Date of Award
Spring 2025
Language
English
Embargo Period
6-11-2025
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
College/School/Department
Department of Chemistry
Program
Chemistry
First Advisor
Alexander Shekhtman
Second Advisor
Alan Chen
Committee Members
Alexander Shekhtman, Alan Chen, Igor Lednev, Mehmet Yigit, Alexey Khodjakov
Keywords
Protein structure, biochemistry, Electron Microscopy
Subject Categories
Biochemistry
Abstract
Human Diaphanous Homologue 1 (DIAPH1) is a 143 kDa functional protein that plays a critical role in the regulation of actin dynamics within eukaryotic cells. As a formin, DIAPH1 facilitates the assembly of actin filaments from globular actin monomers, a process essential for cellular structure, motility, and other fundamental cellular processes. Beyond its structural role in actin polymerization, DIAPH1 is implicated in several diseases, including diabetes, cancer, and inflammation, as well as complications like hearing loss. Despite its significance in human health, the functional and structural properties of DIAPH1, particularly in human cells, remain less explored compared to related proteins, such as the murine homologue mDia1, which has been extensively studied in yeast and other systems.
This study aims to characterize the structure, activity, and ligand interactions of DIAPH1 in vitro using protein expressed from a human vector in human tissue cells. A key feature of DIAPH1 is its regulation by the Rho GTPase family, specifically RhoA, which binds to the G-domain located at the N-terminus. RhoA activation of DIAPH1 involves disrupting the protein's auto-inhibitory mechanisms, which are maintained by the N-terminal diaphanous inhibitory domain (DID) and the C-terminal diaphanous auto-inhibitory domain (DAD). This disruption frees the formin homology 2 (FH2) domain, enabling DIAPH1 to promote the rapid polymerization of actin monomers into filaments. The functional activity of DIAPH1, particularly its ability to assemble actin, can be quantitatively assessed using an actin polymerization assay, where actin monomers are tagged with the fluorophore pyrene to monitor filament formation.
The structural characterization of DIAPH1 presents unique challenges due to its large size and the presence of unstructured regions. Early attempts to capture the structure of full-length DIAPH1 using Cryo-Electron Microscopy (Cryo-EM) have yielded poor resolution, likely due to the flexibility of unstructured domains such as the formin homology 1 (FH1) domain. These unstructured regions, which constitute about 30% of DIAPH1’s structure, obscure particle edges and hinder the resolution of Cryo-EM maps. To overcome this, a recombinant form of DIAPH1, termed DIAPH1/2, has been developed. This variant retains the most important structured domains, such as DID, DAD, G-domain, and FH2, while trimming down the unstructured regions. Preliminary results using negative stain transmission electron microscopy (NSTEM) indicate that DIAPH1/2 provides a significant improvement in particle count and sample clarity, making it more suitable for high-resolution structural studies.
Additionally, the actin polymerization assays performed on DIAPH1/2 have confirmed that this recombinant protein not only exhibits inherent actin polymerization activity but can also be activated by RhoA, mirroring the functionality of the full-length protein. This provides confidence that DIAPH1/2 maintains the critical properties of DIAPH1 while facilitating structural studies.
Beyond its structural characterization, the study also explores the interactions of DIAPH1 with other cellular ligands. An important interaction of DIAPH1 involves the receptor for advanced glycation end-products (RAGE), specifically its C-terminal domain (ctRAGE). Through binding to ctRAGE, DIAPH1 undergoes activation, resulting in enhanced actin polymerization. This interaction has significant implications in inflammation and diseases such as diabetes. In collaboration with researchers at New York University (NYU), two small molecule inhibitors, RAGE 229 and 406R, has been developed to block the ctRAGE/DIAPH1 complex. Preliminary assays show that RAGE 229 successfully inhibits actin polymerization in this system, indicating its potential as a therapeutic agent in regulating DIAPH1 activity.
Another novel binding partner of DIAPH1, the actin-depolymerizing protein Cofilin 1 (CFL1), has been identified through enzyme-linked immunosorbent assay (ELISA) and actin polymerization assays. CFL1, known for its role in actin filament disassembly, may function as an activator of DIAPH1, further influencing the dynamics of actin polymerization and depolymerization within the cell.
MFN2, a mitochondrial GTPase, a protein crucial for mitochondrial fusion and communication with the endoplasmic reticulum (ER). Structurally, DIAPH1 contains domains like the diaphanous inhibitory domain (DID) and formin homology domains (FH1 and FH2), which facilitate actin dynamics, whereas MFN2 features a GTPase domain and heptad-repeat domains that support mitochondrial fusion through GTP hydrolysis. Recent studies have revealed a direct interaction between DIAPH1 and MFN2, suggesting they work together to regulate cellular processes requiring both mitochondrial dynamics and cytoskeletal organization.
This study provides significant advancements in understanding the structure and function of DIAPH1, particularly in its human-relevant form. The development of DIAPH1/2 as a recombinant protein offers a clearer path forward for high-resolution structural analysis. . Identification of key ligand interactions, such as with ctRAGE, MFN2, and RhoA and CFL1, offers new insights into the regulatory mechanisms controlling DIAPH1 activity. Future experiments, including targeted mutagenesis and more refined Cryo-EM studies, will provide further clarity on DIAPH1's structure and its potential as a therapeutic target in diseases associated with actin dysregulation.
License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Premo, Aaron, "Physiological structure, activity, and interactions of the formin, Human Diaphanous Homologue 1" (2025). Electronic Theses & Dissertations (2024 - present). 113.
https://scholarsarchive.library.albany.edu/etd/113
