Our laboratory has a broad interest in understanding the molecular, structural, and cellular mechanisms of how bacterial toxins and effector proteins target and disrupt critical cellular functions. Along these lines of basic research, we are also keen in translating our insights and knowledge toward developing new scientific tools and novel therapeutics.
Our current focus is on a fascinating family of bacterial toxins, botulinum neurotoxins (BoNTs). These toxins pose one of the greatest security challenges as a Category A potential bioterrorism agent. At the same time, they are also one of the most successful bacterial toxins utilized in modern medicine to treat human diseases, with annual sales over one billion dollars. We are seeking to understand the molecular mechanism for the action of BoNTs, and we are also utilizing these toxins as probes to investigate critical membrane trafficking pathways in neurons and epithelial cells, including retrograde transport, synaptic vesicle recycling, and trafficking of amyloid precursor protein (APP).
In addition, we recently expanded to other medically important toxins and pathogens such as Clostridium difficile toxins and urinary tract infections by E.coli (UPEC). We also branched out to investigate how a newly defined family of multi-domain kinases (ROCO kinases) senses internal/external signals and regulates cytoskeleton in critical neuronal processes such as axon guidance, regeneration, and synaptic plasticity, and how their malfunctions contribute to neurological diseases.
(1) Basic research on molecular mechanism of botulinum neurotoxin actions
Humans and animals are usually exposed to BoNTs as a form of food poisoning due to ingesting BoNTs produced by bacteria in food sources. BoNTs are produced in a protein complex with accessory proteins, which protect toxins from degradation in the gastrointestinal environment and may also facilitate the absorption of toxins across the intestine epithelial cell barrier. The toxins then target and enter peripheral nerve terminals via receptor-mediated endocytosis. Once inside the neurons, BoNTs translocate across endosomal membrane and act as proteases cleaving three essential proteins (SNARE proteins) that mediate synaptic vesicle exocytosis. Along this long journey in vivo, BoNTs encounter and interact with two types of highly polarized cells: epithelial cells and neurons. Many questions are currently being investigated to understand how BoNTs cross gut epithelial cells, how these toxins target neurons, how they translocate across endosomal membrane into cytosol, how they maintain their extremely long half-life inside neurons, and how they traffic inside neurons. Specific projects include:
(1) Identifying and characterizing neuronal receptors for BoNTs; (2) Diversity of BoNTs and genetic variations in human receptors; (3) Cellular mechanism of neurodegeneration induced by BoNTs; (4) membrane translocation mechanism of BoNTs; (5) trafficking and half-life of toxins in neurons.
(2) Translational research on developing novel therapeutic toxins
The growing medical use of BoNTs is one of the most exciting successes in utilizing toxins for our own benefit, but shortcomings still exist with current generation of therapeutic toxins. Here we aim to translate our knowledge gained in BoNT research toward developing novel and improved therapeutic toxins. The ongoing projects include: (1) Engineering BoNTs to improve their therapeutic efficacy and safety; (2) Treating chronic pain using engineered BoNTs; (3) Targeting therapeutics toward motor neurons using modified BoNTs; (4) mechanism and application of BoNTs for treating urinary disorders.
(3) Membrane trafficking pathways in neurons
Neurons are highly specialized cells with sophisticated membrane trafficking processes. Our overarching goal is to understand how the membrane trafficking system is specialized and regulated in neurons and how defects in membrane trafficking pathways lead to neurodegeneration and other neurological diseases. Studying how pathogens exploit membrane trafficking systems in cells has been proven to be a powerful approach to understand the molecular mechanism of these critical cellular processes. Our current approach utilizes BoNTs and related tetanus neurotoxin (TeNT) as probes to investigate the following specific questions: (1) Retrograde transport in peripheral neurons; (2) Synaptic vesicle recycling/endocytosis; (3) Amyloid precursor protein (APP) trafficking in neurons.
(4) Regulation of cytoskeleton remodeling by ROCO kinases
We recently branched out to investigate how cytoskeleton remodeling is regulated by a newly defined ROCO family of kinases in neurons. ROCO kinases feature a newly defined ROCO domain composed of a small GTPase domain, and often contain multiple protein-protein interaction domains, as well as domains that sense second messengers such as Ca2+ or cyclic guanosine monosphate (cGMP). The architecture of ROCO kinases presents an intriguing possibility that these kinases might be a fused signaling complex. Importantly, mutations in ROCO kinases have been associated with neurodegenerative diseases, yet the function of ROCO kinases and how they are regulated remain largely unknown. Our current study focuses on a major ROCO kinase, known as death associated protein kinase (DAPK). DAPK is known primarily as a tumor-suppressor protein in cancer studies. Its role in neurons remains unknown, despite that it is highly expressed in the brain. We recently found that DAPK converges various external/internal signals to myosin-mediated actin cytoskeleton remodeling that is critical for many neuronal functions. Built on our DAPK knockout mouse model, we are currently pursuing the following directions: (1) Characterizing the role of DAPK in regulating axon guidance during development and axon growth inhibition during regeneration; (2) Characterizing the role of DAPK in regulating dendritic spines and synaptic functions in mature neurons.