Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Biochemistry & Molecular Biology
Non Technical Summary
As the name implies, dysfunction of the midline1 proteins caused by amino acid mutations in its sequence result in ventral midline abnormalities with the craniofacial region(cleft lip and/or palate, and/or laryngotrachea, and wide-spaced eyes) brain(missing or smaller corpus callosum resulting in mild mental retardation), heart, kidney, genitalia (hypospadias),and anus. Primarily affecting males, patients tend to have a subset of these congenital defects, which are characterized as X-linked Opitz G/BBB syndrome (XLOS). MID1 is also one of three proteins deleted in microphthalmia with linear skin defects (MLS), a X-linked dominant male-lethal syndrome characterized with similar defects but also includes linear patches of missing skin on the face and neck, and anomalies of the heart and skeleton.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
In my lab, we are using NMR spectroscopy to study the structure and function of the Midline-1, a protein that is essential for midline development in human and vertebrates embryogenesis. We intend to solove the structure of each of six domains of midline-1, map their interaction with each other, test how nutations affect their stuctures, establish the function of these domains, and determine their mode of interactions with other proteins. Our structure of B-box1 and B-box2 in tandem reveals that the B-box domains are evolutionarily similar to RING domain, which is located just upstream of these RING dimers, which are shown to be ubiquitin E3 ligases. These results reveals that TRIM proteins, of which Midlin-1 is a member, can have multiple ubiquitin E3 ligase domains either working synergistically or performing independent roles in targeting proteins of degradation. Our work on the purification of proteins will have significant impact because it allows us (and others) to efficiently purify amounts of proteins that are prone to aggregation and precipitation . From the purification experiments, we have applied for two patents. We have been performing ubiquitination assays to test the function of the Midline-1 protein(unpublished data). We anticipate that this research will have significant impact on human health, including cancer. In the past 12 months, I have mentored two post-doctoral fellows, one Ph.D. graduate student, and three undergraduate students, The work was described at the University of Central Arkansas and University of Oklahoma Health Science Center.
Project Methods
To determine the structure of the domains, we will use nuclear magnetic resonance spectroscopy. In order to acquire NMR data,the protein will be 15N and 15N/13C labeled. Proteins will be grown in minimal media supplemented with either 15NH4 or 15NH4/13C-glucose. Two-dimensional (2D) HSQC spectra will allow us to evaluate how well the protein is folded and feasibility to pursue the project. Then multi-dimensional NMR data will be acquired. The data will be processed with interaction studies will be established by HSQC titration experiments. To determine the structures of larger fragments of midline1, we intend to use X-ray crystallography. To establish whether MID1 and its three zinc-finger domains possess ubiquitin E3 ligase activities, proteins will be expressed using the baculoviral systems, whereby sf9 insect cells will be transfected with bacmid and infected with boaculovirus carrying the gene to express midline1 domains. Established with a midline1 has E3 ligase activities because it would be the first such example of a protein with 3 domains that can possess this activities. Results from these studies will be published in peer-reviewed journals, presented at national scientific meetings and at invited seminars at colleges and universities in the US.