Rhomboid intramembrane proteases regulate pathophysiological processes, but their targeting in a disease context has never been achieved. We decoded the atypical substrate specificity of malaria rhomboid PfROM4, but found, unexpectedly, that it results from "steric exclusion": PfROM4 and canonical rhomboid proteases cannot cleave each other's substrates due to reciprocal juxtamembrane steric clashes. Instead, we engineered an optimal sequence that enhanced proteolysis >10-fold, and solved high-resolution structures to discover that boronates enhance inhibition >100-fold. A peptide boronate modeled on our "super-substrate" carrying one "steric-excluding" residue inhibited PfROM4 but not human rhomboid proteolysis. We further screened a library to discover an orthogonal alpha-ketoamide that potently inhibited PfROM4 but not human rhomboid proteolysis. Despite the membrane-immersed target and rapid invasion, ultrastructural analysis revealed that single-dosing blood-stage malaria cultures blocked host-cell invasion and cleared parasitemia. These observations establish a strategy for designing parasite-selective rhomboid inhibitors and expose a druggable dependence on rhomboid proteolysis in non-motile parasites.

• MeSH
• amidy chemická syntéza   chemie   farmakologie   MeSH
• antimalarika chemická syntéza   chemie   farmakologie   MeSH
• HEK293 buňky MeSH
• inhibitory proteas chemická syntéza   chemie   farmakologie   MeSH
• kyseliny boronové chemická syntéza   chemie   farmakologie   MeSH
• lidé MeSH
• malárie krev   farmakoterapie   metabolismus   MeSH
• molekulární struktura MeSH
• parazitické testy citlivosti MeSH
• peptidy chemická syntéza   chemie   farmakologie   MeSH
• Plasmodium falciparum účinky léků   metabolismus   MeSH
• proteasy krev   metabolismus   MeSH
• proteolýza účinky léků   MeSH
• protozoální proteiny antagonisté a inhibitory   krev   metabolismus   MeSH
• racionální návrh léčiv * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Src kinase plays an important role in a multitude of fundamental cellular processes and is often found deregulated in tumors. Active Src adopts an open conformation, whereas inactive Src is characterized by a very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a fluorescence resonance energy transfer (FRET)-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatiotemporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of the Src structure. Surprisingly, we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing an open conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesion assembly, and its activity remains steady and high throughout the life cycle of focal adhesion and decreases during focal adhesion disassembly.

• MeSH
• biosenzitivní techniky metody   MeSH
• fokální adheze metabolismus   MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mutageneze MeSH
• rezonanční přenos fluorescenční energie MeSH
• skupina kinas odvozených od src-genu antagonisté a inhibitory   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Pepsin-family aspartic peptidases are biosynthesized as inactive zymogens in which the propeptide blocks the active site until its proteolytic removal upon enzyme activation. Here, we describe a novel dual regulatory function for the propeptide using a set of crystal structures of the parasite cathepsin D IrCD1. In the IrCD1 zymogen, intramolecular autoinhibition by the intact propeptide is mediated by an evolutionarily conserved exosite on the enzyme core. After activation, the mature enzyme employs the same exosite to rebind a small fragment derived from the cleaved propeptide. This fragment functions as an effective natural inhibitor of mature IrCD1 that operates in a pH-dependent manner through a unique allosteric inhibition mechanism. The study uncovers the propeptide-binding exosite as a target for the regulation of pepsin-family aspartic peptidases and defines the structural requirements for exosite inhibition.

• MeSH
• aktivace enzymů MeSH
• alosterická regulace MeSH
• katalytická doména MeSH
• kathepsin D chemie   metabolismus   MeSH
• kinetika MeSH
• klíšťata enzymologie   MeSH
• koncentrace vodíkových iontů MeSH
• krystalografie rentgenová MeSH
• ligandy MeSH
• peptidy chemie   metabolismus   MeSH
• prekurzory enzymů chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery.

• MeSH
• gramnegativní bakterie enzymologie   MeSH
• grampozitivní bakterie enzymologie   MeSH
• inhibitory serinových proteinas chemická syntéza   chemie   farmakologie   MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• proteasy metabolismus   MeSH
• racionální návrh léčiv * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Despite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 β cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in β cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse β cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated β cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.

• MeSH
• beta-buňky cytologie   účinky léků   metabolismus   sekrece   MeSH
• biosenzitivní techniky MeSH
• buněčné linie MeSH
• fluorescenční mikroskopie MeSH
• glukosa farmakologie   MeSH
• hypoglykemika farmakologie   MeSH
• inzulin metabolismus   sekrece   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• myši MeSH
• rekombinantní fúzní proteiny metabolismus   sekrece   MeSH
• reportérové geny MeSH
• tolbutamid farmakologie   MeSH
• vápník metabolismus   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH