(a) Representative fluorescence micrographs of cells expressing α-syn-YFP after 4 hours (first row) or 48 hours (second row), along with the indicated CP or yeast gene. (b) Representative electron micrographs of cells expressing α-syn-YFP after 4 hours, along with the indicated CP. m = mitochondrion, n = nucleus, v = vacuole, and arrows highlight pools of stalled vesicles. White ovals are lipid droplets.

(a) Sequences of the CPs encoded by CP1 and CP2. Residues found by SAR analysis to be required for function are shown in red, and residues with minor contributions are shown in pink. (b) Spotting assays of normalized, serially diluted cells of the screening strain expressing the negative control HPQ, the selected construct CP1, indicated point mutants of CP1, and the intein-disabled T69A/H72A mutant CP1*. Each construct was verified to express and process in yeast. Similar results were obtained for CP2 (Supplementary Fig. 3). (c) Spotting assays of yeast expressing HPQ, CP1, and additional CP1 variants. These variants illustrate incorporation of a chemical handle (lysine substitution at position seven) as well as minimization of the encoded CP. Each construct was verified to express and process in yeast. Asterisks denote intein-disabled T69A/H72A mutants, which were verified to express but not process.

(a) The SICLOPPS (split-intein mediated circular ligation of peptides and proteins) construct encodes a single protein construct that yields a cyclic peptide (CP) after post-translational splicing5. The dnaE intein C-terminal domain is encoded first, followed by the linker to be cyclized, the intein N-terminal domain, and finally a chitin binding domain affinity tag (not shown). Graphic was generated using the crystal structure of the post-splicing form of the Synechocystis Sp. PCC6803 dnaE intein, PBD ID 1ZD7 26. (b) Western blots against the affinity tag quantitate the expression and processing of the split intein construct. Log-phase yeast cultures expressing the control HPQ construct (HPQ_log) showed robust expression and roughly 50% distribution between the unprocessed 26 kDa construct and the processed 20 kDa byproduct. Yeast blotted at stationary phase (HPQ_stat) showed complete processing, while an HPQ variant with intein-disabling T69A/H72A mutations (HPQ*) showed no processed byproduct in log phase. (c) Blots of log-phase cultures of yeast transformed with 20 randomly picked library members demonstrated that roughly 70% of the library encodes novel CP constructs that express and process in yeast.

(a) The α-synuclein (α-syn) selection strain was transformed with the CP library, allowed to recover, and re-plated on galactose media to induce α-syn expression. Glucose and galactose plates are shown to illustrate how a single robust hit clone was isolated from >150,000 transformants on a single plate. (b) Following isolation and amplification in bacteria, hit plasmids were individually transformed into the screening strain. Transformants were allowed to recover, normalized for cell number, serially diluted, and plated on galactose media. Four hits, named CP1-4, are shown along with a negative control CP plasmid (HPQ) and the positive control Mig1, a genetic repressor of the GAL1 promoter. (c) A GAL1-LacZ reporter assay using the soluble β-galactosidase substrate chlorophenol red-beta-D-galactopyranoside (CPRG) demonstrated that CP1 and CP2 do not affect protein expression. Error bars show standard deviation from five independent trials. (d) Spotting assay, similar to (b), demonstrating that CP1 and CP2 do not affect toxicity in a yeast strain expressing multiple copies of Htt-103Q from the GAL1 promoter. (e) Spotting assay, similar to (b), demonstrating that intein-disabled T69A/H72A mutants of CP1-3 (denoted with asterisks) no longer suppress α-syn toxicity. Thus, for these constructs both specific CP sequences and intein processing are required for activity.

(a–f) Representative fluorescence micrographs of isogenic worm strains with GFP expression in DA neurons. Cell bodies are indicated by arrowheads, with processes visible to the right. (a) At the 7 day-old stage, worms exhibit six intact anterior DA neurons. (b,c) Expression of CP1 and the intein-disabled CP1* constructs in anterior DA neurons had no discernable effect on neuron health or development in the absence of α-syn. (d) Most worms expressing α-syn have missing or degenerated anterior DA neurons. (e) CP1 protected the DA neurons from α-syn-induced neurodegeneration, resulting in a greater proportion of worms with no degeneration in all 4 CEP and 2 ADE neurons. (f) Expression of the intein-disabled CP1* construct does not afford the same protection as CP1. (g) Population analysis revealed that 26% and 24% of worms expressing CP1 and CP2 showed no neurodegeneration, respectively, compared with only ~15% of control worms expressing only α-syn or expressing intein-disabled constructs CP1* and CP2* (**indicates p<0.05, student’s t-test). Error bars show standard deviations of three trials with three independently generated worm lines, n=90 for each line, making a total of 270 animals examined for each transgenic strain. (h) Distributions of worms by number of intact, wild-type DA neurons reveals that selected CPs also reduce the severity of the observed neurodegeneration, preferentially rescuing the two more sensitive ADE neurons.