R35 Investigate structure and function of human amyloid-peptide degrading proteases

/Summary: Aggregates of amyloid peptides are highly cytotoxic and associated with human illnesses such as Alzheimer's disease, Parkinson's disease, and systemic amyloidoses (e.g., AL and AA amyloidosis). These toxic amyloid aggregates form through cross-β-sheet formation between amyloid peptides. The process begins with the slow and reversible formation of small multi-peptide amyloid seeds and advances with rapid and largely irreversible elongation into amyloid fibrils. Monomeric amyloid peptides are crucial in both initiation and progression. Presequence protease (PreP, a metalloprotease from the M16C clan) and angiotensin-1 converting enzyme (ACE, a metalloprotease from the M2 clan) are structurally distinct enzymes that degrade monomeric amyloid peptides, such as amyloid β (Aβ), preventing the formation of toxic amyloid oligomers and fibrils. PreP, located in the mitochondrial matrix, degrades mitochondrial targeting sequences (also known as presequences) and imported Aβ, maintaining mitochondrial proteostasis. Loss-of-function mutations in PreP are linked to human disorders like mental retardation and psychosis; gene deletion in mice is embryonic lethal. In contrast, ACE is found on the plasma membrane or in the extracellular space and processes various bioactive peptides. Though small-molecule ACE inhibitors are a first-line treatment for hypertension, global ACE inhibition can disrupt multiple pathways and cause significant side effects. A deeper understanding of substrate selection mechanisms by these enzymes could lead to improved therapies with fewer off-target effects. Our lab has determined the structures of human PreP in both closed and open states and human full-length ACE dimer. Our integrative structural analysis suggests that these enzymes utilize significant, coordinated motions between separate domains to capture and degrade substrates of diverse sequences. However, the molecular basis for how these metalloprotease families use conformational dynamics to selectively degrade their preferred substrates remains unclear. We hypothesize that these proteins share mechanisms for recognizing and degrading amyloid peptides despite their structural differences. Our long-term goals are to elucidate the mechanistic basis for substrate selectivity by these amyloid peptide-degrading proteases and to understand the role of inter-domain dynamics in their catalytic cycles. To achieve this, we will employ structural, biochemical, and biophysical analyses, molecular dynamics simulations, and cellular assays to identify the molecular determinants governing the recognition and degradation of clinically important bioactive peptides by these enzymes. This work is significant because it will structurally define the key conformational states of clinically relevant metalloproteases and provide novel approaches for manipulating their activities, thereby opening new avenues for treating human diseases. It is innovative because it integrates diverse yet complementary methods of structural analysis to decipher how amyloid peptide-degrading proteases function. Project Number: 1R35GM162573-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: WEI-JEN TANG | Institution: UNIVERSITY OF CHICAGO, CHICAGO, IL | Award Amount: $470,385 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-J (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11260017