I. Ub signaling, autophagy & proteostasis.
In the cell, proteins can undergo up to over 200 types of post-translational modifications (PTMs), which has tremendously expanded the chemical repertoire of 20 amino acids for proteins. Protein ubiquitylation (ubiquitination), the addition of one or multiple ubiquitin (Ub) molecules to the substrate or Ub itself, is one of the most “ubiquitous” post-translational modifications (PTMs) that modulate the activity, localization, stability or its interaction with other proteins. The diverse mechanisms in executing and regulating ubiquitylation collectively constitute Ub signaling. We are keen on dissecting the key molecular events in Ub signaling, e.g. the molecular recognitions of a substrate by specific E3 Ub ligase(s), and their functional consequences. Our research is proceeding with an emphasis on E3s whose mutations or deregulation are closely associated with devastating human diseases such as cancer, autism or Angelman Syndrome. Ub ligases in query include E6-AP/Ube3a and HACE1.
In the cell, every protein is pre-destined to degradation, mainly through either the ubiquitin-proteasome system (UPS) or lysosome-dependent autophagy. Besides routing substrates for proteolysis, Ub signaling also has an emerging role in regulating the functionality of the cellular proteolytic machineries. Currently we are actively pursuing this with periodical reports of the progress that we have made (Liu et al 2014 Cancer Cell 26:106-120).
In tackling these problems, we start with cell or animal models for cancer and metabolic diseases, followed by validation in clinically relevant human samples. We are also developing a battery of systems biology tools and research resources when needs arise, with ProTA (Yu et al 2014; Tao et al in preparation) but one example.
Specific projects:
1. The crosstalk between Ub signaling and selective autophagy (Liu et al 2014 Cancer Cell 26:106 – 120; Peng & Yang in submission; Yang & Peng in preparation)
In selective autophagy, receptors are central for cargo selection and delivery. However, it remains yet unclear whether and how multiple autophagy receptors might form complex and function concertedly to control autophagy. Optineurin (OPTN), implicated genetically in glaucoma and amyotrophic lateral sclerosis, was a recently identified autophagy receptor. Here we report that tumor-suppressor HACE1, a ubiquitin ligase, ubiquitylates OPTN and promotes its interaction with p62/SQSTM1 to form the autophagy receptor complex, thus accelerating autophagic flux. Interestingly, the Lys48-linked polyubiquitin chains that HACE1 conjugates onto OPTN might predominantly target OPTN for autophagic degradation. By demonstrating that the HACE1-OPTN axis synergistically suppresses growth and tumorigenicity of lung cancer cells (Figure 1), our findings may open an avenue for developing autophagy-targeted therapeutic intervention into cancer.
2. Global profiling of protein degradome to elucidate the molecular basis for cellular response to diverse stimuli, such as drug treatment and microbal infection (Yu et al 2014 Cell Research in press; Tao et al in preparation; Sheng et al ongoing).
Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be achieved. Here we developed and applied a dual-fluorescence-based Protein Turnover Assay (ProTA) to quantitatively profile global changes in human protein degradome upon BTZ-induced proteasomal inhibition. ProTA (Figure 2) and subsequent network analyses delineate potential molecular basis for BTZ action and tumor drug resistance in BTZ chemotherapy. Finally, combined use of BTZ with drugs targeting the ProTA-identified key genes or pathways in BTZ action reduced BTZ resistance in multiple myeloma cells. Remarkably, BTZ stabilizes proteasome subunit PSMC1 and proteasome assembly factor PSMD10, suggesting a previously under-appreciated mechanism for regulating proteasome homeostasis. Therefore, ProTA is a novel tool for profiling human protein degradome to elucidate potential mechanisms of drug action and resistance, which might facilitate therapeutic development targeting proteostasis to treat human disorders.
3. Molecular mechanisms underlying diseases caused by deregulated proteolysis or Ub signaling (Shen et al 2014 Cell Reports 7:1-14; Li et al ongoing).
Iron excess is closely associated with tumorigenesis in multiple types of human cancers, with underlying mechanisms yet unclear. Recently, iron deprivation has emerged as a major strategy for chemotherapy, but it exerts tumor suppression only on select human malignancies. Here, we report that the tumor suppressor protein p53 is downregulated during iron excess. Strikingly, the iron polyporphyrin heme binds to p53 protein, interferes with p53-DNA interactions, and triggers both nuclear export and cytosolic degradation of p53. Moreover, in tumorigenicity assay, iron deprivation only suppressed tumor of wild-type p53 signaling, suggesting that upregulation of wild-type p53 signaling underlies the selective efficacy of iron deprivation. Our findings (Figure 3) thus identify a direct link between iron/heme homeostasis and the regulation of p53 signaling, which not only provides mechanistic insights into iron-excess-associated tumorigenesis but also help predict and improve outcomes in iron-deprivation-based chemotherapy.
II. Molecular recognition & Protein engineering
All biological reactions are made possible through specific recognitions between, often time protein-based, molecules in close spatial proximity. We aim to decipher mechanisms underlying such molecular recognitions, develop tools to visualize them, and apply these tools into studies of fundamental biological problems and pathology of diseases. We hope to bring advents in the areas of disease diagnosis and therapeutic methodology, and new insights into fundamental biological problems as well.
One tool that we have developed in last years was Zip-seq, which takes advantage of the engineerability of zinc finger proteins to specifically recognize and enrich trinucleotide repeats (TNR). Expansion of TNR was known to associate with around 30 types of human diseases including Huntington’s Disease and one kind of muscle dystrophy, which are yet incurable. Due to its repetitive nature, TNR has been refractory to genomic mapping, despite the huge advances in sequencing techniques. Application of Zip-seq has led to mapping of tri-nucleotide repeats at unprecedented single-base resolution in mammalian genomes (Figure 4 from Xu et al 2014). Currently, we are developing Zip-seq into formats more facile to clinic diagnosis and genetic screening.
We are also trying to explore applicability of nanotechniques in biomedical uses (Song et al 2012; Shen et al 2014; Li, Song & Ye et al 2014 in preparation).
In collaboration with scientists world-wide, we (Figure 5) are trying to address critical questions in an efficient and original way.
