RESEARCH

Molecular determinants of amyloid peptide clearance and metabolism

 

 

 

Collaborative materials development and drug discovery

Shake it and break it: strategies for amyloid disruption and proteolysis

We are working to better understand and control amyloid peptide accumulation and plaque formation.  This work has implications for amyloid neuropathies and Alzheimer's disease.

Our research is concerned with the development of new materials for targeted manipulation of amyloid fibrils  (a joint project with researchers from the labs of Jillian Smith-Carpenter, Fairfield University and Olin Thompson Mefford III, Clemson university), and with understanding and controlling the function of M16 family metallopeptidases, which metabolize amyloidogenic peptides. Amyloid peptides can accumulate in the brain to form the characteristic plaques which are a hallmark of Alzheimer's disease and other neuropathies.

A graphical summary of our research objectives is presented below:

As part of this work, we are working to understand the molecular basis of M16 peptidase dysfunction in amyloid disease.


We recently demonstrated that a neuropathic mutation in the human pitrilysin (Pitrm1) metallopeptidase results in loss of enzyme function in vitro, Using recombinant protein expression and molecular cloning techniques, we demonstrated that a single amino acid substitution in a human gene product previously associated with neuropathic disease (that is, Pitrm1 Arginine183->Glutamine ) dramatically decreases the activity of the mutant enzyme. Moreover, the disease associated substitution is contained within a conserved M16C metallopeptidase motif that is broadly critical for enzymatic function.

This research was reported in a recently published article (
Functional requirement for human pitrilysin metallopeptidase 1 arginine 183, mutated in amyloidogenic neuropathy. Smith-Carpenter JE, Alper BJ. Protein Sci. 2018 Apr;27(4):861-873. doi: 10.1002/pro.3380. Epub 2018 Feb 23). Excerpts are presented below:

 

We are also working to characterize the molecular determinants of substrate specificity of M16 peptidases


This work will help us to understand how enzymes which degrade amyloid beta peptides and other amyloidogenic peptides recognize, interact with, and degrade their substrates. In a recently reported but as yet unpublished study, we have examined determinants of substrate specificity in human insulin-degrading enzyme. This work lead to the identification of insulin-degrading enzyme mutants which alternatively exhibit enhanced specificity for proteolysis of insulin or amyloid beta peptides.

We hope that this research may have potential therapeutic applications, for example, by identifying engineered amyloid beta peptide specific proteolytic enzymes, guiding the development of insulin variants that are more slowly degraded and metabolized, or informing the development of insulin-degrading enzyme small molecule inhibitors that bind to active site residues which are critical to proteolysis.  This work was led by Lazaros Stefanidis (a former Masters student in the lab), and involved undergraduate student researchers from both Sacred Heart University (Nicholas Fusco) and Fairfield University (Samantha Cooper). The study was conducted in collaboration with material input and mass spectrometric analysis from the lab of Jillian Smith-Carpenter (Fairfield University), and is under review for potential publication in the ACS journal Biochemistry.

With collaborators Jillian Smith-Carpenter (Fairfield University) and Thompson Mefford (Clemson University), we are also working to develop and magnetically active peptide conjugated nanoparticles for the targeted incorporation and mechanical manipulation of elongating amyloid fibrils.  This work is in its early stages, but has shown promising preliminary data.  Our collaborators have succeeded in producing and validating functionalized nanoparticles (MNPs), and an undergraduate student researcher at Sacred Heart University (Nicholas Fusco) recently demonstrated that these MNPs may have the ability to partition amyloid fibrils in solution, in work presented at the 255th National Meeting of the American Chemical Society.

 

Shake it and break it: strategies for amyloid disruption and proteolysis

We are working to better understand and control amyloid peptide accumulation and plaque formation.  This work has implications for amyloid neuropathies and Alzheimer's disease.

Our research is concerned with the development of new materials for targeted manipulation of amyloid fibrils  (a joint project with researchers from the labs of Jillian Smith-Carpenter, Fairfield University and Olin Thompson Mefford III, Clemson university), and with understanding and controlling the function of M16 family metallopeptidases, which degrade amyloidogenic peptides. If not degraded, amyloid peptides can accumulate in the brain to form the characteristic plaques which are a hallmark of Alzheimer's disease and other neuropathies.

A graphical summary of our research objectives is presented below:

As part of this research, we are studying the molecular basis of M16 peptidase dysfunction in amyloid disease.


We recently demonstrated that a neuropathic mutation in the human pitrilysin (Pitrm1) metallopeptidase results in loss of enzyme function in vitro, Using recombinant protein expression and molecular cloning techniques, we demonstrated that a single amino acid substitution in a human gene product previously associated with neuropathic disease (that is, Pitrm1 Arginine183->Glutamine ) dramatically decreases the activity of the mutant enzyme. Moreover, the disease associated substitution is contained within a conserved M16C metallopeptidase motif that is broadly critical for enzymatic function.

This research was reported in a recently published article (
Functional requirement for human pitrilysin metallopeptidase 1 arginine 183, mutated in amyloidogenic neuropathy. Smith-Carpenter JE, Alper BJ. Protein Sci. 2018 Apr;27(4):861-873. doi: 10.1002/pro.3380. Epub 2018 Feb 23). Excerpts are presented below:

 

We are working to characterize the molecular determinants of substrate specificity of M16 peptidases


This research will help us to understand how enzymes which degrade amyloid beta and other amyloidogenic peptides recognize, interact with and degrade their substrates. In a recently reported but as yet unpublished study, we examined determinants of substrate specificity in human insulin-degrading enzyme. This work lead to the identification of insulin-degrading enzyme mutants which alternatively exhibit enhanced specificity for proteolysis of insulin or amyloid beta peptides.

This research may have potential therapeutic applications. For example, identifying engineered amyloid beta peptide specific proteolytic enzymes may inform the development of insulin variants that are more slowly degraded and metabolized or guide the identification of insulin-degrading enzyme small molecule inhibitors that bind to active site residues that are critical to proteolysis.  Our most recent work in this area was led by Lazaros Stefanidis, a former Masters student in the lab, and involved undergraduate student researchers from both Sacred Heart University (Nicholas Fusco) and Fairfield University (Samantha Cooper). Our most recent work related to this project was conducted in collaboration with Jillian Smith-Carpenter (Fairfield University), and was published in the ACS journal Biochemistry (Molecular Determinants of Substrate Specificity in Human Insulin-Degrading Enzyme. Stefanidis L, Fusco ND, Cooper SE, Smith-Carpenter JE, Alper BJ. Biochemistry 2018 Jul 13 4903-4914. doi: 10.1021/acs.biochem.8b00474)

With collaborators Jillian Smith-Carpenter (Fairfield University) and Thompson Mefford (Clemson University), we are also  working to develop and characterize magnetically active peptide conjugated nanoparticles for targeted incorporation and  manipulation of elongating amyloid fibrils. This work is in its early stages, but has yielded promising early results.  Our collaborators have succeeded in producing and validating functionalized nanoparticles (MNPs), while Nicholas Fusco (SHU undergradate) has demonstrated that these MNPs may have the ability to partition amyloid fibrils in solution. Nick presented his work at the 255th National Meeting of the American Chemical Society, and also at the 2018 Sacred Heart University Academic Festival.

 

Rational drug design @ SHU

The Sacred Heart University Drug Discovery Group is a collaborative effort with departmental colleagues and student researchers from the labs of Drs. Joseph Audie (physical biochemistry and in modeling of drug interactions) Todd Sullivan (synthetic organic chemistry and lead optimization) and the Alper lab (experimental biochemistry and target validation).

 

We are working with our colleagues to develop small molecule regulators of enzyme activity with relevance to human disease.  This collaboration recently led to the identification and synthesis of a compound targeting drug detoxification by a eukaryotic parasite, which was reported in Bioorganic and Medicinal Chemistry Letters (Efficient synthesis of α-fluoromethylhistidine di-hydrochloride and demonstration of its efficacy as a glutathione S-transferase inhibitor.Considine KL, Stefanidis L, Grozinger KG, Audie J, Alper BJ. Bioorganic and Medicinal Chemistry Letters. 2017 27(6):1335-1340).  Excerpts from this article are presented below:

The Drug Discovery Group is currently working to identify small molecule inhibitors of human arginase 1, a potential anticancer target, which plays a critical role in amino acid metabolism, and also to identify inhibitors of human insulin-degrading enzyme, which may represent an attractive target for control of glucose metabolism in regulation of metabolic disorders such as diabetes.  Our research was recently presented at the 255th national meeting of the American Chemical Society in New Orleans LA, and also at the 2018 Sacred Heart University academic festival.

©2018

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