta Regev-Rudzki (photo) (http://www.weizmann.ac.il/Biomolecular_Sciences/Regev/home), PhD, Senior Scientist, and her team in the Weizmann’s Biomolecular Sciences Department discovered that tiny sac-like "packages" called extracellular vesicles (EVs), released by Pf, contain peculiar cargo: including a cellular protein-degrading machine called a proteasome (https://en.wikipedia.org/wiki/Proteasome), which normally breaks down misfolded or unneeded proteins. Departmental colleague Professor Michal Sharon (phote below) (http://www.weizmann.ac.il/Biomolecular_Sciences/MichalSharon/), PhD, Principal Investigator, whose team specializes in studying proteasomes, suggested that the two labs combine their expertise to figure out what, if anything, those proteasomes are doing in the malaria EVs. The joint study--led by PhD student Elya Dekel from Dr. Regev-Rudzki's lab and postdoctoral fellow Dr. Dana Yaffe from Professor Sharon's lab--uncovered an extraordinary strategy by which the malaria parasite harnesses the proteasome for its own purpose: priming naïve RBCs for the coming invasion. Their results were published online on February 19, 2021 in Nature Communications (https://www.nature.com/articles/s41467-021-21344-8). The open-access article is titled “20S Proteasomes Secreted by the Malaria Parasite Promote Its Growth.”

RBCs already possess unique properties that make them a particularly attractive host for Pf. First of all, RBCs lose most of their organelles by the time they mature, which means that they lack the means to defend themselves against the parasite. Moreover, because they must squeeze through the smallest blood vessels to deliver oxygen to all body tissues, they possess an enormous facility to undergo structural remodeling, which the parasite turns to its advantage.

After human infection, Pf spends the first two weeks or so in the liver, where it undergoes transformations and multiplies. It then enters the bloodstream, invading mature RBCs, and sends out an advance party--the proteasome encapsulated within the EVs--to soften the membranes of neighboring naïve RBCs, making them easier to penetrate. This preparatory step helps thousands of parasites to invade vast numbers of these RBCs within seconds.

The researchers managed to expose the details of the pre-invasion. They established that malaria parasites’ EVs contain a fully assembled and functional proteasome version known as 20S (image below), which uses less energy and is smaller than another proteasome version (e.g, the larger proteasome 26S).

ACTIVITY OF Pf-RELEASED 20S PROTEASOMES

Working with Dr. Irit Rosenhek-Goldian and Dr. Sidney R. Cohen of the Weizmann Chemical Research Support Department, the scientists developed a special visualization technique based on atomic force microscopy, which showed that the 20S proteasome pokes holes in the RBC’s cytoskeleton, the protein framework that holds the cellular membrane together, and dissolves some of its filaments. The researchers identified four RBC proteins (the phosphorylated cytoskeletal proteins β-adducin, ankyrin-1, dematin, and Epb4.1) targeted for degradation by the proteasome. The scientists also developed a machine-learning algorithm that distinguishes the cytoskeletons of RBCs that had been altered by the secreted 20S proteasome, from those that hadn't.

To confirm that the softening of the RBC membranes had indeed been produced by the proteasomes, the scientists blocked the active site of the 20S proteasome with an inhibitor prior to exposing the cells to the malarial EVs. The inhibitor prevented the softening of the cellular membranes, limiting the invasion of the cells by the parasite and, consequently, the parasite's growth.

BLOCKING PARASITE’S PROTEASOMES

This last result opens up a new direction in the search for malaria therapies: blocking the parasite's proteasome secretion so as to reduce from the level of invasion into red blood cells. The study's findings may also prove relevant to other infectious diseases because subunits of the 20S proteasome have been found in EVs released by different classes of parasites, for example, those causing sleeping sickness (trypanosomiasis) and leishmaniasis. Moreover, the results may also be applicable to cancer, as the 20S proteasome has recently been detected in EVs released by human cells that facilitate tumor growth.

KINASES IN Pf –DERIVED EVs MAY PROVIDE ADDITIONAL TREATMENT TARGETS

The researchers noted that, in addition to the proteasome 20S, the Pf EVs also contain at least 14 Pf kinases, many of which could be involved in the phosphorylation of RBC membrane and cytoskeleton proteins. Thus, the parasite may use the kinases to specifically phosphorylate RBC cytoskeleton proteins. This, in turn, would prime the target proteins towards degradation and, consequently, to cytoskeleton destabilization. Inhibition of the kinase activity detected in Pf-derived EVs will be an exciting avenue for testing the potential of specific kinase inhibitors in preventing parasite Invasion, the authors stated.

SEQUENE OF EVENTS REDUCING RBC MEMBRANE STIFFNESS

In their Nature Communications article, the authors wrote that “We found that following Pf-derived EV introduction, two sequential steps take place. First, RBC proteins are subjected to specific phosphorylation, including many cytoskeletal proteins. Then, the delivered 20S proteasome mediates the degradation of the phosphorylated cytoskeleton proteins by an ubiquitin-independent process. This mechanism, reduces the stiffness of the membrane, and therefore primes naïve RBCs for parasite invasion, eventually increasing the parasite’s growth capacity. Put together, our results combine to reveal a previously unidentified pathway of Pf survival in the human host RBC.”

PREFERENCE FOR 20S PROTEOSOME OVER 26S PROTEOSOME

The authors also noted that “The parasite’s specific preference for the 20S proteasome is interesting, given that the degradation of proteins is predominantly mediated by the ubiquitin-dependent 26S proteasomal pathway, which comprises the 20S catalytic core particle and the 19S regulatory complex. The existence of the alternative, simpler degradation route mediated solely by the 20S proteasome is a recent discovery.”

“Unlike its 26S counterpart, wherein substrate selectivity is achieved by ubiquitin tagging, the 20S proteasome cleaves proteins containing partially unfolded regions that can enter into its catalytic chamber. Although this field of research is still in its infancy, accumulating evidence indicates that the 20S proteasome degradation route is tightly regulated, influencing various cellular processes, such as neuronal stimulation, antigenic peptide production, and post-translational processing.”

“When considering the requirements of 26S proteasome-mediated degradation, i.e., the dependency on ATP hydrolysis and ubiquitinylating enzymes (E1, E2, and E3), the preference of the parasite to harness the 20S-mediated degradation route is reasonable.

Another critical factor is the restricted size of the malaria-derived EVs, 50–200 nm in diameter, which limits the encapsulation [of] 26S proteasomes (~45 nm × 20 nm), to a greater degree than 20S particles (~15 nm × 12 nm). Thus, degradation via the 20S proteasome offers a relatively compact system for eliminating proteins, one that reduces energy costs and is not dependent on an enzymatic cascade.”

PREVIOUSLY UNKNOWN 20S PROTEASOME SECRETION MECHANISM

Overall, the scientists said that their findings reveal a previously unknown 20S proteasome secretion mechanism employed by the human malaria parasite, which primes RBCs for parasite invasion by altering membrane stiffness, to facilitate malaria parasite growth.

CONCLUSIONS

In conclusion, the authors noted that “We demonstrated that functional cellular machinery, the 20S proteasome, is transported by EVs in order to reshape the host membrane. Along these lines, recent proteomic data indicated the presence of subunits of the 20S proteasome in EVs derived from many pathogens, including pathogenic protozoans such as Acanthamoeba castellanii and Toxoplasma gondii, helminth parasites such as Echinococcus granulosus, as well as Leishmania, Trichuris muris, Trichomonas vaginalis, and even fungus. These results may therefore hint at a more general mechanism, in which pathogen-derived EVs use the degradation machinery to reshape the host proteome.”

“From a broader perspective, active 20S proteasomes, and not 26S proteasomes, were recently discovered within EVs secreted from tumor-associated macrophages, mesenchymal stem cells, and endothelial cells. Hence, it is possible that EV-mediated delivery of functional 20S proteasome complexes for cellular remodeling is a more common phenomenon than currently believed.”
RESEARCH TEAM

In addition to those already mentioned, the research team Included Dr. Gili Ben-Nissan, Dr. Yifat Ofir-Birin, Dr. Mattia Morandi, Yael Ohana Daniel, Paula Abou Karam, Daniel Alfandari, Shimrit Malihi, Tal Temin Block, Dr. Or-Yam Revach, Ariel Rudik and Dr. Ori Avinoam in the Weizmann’s Biomolecular Sciences Department; Dr. Ron Rotkopf, Dr. Ido Azuri, and Dr. Ziv Porat of the Weizmann’s Life Sciences Core Facilities Department; Debakshi Mullick and Professor Nir S. Gov of the Weizmann’s Chemical and Biological Physics Department; Dr. Tamar Ziv of The Technion--Israel Institute of Technology; Dr. Xavier Sisquella, Dr. Matthew A. Pimentel, Dr. Thomas Nebl, and Dr. Eugene Kapp of the University Of Melbourne and the Walter and Eliza Hall Institute of Medical Research; Giulia Bergamaschi and Professor Gijs J.L Wuite of Vrije Universiteit Amsterdam; Dr. Raya Sorkin of Tel Aviv University; and Dr. Teresa Carvalho of La Trobe University, Melbourne.

This posting was based on a news release prepared by the Weizmann Institute of Science, and has been modified slightly by BioQuick News to clarify and/or amplify certain points.

[Nature Communications article]

IMAGES

Professor Michal Sharon and image of proteasome 20S.