Project Summary Our research objective is to engineer variants of a human tripartite motif (TRIM) protein, TRIM11, with enhanced ability to mitigate alpha-synuclein (alpha-syn) misfolding and toxicity that underlies synucleinopathies. Synucleinopathies are common, debilitating neurodegenerative disorders that afflict millions of people worldwide and include Parkinson's Disease, Multiple System Atrophy and Lewy Body Dementia, an Alzheimer's Disease-Related Dementia [ADRD]). In all synucleinopathies, the proteostasis network fails to counter the misfolding of the small presynaptic protein alpha-syn. alpha-Syn populates a range of misfolded conformers ranging from soluble toxic oligomers to self-templating amyloid fibrils capable of initiating and propagating disease de novo. alpha-Syn fibrils cluster into large cytoplasmic inclusions termed Lewy Bodies, a pathological hallmark of synucleinopathies. Unfortunately, there are no effective therapeutics for synucleinopathies. Remarkably, we have discovered that human TRIM11 can prevent and reverse the formation of alpha-syn fibrils and reduce their toxicity in vitro and in vivo. However, we suspect that TRIM11 becomes overwhelmed and fails to counter alpha-syn misfolding in synucleinopathies. Indeed, elevating expression of TRIM11 partially mitigates alpha-syn-mediated neurodegeneration in vivo, but neuroprotection is incomplete and significant neurodegeneration still occurs. Thus, methods to enhance TRIM11 disaggregase activity in the degenerating neurons of synucleinopathy patients could reverse deleterious accumulation of alpha-syn and offer a solution for synucleinopathies. We hypothesize that engineering TRIM11 with enhanced disaggregase activity will enable more effective disassembly of toxic oligomeric and amyloid forms of alpha-syn, which could confer therapeutic benefits in synucleinopathies. Thus, we will engineer TRIM11 with enhanced disaggregase activity against neurotoxic alpha-syn conformers. We will then assess the ability of these enhanced TRIM11 variants to antagonize alpha-syn aggregation and toxicity elicited by alpha-syn oligomers and fibrils in mammalian primary neurons. To do so, we will pursue three aims: (1) Engineer enhanced TRIM11 variants to mitigate alpha-syn aggregation and toxicity in yeast; (2) Define optimal TRIM11 variants that disassemble alpha-syn fibrils and oligomers in vitro; and (3) Define enhanced TRIM11 variants that mitigate alpha-syn aggregation and toxicity in primary neurons. The proposed project establishes a pipeline that begins by leveraging the power of yeast genetics to pinpoint optimal TRIM11 disaggregases from mutant libraries and culminates with their validation at the pure protein level and in mammalian neurons. By the end of these studies, there will be a clear “go/no go” decision for moving enhanced TRIM11 variants into mouse synucleinopathy models and ultimately synucleinopathy patients. Moreover, by revealing how TRIM11 disaggregase activity can ...