Polyphosphate and tyrosine phosphorylation in the N-terminal domain of the human mitochondrial Lon protease disrupts its functions.

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.

Mitochondrial HSP70 Chaperone System-The Influence of Post-Translational Modifications and Involvement in Human Diseases.

Since their discovery, heat shock proteins (HSPs) have been identified in all domains of life, which demonstrates their importance and conserved functional role in maintaining protein homeostasis. Mitochondria possess several members of the major HSP sub-families that perform essential tasks for keeping the organelle in a fully functional and healthy state. In humans, the mitochondrial HSP70 chaperone system comprises a central molecular chaperone, mtHSP70 or mortalin (HSPA9), which is actively involved in stabilizing and importing nuclear gene products and in refolding mitochondrial precursor proteins, and three co-chaperones (HSP70-escort protein 1-HEP1, tumorous imaginal disc protein 1-TID-1, and Gro-P like protein E-GRPE), which regulate and accelerate its protein folding functions. In this review, we summarize the roles of mitochondrial molecular chaperones with particular focus on the human mtHsp70 and its co-chaperones, whose deregulated expression, mutations, and post-translational modifications are often considered to be the main cause of neurological disorders, genetic diseases, and malignant growth.

The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.

Mutations to a glycine loop in the catalytic site of human Lon changes its protease, peptidase and ATPase activities.

UNLABELLED: Lon, also called protease La, is an ATP-dependent protease present in all kingdoms of life. It is involved in protein quality control and several regulatory processes. Eukaryotic Lon possesses three domains, an N-terminal domain, an ATPase domain and a proteolytic domain. It requires ATP hydrolysis to digest larger, intact proteins, but can cleave small, fluorogenic peptides such as Glu-Ala-Ala-Phe-MNA by only binding, but not hydrolyzing, ATP. Both ATPase and peptidase activities can be stimulated by the binding of a larger protein substrate, such as ß-casein. To better understand its mechanism of action, we have prepared several point mutants of four conserved residues of human Lon (G893A, G893P, G894A, G894P, G894S, G893A-G894A, G893P-G894A, G893A-G894P, T880V, W770A, W770P) and studied their ATPase, protease and peptidase activities. Our results show that mutations to Gly894 enhance its basal ATPase activity but do not change its ß-casein-stimulated activity. The loop containing Gly893 and Gly894, which flanks Lon's proteolytic active site, therefore appears to be involved in the conformational change that occurs upon substrate binding. Furthermore, mutations to Trp770 have the same general effects on the ATPase activity as mutations to Gly893, indicating that Trp770 is involved in ATPase stimulation. We have also established that this loop does not need to move in order to cleave small, fluorogenic peptides, but does move during the digestion of ß-casein. Finally, we also noted that Lon's ability to digest small peptides can be inhibited by moderate ATP concentrations. DATABASE: Lon (Endopeptidase La), EC 4.4.21.53 STRUCTURED DIGITAL ABSTRACT: • hLonP cleaves beta casein by protease assay (1, 2, 3, 4, 5, 6) • hLon and hLon bind by cross-linking study (View interaction).