Source: OKLAHOMA STATE UNIVERSITY submitted to NRP
STRUCTURE-FUNCTION OF THE MIDLINE-1 PROTEIM ASSOCIATED WITH OPITZ G SYNDROME.
Sponsoring Institution
State Agricultural Experiment Station
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0220988
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Dec 1, 2009
Project End Date
Sep 30, 2010
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
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)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
72370101000100%
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.

Progress 12/01/09 to 09/30/10

Outputs
OUTPUTS: In my lab, we use NMR spectroscopy and other biochemical techniques to study the structure and function of proteins associated with how cells differentiate and sense the amount of nutrients. Specifically, our overall goal is to understand how protein phosphatase 2A (PP2A), a central protein that control metabolism and cell cycle, is regulated. PP2A is present in all eukaryotic cells and the two proteins in vertebrates shown to regulate its location and concentration are MID1 and alpha4. The research focused on the enzymatic function of the three non-homologous N-terminal zinc-binding domains (RING, Bbox1 and Bbox2) of MID1 and the interaction between Bbox1 and a portion of the C-terminal domain of alpha4. We conducted ubiquitination experiments to test whether the three zinc-binding domains possess ubiquitin E3 ligase activities. The mechanism of action of facilitating the modification of proteins with ubiquitin is poorly understood even though it is essential for protein homeostasis in eukaryotic cells. Interestingly, plants contain far more proteins with E3 ligase domains than animals. To gain insight into the mechanism, we acquired NMR data of Bbox1 in the presence of the other proteins associated ubiquitination to identify which amino acid is modified and what effect it has on structure. We also collected mass spectrometry data to identify and/or confirm the residues that would be modified. We also collected NMR data on a 45-amino acid region of alpha4 that was shown to interact with the Bbox1 domain. The project in my lab has attracted an OSU undergraduate student, who has won two university-wide scholarships based on proposals he has written. He worked on the fibronectin domain from MID1 that we believe also binds alpha4. I have presented results from my lab to National Science Foundation. PARTICIPANTS: Xiaofeng Han, Ph.D., Post-doc: performed ubiquitination assays to test whether the zinc-binding domains have E3 ligase activities Haijuan Du, Ph.D., Post-doc; expressed, purified and analyzed the structure of the 45-amino acid peptide from alpha4. Also performed binding studies between Bbox1 and alpha4. Wenjun Liu - graduate student, worked on purification protocol to isolate soluble functional proteins from inclusion bodies Val Sluch, undergraduate student, worked on expressing and purifying FNIII domain to determine whether it binds to alpha4. TARGET AUDIENCES: Currently, the project impacts other scientists who are trying to understand structure and function of TRIM proteins. These proteins are important for proper development of vertebrates and for innate immunity. The lab has been successful in using results to educate undergraduate students about benefits of pursuing a Ph.D. degree. PROJECT MODIFICATIONS: None

Impacts
Ubiquitination assays reveal that the three zinc-binding domains possess E3 ligase activity. We observed evidence that the RING-Bbox1 and RING-Bbox1-Bbox2 in tandem have enhanced activities compared to each domain alone. These are novel findings and a manuscript describing these finds have been submitted to the Journal of Molecular Biology. The results are novel because MID1 comes from a large family of poorly understood TRIM proteins that are essential for the proper development of most, if not all, vertebrates. We studied the structural properties of a 45-amino acid region of alpha4 by NMR and circular dichroism spectroscopy. The data reveal a highly extended structure in aqueous solution. However, the backbone atoms maintained helical properties. We postulate that the C-terminus is intrinsically unstructured, with the capacity to become structured in the presence of its binding partner. Indeed, we see evidence that alpha45 binds Bbox1 and becoming structured. The intrinsic properties may be important in regulating ubiquitination of PP2A and dephosphorylation of MID1 by PP2A. Results from these studies were included in a OCAST application that was funded for three years to support a post-doctoral fellow. Results from these studies were submitted for publication.

Publications

  • Submitted Xiaofeng Han, Haijuan Du, Janet Rogers, Steven D. Hartson, Michael A. Massiah, (2010) Human MID1 Protein Contains Three Consecutive E3 Ligase Domains That Recognize Multiple E2 Conjugating Enzymes.
  • Haijuan Du, Michael A. Massiah,(2010) Structural studies of the C-terminal domain of alpha4 suggest how it may function as a regulator of PP2A and MID1.