A Transcriptome Study of Progeroid Neurocutaneous Syndrome Reveals POSTN As a New Element in Proline Metabolic Disorder

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  Figure 1.. Intracellular proline levels of ARCL2B patient primary skin fibroblasts are lower than healthy controls. (A) Overview of proline biosynthesis. Proline is synthesized via glutamate and ornithine routes. The enzymes P5CS and OAT catalyze the conversion of glutamate and ornithine, respectively, to P5C which is subsequently converted to proline by PYCR1 and PYCR2. (B) Intracellular proline levels in the fibroblasts from healthy controls and patients with PYCR1 mutations (n=4). P5C: Δ1-pyrroline-5-carboxylate; GLS: Glutaminase; GAD: Glutamate decarboxylase; GABA: γ- aminobutyric acid; P5CS: P5C synthase; P5CDH: P5C dehydrogenase; OAT: Ornithine aminotransferase; POX: Proline oxidase; PEPD: Proline dipeptidase; ***: p < 0.001

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  Figure 2.. PYCR1 mutations affected its mRNA and protein levels in patients’ primary skin fibroblasts. (A) Immunoblot showing the truncated PYCR1 protein p.P115fsX7 is undetectable in fibroblasts from patients (P2 and P3) with homozygous c.345delC mutation, whereas the mutant proteins p.G248E (P1) and p.A187T (P4) are detected in samples with heterozygous mutations. (B) PYCR1 levels relative to GAPDH in four each of healthy control and patients were determined by SYBR Green based qRT-PCR. Data is expressed as mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (C) Different single amino acid substitutions in P1 and P4 impeded the PYCR1 half-life as determined by cycloheximide (CHX) chase assay. Cells were harvested and lysed at 0, 4, 8, 16, and 24 h after treatment in medium containing 20 µg/ml CHX. (D) Results in (C) were quantified using the IMAGE J software. α-tubulin was used to normalize the PYCR1 protein levels. The relative levels at 0 hr were defined as 1 for each sample.

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  Figure 3.. qRT-PCR validated the expression changes of PYCR1 affected extracellular space protein genes. (A) Relative change of the most up- and downregulated expressed genes as determined by RNA-Seq analysis. (B) qRT-PCR validation of the two candidates in (A). (C) Gene ontology (GO) analysis shows enrichment of genes related to extracellular marix/region components are downregulated in patients’ fibroblasts. (D) The expression levels of ten extracellular space protein genes (fold-change > 2, TPM > 10) in the skin fibroblasts of all four patients and three different healthy donors were quantified by qRT-PCR. Data is expressed as mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01.

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  Figure 4.. IPA analysis indicated differentially expressed gene candidates are associated with cardiovascular, ophthalmic, and dermatological diseases. Genes with fold change > 2 and TPM > 10 were analyzed. (A) Genes implicated in the principal biological function categories related to the catalogues “Diseases and Disorders” and “Physiological System Development and Function”. (B) List of the genes implicated in top five catalogues. The input putatively up- and downregulated genes are indicated by red and green texts, respectively. The molecules located in extracellular space are bold and underlined.

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  Figure 5.. Perturbations of PYCR1 expression modulate the expression of extracellular space genes in skin fibroblast. (A) Western blot (left) and quantification (right) of PYCR1 and POSTN protein levels in primary skin fibroblasts from controls and patients. Data was expressed as means ± SD from four biological replicates. (B) Evaluation of knockdown efficiencies of pLKO.1 lentiviruses expressing five independent PYCR1 shRNAs in primary skin fibroblasts. At 48 h post-transduction, skin fibroblasts were collected for RNA isolation and qRT-PCR. (C) At 72 h post-transduction, proteins were isolated from fibroblasts and analyzed by Western blot. Scramble shRNA was used as negative control. (D) Knockdown of PYCR1 (shPYCR1 #3, 2MOI) in skin fibroblast modulates the expression of extracellular space genes. (E) Evaluation of the overexpression of PYCR1 in human fibroblasts infected with 2, 5, and 30 MOI of recombinant PYCR1 lentiviruses relative to the empty vector control. (F) Western blot analyses revealed that PYCR1 overexpression (30 MOI) induce POSTN expression in skin fibroblast cells. (G) PYCR1 overexpression in patient’s skin fibroblasts (P2) modulates expression of extracellular space…