Purdue University Graduate School
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posted on 2024-04-24, 02:32 authored by Shanica Mariah BrownShanica Mariah Brown

The yeast zinc metalloprotease, Ste24, is involved in the maturation of the yeast mating pheromone a-factor by performing two distinct cleavages in the same precursor peptide substrate. Firstly, during the CaaX processing, Ste24 cleaves the three terminal residues of a-factor. CaaX processing is a well-studied process that involves the prenylation, proteolysis, and carboxyl-methylation of proteins ending with a cysteine (C), two aliphatic residues (aa), and one of several amino acids (X). The second cleavage step by Ste24 occurs after CaaX processing and involves an upstream cleavage N-terminal to the CaaX site. Another cleavage is performed by the enzyme Axl1 before the precursor peptide is transported from the cell to initiate mating processes. Inhibition of Ste24 typically results in ‘sterile’ cells which is how the term ‘Sterile 24’ was coined. In humans, defects in this metalloprotease or its substrate, Prelamin A, typically result in a range of progeroid disorders. Furthermore, the severity of these diseases has been directly linked to the catalytical activity of the enzyme. Treatments for these diseases are difficult to develop due to the limited knowledge available on the catalysis, substrate recognition, and functions of Ste24 and its homolog.

As such, these studies aim to define the substrate specificity of Ste24 and elucidate the binding site of Ste24. Identifying the substrate requirements of Ste24 has been an increasingly interesting topic due to the implication of Ste24 in a variety of unrelated functions. Previously, it has only been shown that yeast Ste24 is able to cleave the native substrate, the precursor of a-factor, and the substrate of its human homolog, prelamin A. This is an interesting finding because both substrates have dissimilar sequences at each cleavage site; so, it could be hypothesized that Ste24 may be able to recognize a wider range of sequences than expected. Further research has provided evidence that Ste24 is able to cleave both prenylated and non-prenylated substrates. It is also able to act as a translocon unclogger which may support its function in cleaving toxic islet amyloid polypeptides involved in cell failure in diabetes. Surprisingly, it was shown that this ‘unclogger ability’ was directly correlated to the activity level of Ste24, suggesting that the active site is directly involved in cleaving these peptides. With this information, it is clear that Ste24 has a broader substrate recognition ability than previously believed.

To elucidate the substrate specificity of Ste24, short peptide sequences containing varying CaaX sequences were developed and tested for C-terminal activity through a radioactive methyltransferase-coupled diffusion assay. Ste24 was able to recognize several sequences, however, a larger library is necessary to identify the specific requirements necessary for cleavage. Secondly, we tested the necessity of carboxylmethylation for the upstream N-terminal cleavage. The Distefano group designed three 33-mer analogs of a-factor, developed to mimic the C-terminally cleaved peptide. These peptides had either a) a methyl ester terminus representing the native substrate, b) a free carboxyl terminus representing the unmethylated precursor, and c) an amide terminus representing an unnatural end. All three peptides were tested using a FRET-based assay that allowed for the kinetic parameters of each peptide to be evaluated. We demonstrated that carboxylmethylation was not necessary for the upstream N-terminal cleavage; all three peptides presented similar kinetics. Finally, we interrogated the binding site of Ste24 through the use of a radioactive methyltransferase-coupled diffusion assay (C-terminal cleavage), a FRET-based assay (N-terminal cleavage), and photocrosslinking assays (binding). Together, these data presented a clearer image of residues necessary for the cleavage and binding of substrates within Ste24.


Degree Type

  • Doctor of Philosophy


  • Chemistry

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Christine A. Hrycyna

Advisor/Supervisor/Committee co-chair

Shalini Low-Nam

Additional Committee Member 2

Elizabeth Parkinson

Additional Committee Member 3

Angeline Lyon

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