A myeloid leukemia factor homolog is involved in tolerance to stresses and stress-induced protein metabolism in Giardia lamblia

by Selleckchem | May 16 2023

Background: The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia.

Results: In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF.

Conclusions: Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments.

Comments：

In summary, this study investigated the function of MLF vesicles (MLFVs) in Giardia lamblia, an organism that lacks some autophagy machineries. The researchers found that six stress inducers increased reactive oxygen species production, vesicle number, and levels of MLF, FYVE, and ATG8-like protein in G. lamblia. They also found that CDK2m3, a mutant protein of cyclin-dependent kinase 2, colocalized with MLF in the same vesicles and was positively regulated by MLF. Knockdown of MLF with the CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers, and complementation of MLF restored cell survival. Finally, the study found that human MLF2 can increase cyst wall protein expression and cyst formation in G. lamblia and colocalize with MLFVs and interact with MLF, suggesting that MLF family proteins are functionally conserved in evolution. Overall, the study provides insights into the important role of MLF in survival in stress conditions and suggests that MLFVs share similar stress-induced characteristics with autophagy compartments.