Tripeptidyl Peptidase 1 (TPP1) Deficiency in a 36-Year-Old Patient with Cerebellar-Extrapyramidal Syndrome and Dilated Cardiomyopathy

We report on a 36-year-old man with cerebellar-extrapyramidal syndrome and severe heart failure because of dilated cardiomyopathy of unknown origin. Dysarthria and cardiac arrhythmia began at early childhood (4 years of age). Brain MRI (28 years of age) demonstrated severe cerebellar atrophy. At the age 32, he presented with dysarthria, ataxia, dystonia, and tremor of the right hand, bilateral slowed neural conduction in the visual pathways, and decreased mental acuity. At the age of 33 years, the patient underwent cardiac transplantation because of severe dilated cardiomyopathy. In the TPP1 gene, biallelic variants were identified: previously reported p.(Leu13Pro) and novel p.(Tyr508Cys) variant. Additionally, hemizygous novel missense variant in the ABCD1 gene was inherited from the mother p.(Arg17His). Normal very-long-chain fatty acids (VLCFA) levels both in patient and his mother excluded ABCD1 mutation as the pathogenic one. Tripeptidyl peptidase 1 (TPP1) activity was reduced (8,8 U/mg protein/h; reference range: 47.4 ± 10.7). In light microscopy the biopsy specimens obtained from explanted heart showed severe myocyte hypertrophy with perinuclear vacuolization with inclusions. Electron microscopy revealed absence of lipofuscin accumulation, no ultrastructural curvilinear profiles, fingerprint bodies, or granular osmiophilic deposits (GRODs) in lysosomes. As described here, the patient presents clinical symptoms observed in benign forms of ceroid lipofuscinosis type 2 (CLN2) and simultaneously some features of autosomal recessive spinocerebellar ataxia type 7 (SCAR7), which is also caused by mutations in the TPP1 gene.

A survival analysis of ventricular access devices for delivery of cerliponase alfa

OBJECTIVE Late infantile neuronal ceroid lipofuscinosis type 2 (CLN2) is a rare autosomal recessive disease caused by tripeptidyl peptidase 1 enzyme deficiency. At the authors’ center, the medication cerliponase alfa is administered every 2 weeks via the intracerebroventricular (ICV) route. This requires the placement of a ventricular access device (VAD) or reservoir and frequent percutaneous punctures of this device over the child’s lifetime. In this study, the authors audited the longevity and survival of these VADs and examined the causes of device failure. METHODS A single-center survival analysis of VAD insertions and revisions (January 2014 through June 2020) was conducted. All children received cerliponase alfa infusions through a VAD. Patient characteristics and complications were determined from a prospectively maintained surgical database and patient records. For the VAD survival analysis, the defined endpoint was when the device was removed or changed. Reservoir survival was assessed using Kaplan-Meier curves and the log-rank (Cox-Mantel) test. RESULTS A total of 17 patients had VADs inserted for drug delivery; median (range) age at first surgery was 4 years 4 months (1 year 8 months to 15 years). Twenty-six VAD operations (17 primary insertions and 9 revisions) were required among these 17 patients. Twelve VAD operations had an associated complication, including CSF infection (n = 6) with Propionibacterium and Staphylococcus species being the most prevalent organisms, significant surgical site swelling preventing infusion (n = 3), leakage/wound breakdown (n = 2), and catheter obstruction (n = 1). There were no complications or deaths associated with VAD insertion. The median (interquartile range) number of punctures was 59.5 (7.5–82.0) for unrevised VADs (n = 17) versus 2 (6–87.5) for revised VADs (n = 9) (p = 0.70). The median survival was 301 days for revisional reservoirs (n = 9) versus 2317 days for primary inserted reservoirs (n = 17) (p = 0.019). CONCLUSIONS In the context of the current interest in intrathecal drug delivery for rare metabolic disorders, the need for VADs is likely to increase. Auditing the medium- to long-term outcomes associated with these devices will hopefully result in their wider application and may have potential implications on the development of new VAD technologies. These results could also be used to counsel parents prior to commencement of therapy and VAD implantation.

Shotgun Proteomics of Isolated Urinary Extracellular Vesicles for Investigating Respiratory Impedance in Healthy Preschoolers

Urine proteomic applications in children suggested their potential in discriminating between healthy subjects from those with respiratory diseases. The aim of the current study was to combine protein fractionation, by urinary extracellular vesicle isolation, and proteomics analysis in order to establish whether different patterns of respiratory impedance in healthy preschoolers can be characterized from a protein fingerprint. Twenty-one 3–5-yr-old healthy children, representative of 66 recruited subjects, were selected: 12 late preterm (LP) and 9 full-term (T) born. Children underwent measurement of respiratory impedance through Forced Oscillation Technique (FOT) and no significant differences between LP and T were found. Unbiased clustering, based on proteomic signatures, stratified three groups of children (A, B, C) with significantly different patterns of respiratory impedance, which was slightly worse in group A than in groups B and C. Six proteins (Tripeptidyl peptidase I (TPP1), Cubilin (CUBN), SerpinA4, SerpinF1, Thy-1 membrane glycoprotein (THY1) and Angiopoietin-related protein 2 (ANGPTL2)) were identified in order to type the membership of subjects to the three groups. The differential levels of the six proteins in groups A, B and C suggest that proteomic-based profiles of urinary fractionated exosomes could represent a link between respiratory impedance and underlying biological profiles in healthy preschool children.

Proteomics of serum extracellular vesicles identifies a novel COPD biomarker, fibulin-3 from elastic fibres

There is an unmet need for novel biomarkers in the diagnosis of multifactorial COPD. We applied next-generation proteomics to serum extracellular vesicles (EVs) to discover novel COPD biomarkers.EVs from 10 patients with COPD and six healthy controls were analysed by tandem mass tag-based non-targeted proteomics, and those from elastase-treated mouse models of emphysema were also analysed by non-targeted proteomics. For validation, EVs from 23 patients with COPD and 20 healthy controls were validated by targeted proteomics.Using non-targeted proteomics, we identified 406 proteins, 34 of which were significantly upregulated in patients with COPD. Of note, the EV protein signature from patients with COPD reflected inflammation and remodelling. We also identified 63 upregulated candidates from 1956 proteins by analysing EVs isolated from mouse models. Combining human and mouse biomarker candidates, we validated 45 proteins by targeted proteomics, selected reaction monitoring. Notably, levels of fibulin-3, tripeptidyl-peptidase 2, fibulin-1, and soluble scavenger receptor cysteine-rich domain-containing protein were significantly higher in patients with COPD. Moreover, six proteins; fibulin-3, tripeptidyl-peptidase 2, UTP-glucose-1-phosphate uridylyl transferase, CD81, CD177, and oncoprotein-induced transcript 3, were correlated with emphysema. Upregulation of fibulin-3 was confirmed by immunoblotting of EVs and immunohistochemistry in lungs. Strikingly, fibulin-3 knockout mice spontaneously developed emphysema with age, as evidenced by alveolar enlargement and elastin destruction.We discovered potential pathogenic biomarkers for COPD using next-generation proteomics of EVs. This is a novel strategy for biomarker discovery and precision medicine.

Harnessing affinity-based protein profiling to reveal a novel target of nintedanib

We identified tripeptidyl-peptidase 1 (TPP1) as one of the direct targets of nintedanib (NDNB) employing clickable photoaffinity probes, which provides insights into the functional meaning of the well-known IPF therapeutic drug.

Tripeptidyl peptidase I promotes human endometrial epithelial cell adhesive capacity implying a role in receptivity

The endometrium undergoes cyclic remodelling throughout the menstrual cycle in preparation for embryo implantation which occurs in a short window during the mid-secretory phase. It is during this short ‘receptive window’ that the endometrial luminal epithelium acquires adhesive capacity permitting blastocysts firm adhesion to the endometrium to establish pregnancy. Dysregulation in any of these steps can compromise embryo implantation resulting in implantation failure and infertility. Many factors contribute to these processes including TGF-β, LIF, IL-11 and proteases. Tripeptidyl peptidase 1 (TPP1) is a is a lysosomal serine-type protease however the contribution of the TPP1 to receptivity is unknown. We aimed to investigate the role of TPP1 in receptivity in humans.In the current study, TPP1 was expressed in both epithelial and stromal compartments of the endometrium across the menstrual cycle. Expression was confined to the cytoplasm of luminal and glandular epithelial cells and stromal cells. Staining of mid-secretory endometrial tissues of women with normal fertility and primary unexplained infertility showed reduced immunostaining intensity of TPP1 in luminal epithelial cells of infertile tissues compared to fertile tissues. By contrast, TPP1 levels in glandular epithelial and stromal cells were comparable in both groups in the mid-secretory phase. Inhibition of TPP1 using siRNA compromised HTR8/SVneo (trophoblast cell line) spheroid adhesion on siRNA-transfected Ishikawa cells (endometrial epithelial cell line) in vitro. This impairment was associated with decreased sirtuin 1 (SIRT1), BCL2 and p53 mRNA and unaltered, CD44, CDH1, CDH2, ITGB3, VEGF A, OSTEOPONTIN, MDM2, CASP4, MCL1, MMP2, ARF6, SGK1, HOXA-10, LIF, and LIF receptor gene expression between treatment groups. siRNA knockdown of TPP1 in primary human endometrial stromal cells did not affect decidualization nor the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Taken together, our data strongly suggests a role for TPP1 in endometrial receptivity via its effects on epithelial cell adhesion and suggests reduced levels associated with unexplained infertility may contribute to implantation failure.

Targeting prolyl tripeptidyl peptidase from Porphyromonas gingivalis with the bioactive compounds from Rosmarinus officinalis

BackgroundComplications in periodontitis and other systemic infections related to Porphyromonas gingivalis poses a serious impediment in the treatment process. This leads to the search of novel target proteins to develop newer drugs against P. gingivalis. Prolyl tripeptidyl peptidase (ptp-A) seem to be a vital protein in P. gingivalis virulence and can be a good target for the novel natural bioactive compounds.ObjectivesTo explore the inhibitory potential of Rosmarinus officinalis biocompounds against the ptp-A of P. gingivalis.MethodsThree-dimensional structure of ptp-A was retrieved from the Protein Data Bank with further optimization of both the protein and ligands. In silico inhibitory potential of the selected ligands against ptp-A was done by AutoDock 2.0 and was visualized with Biovia discovery studio visualizing tool with the assessment of the molecular properties of the ligands against ptp-A by molinspiration calculations and drug likeliness.ResultsHigh ptp-A inhibitory effect was observed using rosmarinic acid and luteolin with a bonding energy of −9.81 kcal/mol with 10 hydrogen bond interactions and −9.99 kcal/mol with 7 hydrogen bond interactions, respectively. Carnosic acid and p-coumaric acid showed a binding energy of −7.14 kcal/mol and −6.34 kcal/mol, respectively, with 5 hydrogen bond interactions. Molinspiration assessments showed R. officinalis compounds as the best drug candidates with the topological polar surface area scores <140 Å toward the best oral bioavailability.ConclusionThe carnosic acid, rosmarinic acid, p-coumaric acid, and luteolin from R. officinalis seem to possess a promising inhibitory effect against ptp-A of Candida albicans suggesting ptp-A as the best target to combat P. gingivalis with further in vivo validation.

Lysosomal protein thermal stability does not correlate with cellular half-life: global observations and a case study of tripeptidyl-peptidase 1

Late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurodegenerative lysosomal storage disorder caused by mutations in the gene encoding the protease tripeptidyl-peptidase 1 (TPP1). Progression of LINCL can be slowed or halted by enzyme replacement therapy, where recombinant human TPP1 is administered to patients. In this study, we utilized protein engineering techniques to increase the stability of recombinant TPP1 with the rationale that this may lengthen its lysosomal half-life, potentially increasing the potency of the therapeutic protein. Utilizing multiple structure-based methods that have been shown to increase the stability of other proteins, we have generated and evaluated over 70 TPP1 variants. The most effective mutation, R465G, increased the melting temperature of TPP1 from 55.6°C to 64.4°C and increased its enzymatic half-life at 60°C from 5.4 min to 21.9 min. However, the intracellular half-life of R465G and all other variants tested in cultured LINCL patient-derived lymphoblasts was similar to that of WT TPP1. These results provide structure/function insights into TPP1 and indicate that improving in vitro thermal stability alone is insufficient to generate TPP1 variants with improved physiological stability. This conclusion is supported by a proteome-wide analysis that indicates that lysosomal proteins have higher melting temperatures but also higher turnover rates than proteins of other organelles. These results have implications for similar efforts where protein engineering approaches, which are frequently evaluated in vitro, may be considered for improving the physiological properties of proteins, particularly those that function in the lysosomal environment.