The present disclosure provides a method of detecting the presence or amount of a UBE3A protein in a sample, such as a human sample, using mass spectrometry based techniques. The methods described herein are useful for diagnosing Angelman syndrome, as well as monitoring disease progression and treatment effectiveness.

Parkin protein variants having activating mutations and/or fused to a mitochondrial targeting sequence are provided. The engineered Parkin may be a fusion protein including a mitochondrial targeting sequence (MTS); a transmembrane domain; and a Parkin protein or functional variant or fragment thereof, such as a Parkin having an N-terminal deletion. The MTS may be the MTS of PINK1 or a functional variant thereof. Alternatively or in addition, the engineered Parkin may have one or more activating mutations, such as single amino-acid substitutions. The engineered Parkin may be delivered in a vector, such as an adeno-associated virus (AAV) vector, and may be used to treat a disease or disorder, such as Parkinson’s disease or any of various neurodegenerative diseases.

The present invention concerns compounds that are capable of covalently entrapping adenylating enzymes. The present invention is essentially based on the discovery that analogues of adenylating enzyme (AE) substrates, wherein a methylene group has been incorporated at the carbon atom in the α-position relative to the carboxylate group involved in the adenylation, are capable of undergoing the adenylation reaction, thereby creating an activated methylene group in situ. The resulting ‘armed’ acyladenylate can interact with the enzyme resulting in covalent entrapment. Interestingly, the acyladenylate can alternatively be transferred to the next step in the enzyme cascade, following which the activated methylene group can interact with the next (active site cysteine containing) enzyme in the enzymatic cascade. The AE substrate analogues, based on their capability of ‘entrapping’ the respective enzymes, will have utility as activity based probes in biological research and also as diagnostic and/or therapeutic agents.

The present disclosure provides compositions and methods for treating subjects at risk for or with sensorineural hearing loss by modulating the rate of Atoh1 protein degradation to increase levels of Atoh1 protein.

The invention relates to bifunctional stapled or stitched peptides comprising a targeting domain, a linker moiety, and an effector domain, that can be used to tether, or to bring into close proximity, at least two cellular entities (e.g., proteins). Certain aspects relate to bifunctional stapled or stitched peptides that bind to an effector biomolecule through the effector domain and bind to a target biomolecule through the targeting domain. Polypeptides and/or polypeptide complexes that are tethered by the bifunctional stapled or stitched peptides of the invention, where the effector polypeptide bound to the effector domain of the bifunctional stapled or stitched peptide modifies or alters the target polypeptide bound to the targeting domain of the bifunctional peptide. Uses of the inventive bifunctional stapled or stitched peptides including methods for treatment of disease (e.g., cancer, inflammatory diseases) are also provided.

Methods are provided for modulating the activity of multimeric ubiquitin-protein E3 ligases including, but not limited to, E6AP ligase activities. The methods reduce the level of oligomer formation such as homotrimeric E6AP ligase to reduce the enzyme activity. Alternatively, agents are provided that can promote the association of the ligase monomers, thereby increasing the ligase activity. Accordingly, novel therapeutic strategies are provided that are useful for the treatment of pathologies resulting from mutations in the genes encoding the ligases and which adversely increase or decrease a ubiquitination reaction.

Methods and compositions disclosed herein generally relate to compositions and methods for suppressing hematopoietic stem and progenitor cells (HSPCs) and the treatment of diseases or disorders involving UBE2N, such as cancers, including disorders such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) and chronic inflammatory disorders. Particular aspects relate to treating, e.g. acute myelomonocytic leukemia (AML-M4) and acute monocytic leukemia (AML-M5). Particular aspects of the invention relate to determining an individual in need of treatment who can be treated with a UBE2N inhibitor, such as an individual having AML-M4 and/or AML-M5. The invention further relates to using a UBE2N inhibitor to treat a disease or disorder characterized by malignant hematopoietic cells, as well as other cancers, and chronic inflammatory disorders, and as immune checkpoint regulators.

The invention provides ubiquitin variants that specifically bind to HECT E3 ligases, and methods of using these variants to modulate the activity of HECT E3 ligases.

The invention provides ubiquitin variants that specifically bind to SCF E3 ligases, and use of these variants to modulate the activity of SCF E3 ligases.

Disclosed herein are methods for identifying a ubiquitin ligase agonist, and the methods include (a) contacting a ubiquitin ligase with a candidate agonist and a neo-substrate; and (b) determining whether the candidate agonist is effective to result in binding the ubiquitin ligase to the neo-substrate, wherein binding of the ubiquitin substrate to the neo-substrate identifies the candidate agonist as a ubiquitin ligase agonist.