About this project

Unraveling the three-dimensional (3D) structure of the ovarian tumor deubiquitinase 6B (OTUD6B) is important to understanding its function in a variety of human diseases, including rare forms of intellectual disability. OTUD6B is an enzyme that removes ubiquitin tags from proteins, thereby influencing how rapidly the cell replaces those proteins. Here, we aim to determine the atomic structure of OTUD6B and its variants by x-ray crystallography and use those structures to obtain insights into OTUD6B’s mechanism of action, regulation, and its role in intellectual impairment. Ultimately, such studies may lead to the development of therapeutic strategies for management of the diseases associated with OTUD6B and its variants.

What is the context of this research?

To date, no structure for OTUD6B, or its variants, have been published. The lab of Dr. Andrew Howard at Illinois Tech is in the process of obtaining structural data for the wild-type OTUD6B and we now aim to obtain the structure of the variant implicated in a rare form of an intellectual disability syndrome associated with variants of OTUD6B (1). Patients carrying such variants have displayed clinical symptoms such as intellectual disability, speech delay, seizures, feeding difficulties, and dysmorphic features of the face, hands, or feet (1). OTUD6B is a deubiquitinating (DUB) enzyme that reverses the process by which a ubiquitin molecule binds to a target protein, leading to protein degradation and changes in cell signaling and trafficking. OTUD6B mutations and DUBs are implicated in cancers, neurodegeneration, and inflammation (1); therefore, we anticipate broad applicability to human health for studies of this enzyme.

Modern sequencing methods have uncovered 11 OTUD6B variants implicated in intellectual disability syndromes of varying degrees of severity. With the Tyr216Cys variant arising from a single-site mutant (2), Tyr216Cys is a great candidate for initial studies on the pathogenicity of OTUD6B mutants and their role in the development of intellectual disorders. We propose to obtain crystallographic structures for wild-type OTUD6B and this Tyr216Cys mutant (2) to probe structural variations in the vicinity of the replaced tyrosine. Furthermore, we will co-crystallize Tyr216Cys with ubiquitin and enhance the structural analysis using modeling tools in silico (2-4).

Our research strategy is to obtain 1–3mg of commercially available OTUD6B protein and the Tyr216Cys mutant at >95% purity. By acquiring the protein from vendors, we will be able to conduct structural studies without the excessive cost and labor associated with the processes of generating the proteins in-house through protein expression and purification experiments and the uncertainty of obtaining a high-purity product in a short-time period. Following acquisition of the model protein, trials will begin by using commercially available crystallization screening kits. Each kit comprises 48 conditions of various precipitants and cryo-protectants at multiple pH values; these are used to screen for conditions that produce crystals. These conditions are then optimized by varying the ionic strength, pH, protein concentration, and precipitant concentration to generate a sufficient amount of crystals for X-ray crystallography experiments at a storage-ring beamline and downstream analyses.

Once experimental structure amplitudes are obtained, we will obtain phase angles by molecular replacement to generate three-dimensional atomic models of OTUD6B, Tyr216Cys, and Tyr216Cys co-crystalized with ubiquitin. The 3D structures will be refined through a variety of software tools, such as Phenix and Coot, to improve the agreement between the model and the experimental data. Then, the structure of the wild-type OTUD6B and the Tyr216Cys variant can be correlated to their function in intellectual disability.

We hypothesize that crystallographic structure determinations of OTUD6B, Tyr216Cys, and Tyr216Cys co-crystallized with ubiquitin will yield insight into the structural basis of the enzyme’s catalytic activity, its substrate specificity, and its role in human diseases, including intellectual disability syndrome.

What is the significance of this project?

It is known that OTUD6B regulates protein synthesis, immune response, and tumor progression an intellectual disability in humans. However, the role played by OTUD6B on the latter condition remains to be discovered. Thus, a deeper insight into the three-dimensional structure of OTUD6B and its variants is seen as critical for understanding OTUD6B’s function and its interactions with other molecules, potentially leading to therapeutics and disease management.

What are the goals of the project?

The primary outcome of the X-ray crystallography study is a determination of the three-dimensional structure of OTUD6B and the Tyr216Cys variant to understand the protein's function and its interactions with other molecules; ultimately it could enable the design of inhibitors or drugs that target the protein.

Stretch Goal

If we surpass our target of $1,791.61 and can raise an additional $1,400, we will purchase a protein of another project to determine the structure of Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-3 (GNB3), mutations of which are correlated to a rare human disease called Congenital Stationary Night Blindness.

The team

This work will be performed by J. Ritu Chander (M.A.S. BIOL ’11) as part of her doctoral project in the laboratory of Dr. Andrew Howard at Illinois Tech. Ritu has a bachelor’s degree in genetics and a master’s degree in cellular and molecular biology from Illinois Tech.

Dr. Andrew Howard is the principal investigator on this project. Dr. Howard obtained his Ph.D. in physics from the University of California, San Diego, and is an Associate Professor of Biology and Physics at Illinois Tech.

We greatly appreciate the time you have taken to consider donating to our cause. No donation is too small for us to return value to the cause of uncovering the science behind severe human ailments. We thank you for your generosity in advance.

Project budget

2mg of Tyr216Cys >95% purity (Bon Opus Biosciences): $1,400.00

Shipping – Domestic Dry Ice 10lbs: $150.00

1mg of Ubiquitin >95% purity (Fisher Scientific): $111.36

Shipping/Handling: $6.25

Hampton AlumaSeal™ II Sealing Film and Applicator: $124.00

Total Project Budget: $1,791.61

Budget justification

The items listed in the project budget include the biomolecules that we propose to study, and a crystallization screening kit to be specifically used for screening for optimal crystallization conditions of OTUD6B. Typically, mutant proteins and conjugated molecules crystallize under the same conditions as the parent molecule, but the single screening kit will be sufficient even if we need to undertake an additional screen for the variant.

All other supplies and reagents required to perform these studies are available in the laboratory of Dr. Andrew Howard at Illinois Tech. Using a storage-ring beamline facility at a national laboratory to obtain X-ray diffraction data carries no cost.

Rewards/Incentives

Donations up to $100:

an update on our progress by email every quarter until publication will be provided

Donations of $101 - $500:

an update on our progress by email every quarter until publication will be provided

an acknowledgement of funding in publications

Donations up to $500 - $1,000:

an update on our progress by email every quarter until publication will be provided

acknowledgement of funding in publications

a plastic 3D model of an OTUD6B based on the predicted structure in AlphaFold 2 will be provided

References

Teresa Santiago-Sim, et al (2017) Biallelic Variants in OTUD6B Cause an Intellectual Disability Syndrome Associated with Seizures and Dysmorphic Features. The American Journal of Human Genetics 100(4): 676-688

Sultan Cingöz, et al (2022) Novel biallelic variants affecting the OTU domain of the gene OTUD6B associate with severe intellectual disability syndrome and molecular dynamics simulations. European Journal of Medical Genetics 65(6): 104497

Straniero Letizia, Rimoldi Valeria, Soldà Giulia, Bellini Melissa, Biasucci Giacomo, Asselta Rosanna, Duga Stefanon (2018) First Replication of the Involvement of OTUD6B in Intellectual Disability Syndrome with Seizures and Dysmorphic Features. Frontiers in Genetics 9: 1-7