Cln5 represents a new type of cysteine-based S-depalmitoylase linked to neurodegeneration.

Genetic CLN5 variants are associated with childhood neurodegeneration and Alzheimer's disease; however, the molecular function of ceroid lipofuscinosis neuronal protein 5 (Cln5) is unknown. We solved the Cln5 crystal structure and identified a region homologous to the catalytic domain of members of the N1pC/P60 superfamily of papain-like enzymes. However, we observed no protease activity for Cln5; and instead, we discovered that Cln5 and structurally related PPPDE1 and PPPDE2 have efficient cysteine palmitoyl thioesterase (S-depalmitoylation) activity using fluorescent substrates. Mutational analysis revealed that the predicted catalytic residues histidine-166 and cysteine-280 are critical for Cln5 thioesterase activity, uncovering a new cysteine-based catalytic mechanism for S-depalmitoylation enzymes. Last, we found that Cln5-deficient neuronal progenitor cells showed reduced thioesterase activity, confirming live cell function of Cln5 in setting S-depalmitoylation levels. Our results provide new insight into the function of Cln5, emphasize the importance of S-depalmitoylation in neuronal homeostasis, and disclose a new, unexpected enzymatic function for the N1pC/P60 superfamily of proteins.

CD9 and folate receptor overexpression are not sufficient for VSV-G-independent lentiviral transduction.

Extracellular vesicles have become a research focus for their potential as therapeutic vehicles that carry cargo substances. Extracellular vesicles may origin from the endosomal compartment and share several characteristics with the envelope of lentiviruses. A previous study reported that constitutive expression of the tetraspanin CD9, an extracellular vesicle marker, not only increases vesicle secretion from cells, but has also a positive effect on lentiviral transduction efficiency. Moreover, it was shown that expression of CD9 on the viral envelope in absence of viral glycoproteins was sufficient for the transduction of mammalian cells. In this study, we investigate the effect of CD9 and folate receptor alpha, a GPI-anchored protein, on biosynthesis and transduction efficiency of vesicles carrying lentiviral vectors. We demonstrate that neither CD9 nor FRα nor the combination of both were able to mediate a significant transduction of therapeutic vesicles carrying lentiviral RNA. Further studies are required to identify endogenous mammalian proteins that can be used for pseudotyping of viral envelopes to improve viral targeting without inducing immune responses.