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In addition to vaccines, there is an urgent need for supplemental antiviral therapeutics to dampen the persistent COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS–CoV-2). The transmembrane protease serine 2 (TMPRSS2), which is responsible for the proteolytic processing of the SARS-CoV-2 spike protein as virus priming for cell entry, appears as a rational therapeutic target for the clearance of SARS-CoV-2 infection. Accordingly, selective inhibitors of TMPRSS2 represent potential tools for prevention and treatment of COVID-19. Here, we tested the inhibitory capacities of the human milk glycoprotein lactoferrin and its N-terminal peptide pLF1, which we identified as inhibitors of plasminogen, a serine protease homologous to TMPRSS2. In vitro proteolysis assays revealed that, unlike full-length lactoferrin, pLF1 significantly inhibited the proteolytic activity of TMPRSS2. pLF1 inhibited both the proteolytic processing of the SARS-CoV-2 spike protein and the SARS-CoV-2 infection of simian Vero cells. Because lactoferrin is a natural product and several biologically active peptides, such as the N-terminally derived lactoferricins, are produced naturally by pepsin-mediated digestion, natural or synthetic peptides from lactoferrin represent well-achievable candidates for supporting prevention and treatment of COVID-19.

Mitochondrial proteins are encoded by both nuclear and mitochondrial DNA. While some of the essential subunits of the oxidative phosphorylation (OXPHOS) complexes responsible for cellular ATP production are synthesized directly in the mitochondria, most mitochondrial proteins are first translated in the cytosol and then imported into the organelle using a sophisticated transport system. These proteins are directed mainly by targeting presequences at their N-termini. These presequences need to be cleaved to allow the proper folding and assembly of the pre-proteins into functional protein complexes. In the mitochondria, the presequences are removed by several processing peptidases, including the mitochondrial processing peptidase (MPP), the inner membrane processing peptidase (IMP), the inter-membrane processing peptidase (MIP), and the mitochondrial rhomboid protease (Pcp1/PARL). Their proper functioning is essential for mitochondrial homeostasis as the disruption of any of them is lethal in yeast and severely impacts the lifespan and survival in humans. In this review, we focus on characterizing the structure, function, and substrate specificities of mitochondrial processing peptidases, as well as the connection of their malfunctions to severe human diseases.

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.

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.

Mitochondrial nucleoids consist of several different groups of proteins, many of which are involved in essential cellular processes such as the replication, repair and transcription of the mitochondrial genome. The eukaryotic, ATP-dependent protease Lon is found within the central nucleoid region, though little is presently known about its role there. Aside from its association with mitochondrial nucleoids, human Lon also specifically interacts with RNA. Recently, Lon was shown to regulate TFAM, the most abundant mtDNA structural factor in human mitochondria. To determine whether Lon also regulates other mitochondrial nucleoid- or ribosome-associated proteins, we examined the in vitro digestion profiles of the Saccharomyces cerevisiae TFAM functional homologue Abf2, the yeast mtDNA maintenance protein Mgm101, and two human mitochondrial proteins, Twinkle helicase and the large ribosomal subunit protein MrpL32. Degradation of Mgm101 was also verified in vivo in yeast mitochondria. These experiments revealed that all four proteins are actively degraded by Lon, but that three of them are protected from it when bound to a nucleic acid; the Twinkle helicase is not. Such a regulatory mechanism might facilitate dynamic changes to the mitochondrial nucleoid, which are crucial for conducting mitochondrial functions and maintaining mitochondrial homeostasis.

The major role of mitochondria is to provide cells with energy, but no less important are their roles in responding to various stress factors and the metabolic changes and pathological processes that might occur inside and outside the cells. The post-translational modification of proteins is a fast and efficient way for cells to adapt to ever changing conditions. Phosphorylation is a post-translational modification that signals these changes and propagates these signals throughout the whole cell, but it also changes the structure, function and interaction of individual proteins. In this review, we summarize the influence of kinases, the proteins responsible for phosphorylation, on mitochondrial biogenesis under various cellular conditions. We focus on their role in keeping mitochondria fully functional in healthy cells and also on the changes in mitochondrial structure and function that occur in pathological processes arising from the phosphorylation of mitochondrial proteins.

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions.

TheS-adenosyl-L-methionine (SAM)-dependent methyltransferase CcbJ fromStreptomyces caelestiscatalyzes one of the final steps in the biosynthesis of the antibiotic celesticetin, methylation of the N atom of its proline moiety, which greatly enhances the activity of the antibiotic. Since several celesticetin variants exist, this enzyme may be able to act on a variety of substrates. The structures of CcbJ determined by MAD phasing at 3.0 Å resolution, its native form at 2.7 Å resolution and its complex withS-adenosyl-L-homocysteine (SAH) at 2.9 Å resolution are reported here. Based on these structures, three point mutants, Y9F, Y17F and F117G, were prepared in order to study its behaviour as well as docking simulations of both CcbJ–SAM–substrate and CcbJ–SAH–product complexes. The structures show that CcbJ is a class I SAM-dependent methyltransferase with a wide active site, thereby suggesting that it may accommodate a number of different substrates. The mutation results show that the Y9F and F117G mutants are almost non-functional, while the Y17F mutant has almost half of the wild-type activity. In combination with the docking studies, these results suggest that Tyr9 and Phe117 are likely to help to position the substrate for the methyl-transfer reaction and that Tyr9 may also facilitate the reaction by removing an H+ion. Tyr17, on the other hand, seems to operate by helping to stabilize the SAM cofactor.

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 β-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 β-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 β-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)

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.

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18–36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates. Background: The plasminogen system is central in cell migration and is thus involved in many patho/physiological processes. Results: M6P-IGF2R is a regulatory factor in plasminogen-associated complexes and mediates plasminogen internalization. Conclusion: The uptake of plasminogen by M6P-IGF2R might be an important pathway to control plasminogen activation in cells. Significance: M6P-IGF2R restricts plasmin activity and its loss might lead to rampant fibrinolysis.

Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.

The ATP-dependent Lon protease belongs to a unique group of proteases that bind DNA. Eukaryotic Lon is a homo-oligomeric ring-shaped complex localized to the mitochondrial matrix. In vitro, human Lon binds specifically to a single-stranded GT-rich DNA sequence overlapping the light strand promoter of human mitochondrial DNA (mtDNA). We demonstrate that Lon binds GT-rich DNA sequences found throughout the heavy strand of mtDNA and that it also interacts specifically with GU-rich RNA. ATP inhibits the binding of Lon to DNA or RNA, whereas the presence of protein substrate increases the DNA binding affinity of Lon 3.5-fold. We show that nucleotide inhibition and protein substrate stimulation coordinately regulate DNA binding. In contrast to the wild type enzyme, a Lon mutant lacking both ATPase and protease activity binds nucleic acid; however, protein substrate fails to stimulate binding. These results suggest that conformational changes in the Lon holoenzyme induced by nucleotide and protein substrate modulate the binding affinity for single-stranded mtDNA and RNA in vivo. Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase gamma and the Twinkle helicase, which are components of mitochondrial nucleoids. Taken together, these results suggest that Lon participates directly in the metabolism of mtDNA.

Rationale: The urokinase plasminogen activator (uPA) system is among the most crucial pericellular proteolytic systems associated with the processes of angiogenesis. We previously identified an important regulator of the uPA system in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R). Objective: Here, we wanted to clarify whether and how did the soluble form of M6P/IGF2R (sM6P/IGF2R) contribute to modulation of the uPA system. Methods and Results: By using specific inhibitors and RNA interference, we show that the tumor necrosis factor α convertase (TACE, ADAM-17) mediates the release of the ectodomain of M6P/IGF2R from human endothelial cells. We demonstrate further that sM6P/IGF2R binds plasminogen (Plg) and thereby prevents Plg from binding to the cell surface and uPA, ultimately inhibiting in this manner Plg activation. Furthermore, peptide 18-36 derived from the Plg-binding site of M6P/IGF2R mimics sM6P/IGF2R in the inhibition of Plg activation and blocks cancer cell invasion in vitro, endothelial cell invasion in vivo, and tumor growth in vivo. Conclusions: The interaction of sM6P/IGF2R with Plg may be an important regulatory mechanism to inhibit migration of cells using the uPA/uPAR system.