The presenilin homologue signal peptide peptidase-like 2a (SPPL2a) is an intramembrane protease of lysosomes/late endosomes which cleaves type II transmembrane proteins. We recently identified CD74, the invariant chain of the MHCII complex, as the first in vivo validated substrate of this protease. In endosomal compartments, CD74 undergoes sequential proteolysis leading to the generation of a membrane-bound N-terminal fragment (NTF) that requires cleavage by SPPL2a for its turnover. In SPPL2a(-/-) mice, this fragment accumulates in B-cells and significantly disturbs their maturation and functionality. To date, the substrate requirements of the protease SPPL2a have not been investigated. In the present study, we systematically analysed the molecular determinants of CD74 with regard to the intramembrane cleavage by SPPL2a. Using domain-exchange experiments, we demonstrate that the intracellular domain (ICD) of CD74 can be substituted without affecting cleavability by SPPL2a. Based on IP-MS analysis of the cleavage product, we report identification of the primary SPPL2a cleavage site between Y52 and F53 within the CD74 transmembrane segment. Furthermore, systematic alanine-scanning mutagenesis of the transmembrane and membrane-proximal parts of the CD74 NTF has been performed. We show that none of the analysed determinants within the CD74 NTF including the residues flanking the primary cleavage site are absolutely essential for SPPL2a cleavage. Importantly, we found that alanine substitution of helix-destabilizing glycines within the transmembrane segment and distinct residues within the luminal membrane-proximal segment led to a reduced efficiency of SPPL2a-mediated processing. Therefore we propose that elements within the transmembrane segment and the luminal juxtamembrane domain facilitate intramembrane proteolysis of CD74 by SPPL2a.

In murine and canine animal models, mutations in the Arylsulfatase G gene (ARSG) cause a particular lysosomal storage disorder characterized by neurological phenotypes. Recently, two variants in the same gene were found to be associated with an atypical form of Usher syndrome in humans, leading to visual and auditory impairment without the involvement of the central nervous system. In this study, we identified three novel pathogenic variants in ARSG, which segregated recessively with the disease in two families from Portugal. The probands were affected with retinitis pigmentosa and sensorineural hearing loss, generally with an onset of symptoms in their fourth decade of life. Functional experiments showed that these pathogenic variants abolish the sulfatase activity of the Arylsulfatase G enzyme and impede the appropriate lysosomal localization of the protein product, which appears to be retained in the endoplasmic reticulum. Our data enable to definitely confirm that different biallelic variants in ARSG cause a specific deaf‐blindness syndrome, by abolishing the activity of the enzyme it encodes.

Usher syndrome (USH) is an autosomal recessively inherited disease characterized by sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP) with or without vestibular dysfunction. It is highly heterogeneous both clinically and genetically. Recently, variants in the arylsulfatase G (ARSG) gene have been reported to underlie USH type IV. This distinct type of USH is characterized by late-onset RP with predominantly pericentral and macular changes, and late onset SNHL without vestibular dysfunction. In this study, we describe the USH type IV phenotype in three unrelated subjects. We identified three novel pathogenic variants, two novel likely pathogenic variants, and one previously described pathogenic variant in ARSG. Functional experiments indicated a loss of sulfatase activity of the mutant proteins. Our findings confirm that ARSG variants cause the newly defined USH type IV and support the proposed extension of the phenotypic USH classification.

Skeletal muscle requires functional mitochondria to provide it with its energy needs. The quality of the organelle is dependent on the synthesis of new mitochondria and the degradation of those that are no longer operative, via the mitophagy pathway. Current research is examining mitophagy impairments at the autophagosome level, yet little is known about the degradation of the organelle at the level of the lysosome. Dysfunctional mitochondria produce high amounts of ROS and have a lower membrane potential, targeting them for degradation. Tagged mitochondria are engulfed in autophagosomes, which then fuse with lysosomes containing hydrolytic enzymes. The fusion of the lysosome and autophagosome is mediated by the lysosomal protein Lamp2, and is essential for the final stage of mitophagy. The purpose of this project is to evaluate the consequences of Lamp2 deficiency on mitochondrial and lysosomal proteins, as well as potential compensatory signaling responses within muscle. Gastrocnemius and quadriceps muscles of Lamp2 KO mice, compared to WT mice, were used for western blotting and enzymatic analyses. Lamp2 KO mice exhibited a significant 1.4‐fold increase in the adapter protein p62, responsible for tethering the autophagosome to the dysfunctional cargo. Similarly, the LC3‐II to LC3‐I ratio was increased by 2.4‐fold in mice lacking Lamp2. COX activity, a measurement of mitochondrial content, was also elevated by 1.3‐fold in KO mice compared to WT counterparts. These results suggest that the absence of Lamp2 leads to an accumulation of autophagosomes containing mitochondria that are not properly degraded. Protein levels of Beclin1 and the E3 ubiquitin ligase Parkin, were augmented in KO mice, possibly in an attempt increase signaling towards autophagosome formation, and the targeting of dysfunctional mitochondria. TFEB, the master regulator of lysosomal and autophagy genes, was increased 2.2‐fold in KO mice compared to WT animals. Interestingly, mTOR phosphorylation was also increased 2‐fold in KO mice. This activation of mTOR suggests that the elevated TFEB levels are largely confined to the cytosol, and may be less transcriptionally active. However, a downstream target of TFEB, Cathepsin D was increased in KO mice, but no changes were observed in the lysosomal marker V‐ATPase between genotypes. PGC‐1a, the master regulator of mitochondrial biogenesis was elevated by 1.8‐fold in the KO mice, suggesting increased signaling towards mitochondrial biogenesis. However, levels of Transcription factor A (Tfam) were similar between genotypes, suggesting that the absence of Lamp2 may dysregulate the coordinated expression of nuclear and mtDNA encoded gene products. This was evident from an increase in UQCRC2, a nuclear‐derived protein, with no changes observed in mtDNA encoded COXI. Thus, these data suggest that Lamp2 is required for the clearance of mitochondria. In its absence, mitochondrial degradation is defective, initiating compensatory signaling responses to mitochondrial biogenesis and autophagy induction to promo...