The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired.
Keywords: ALF, artificial lysosomal fluid; APC, antigen-presenting cell; B lymphocytes; CMA, chaperone-mediated autophagy; CPZ: chlorpromazine; CTSD, cathepsin D; CoIP, coimmunoprecipitation; DAPI, 4′, 6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HCQ, hydroxychloroquine; HSPA8/HSC70; LAMP2A, lysosomal-associated membrane protein 2A; LC-MS, liquid chromatography-mass spectrometry; LC3-II, MAP1LC3-II; MHCII, major histocompatibility complex class II; NBD, nucleotide binding domain; PBS, phosphate-buffered saline; RP-HPLC, reversed-phase high-performance liquid chromatography; RPL5, ribosomal protein L5; SBD, substrate binding domain; SD, standard deviation; SEM, standard error of the mean; SLE, systemic lupus erythematosus; SNRNP70/U170K: small nuclear ribonucleoprotein 70kDa; SQSTM1/p62, sequestosome 1; TF, transferrin; TFA, trifluoroacetic acid; antigen-presenting cells; autophagy; bodipy: BODIPY FL C5 Lactosylceramide/bovine serum albumin; chaperone-mediated autophagy; class II MHC molecules; heat shock proteins; iv, intravenous; lupus; lysosomal chaperones; lysosomes; paraquat, 1, 1′-dimethyl-4, 4′-bipyridyldinium dichloride; qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction.