OBJECTIVE: To demonstrate that mutations in the phosphatidylglycerol remodelling enzyme SERAC1 can cause juvenile-onset complicated hereditary spastic paraplegia (cHSP) clusters, thus adding SERAC1 to the increasing number of complex lipid cHSP genes. METHODS: Combined genomic and functional validation studies (whole-exome sequencing, mRNA, cDNA and protein), biomarker investigations (3-methyl-glutaconic acid, filipin staining and phosphatidylglycerols PG34:1/PG36:1), and clinical and imaging phenotyping were performed in six affected subjects from two different branches of a large consanguineous family. RESULTS: 5 of 6 affected subjects shared cHSP as a common disease phenotype. Three subjects presented with juvenile-onset oligosystemic cHSP, still able to walk several miles at age >10-20 years. This benign phenotypic cluster and disease progression is strikingly divergent to the severe infantile phenotype of all SERAC1 cases reported so far. Two family members showed a more multisystemic juvenile-onset cHSP, indicating an intermediate phenotype between the benign oligosystemic cHSP and the classic infantile SERAC1 cluster. The homozygous splice mutation led to loss of the full-length SERAC1 protein and impaired phosphatidylglycerol PG34:1/PG36:1 remodelling. These phosphatidylglycerol changes, however, were milder than in classic infantile-onset SERAC1 cases, which might partially explain the milder SERAC1 phenotype. CONCLUSIONS: Our findings add SERAC1 to the increasing list of complex lipid cHSP genes. At the same time they redefine the phenotypic spectrum of SERAC1 deficiency. It is associated not only with the severe infantile-onset 'Methylglutaconic aciduria, Deafness, Encephalopathy, Leigh-like' syndrome (MEGDEL syndrome), but also with oligosystemic juvenile-onset cHSP as part of the now unfolding SERAC1 deficiency spectrum.
The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order-disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253-Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains. This network can only form if at least half of the carboxylate groups involved are protonated, which explains the acidic pH optimum of the enzyme.
Cathepsin A (CatA) is a serine carboxypeptidase distributed between lysosomes, cell membrane, and extracellular space. Several peptide hormones including bradykinin and angiotensin I have been described as substrates. Therefore, the inhibition of CatA has the potential for beneficial effects in cardiovascular diseases. Pharmacological inhibition of CatA by the natural product ebelactone B increased renal bradykinin levels and prevented the development of salt-induced hypertension. However, so far no small molecule inhibitors of CatA with oral bioavailability have been described to allow further pharmacological profiling. In our work we identified novel beta-amino acid derivatives as inhibitors of CatA after a HTS analysis based on a project adapted fragment approach. The new inhibitors showed beneficial ADME and pharmacokinetic profiles, and their binding modes were established by X-ray crystallography. Further investigations led to the identification of a hitherto unknown pathophysiological role of CatA in cardiac hypertrophy. One of our inhibitors is currently undergoing phase I clinical trials.
