Analysis of conjunctival fibroblasts from a proband with Schnyder corneal dystrophy

Molecular Vision141277-128

Molecular analysis of CHX10 and MFRP in Chinese subjects with primary angle closure glaucoma and short axial length eyes

Molecular Vision141313-131

TGFBIp-associated corneal dystrophy : exploring structure-based drug development strategies for disease prevention and treatment

Corneal Dystrophies are a group of inherited disorders localized to various layers of the cornea that affect corneal transparency and visual acuity. The deposition of insoluble protein materials in the form of extracellular amyloid fibrils or intracellular cysts is pathognomonic. The gene associated with the stromal and Bowman’s related Corneal Dystrophy is TGFBI. The protein product of the gene is known as Transforming Growth Factor-induced protein (TGFBIp). This protein is a 68 KDa protein with 683 amino acids with 4 Fasciclin-like domains. In pathological conditions, the protein accumulates as insoluble deposits in various forms. The severity, clinicopathologic variations, age of the onset, and location of the deposits depend on the type of amino acid alterations in the protein. Until 2006, 38 different pathogenic mutants were reported in the TGFBI gene associated with various phenotypes of corneal dystrophy. To date, there are more than 65 reported mutations in the gene, and 84% of the mutations are found in the 4th FAS1 domain, making it a mutational hotspot. Hence our research focused mainly on the 4th FAS1 domain and mutations involved in the region. There is no effective treatment to prevent, halt, or reverse the deposition of TGFBIp. The main aim of this project is to understand the aggregation mechanism of mutant TGFBIp in the cornea. We have sought different approaches to understand the disease pathology and mechanism of protein aggregation and deposition. The first was a proteomics-based approach where we aimed to identify the composition of various proteins present in the amyloid corneal deposits of the patients with different mutations reported in TGFBI. The study aimed to understand the clearance mechanism of the mutant protein and compare it with the wild-type (WT) protein. The study also aimed to identify specific proteases and proteolytic mechanism involved in clearing the mutant protein in the cornea. The proteolytic processing of mutant TGFBIp protein compared to the WT protein may provide insight to the disease pathology. The proteomics data was vital to understand the role of specific proteases and the proteolytic clearance activity in WT and mutant protein. We identified enrichment of serine protease HTRA1 in the amyloid deposits of corneal dystrophy patients compared to the normal healthy controls. The corneal amyloid deposits are also enriched with amyloid-associated proteins, non-fibrillar amyloid proteins and TGFBIp itself. The study also showed that the patient corneal amyloid deposit samples showed enrichment of certain tryptic peptide fragments like TGFBIp 515-533, TGFBIp 571-588 and TGFBIp 611-642, which are either not present or present only in low amounts in healthy controls. The study showed the possibility of mutant protein processing by serine protease HTRA1, which may give rise to peptides that potentially act as amyloid fibril seeds and enhance the aggregation process. The second approach was to understand the mechanism of fibril formation using a peptide model. Based on the mass spectrometry analysis, we identified many short peptide residues (TGFBIp 515-533, TGFBIp 571-588 and TGFBIp 611-642) enriched in the amyloid corneal deposits of patients compared to the healthy control cornea. The main aim of the study was to understand the molecular-functional relationship of TGFBIp-derived mutant peptides where the amino acid substitutions decreased the overall net charge and their effects on the propensity of the peptides to form amyloid fibrils. We modified the 23 residue peptide (TGFBIp E611PVAEPDIMATNGVVHVITNVLQ633) with clinically relevant cationic charged amino acid substitution and investigated the amyloid-forming properties of the mutant peptides using various biophysical approaches. The peptide sequences were predicted to have a very high propensity to form amyloid fibres. The study also aimed to investigate the structure of amyloid fibrils derived from the peptides by Solid-state NMR. The study may be used to understand the fibrillation process, intermediate states of fibril formation, and will be useful for drug discovery and treatment of TGFBIassociated Corneal Dystrophy. The study provides an insight into the effects of substitution of individual amino acid on the rate of amyloid formation in the peptides, the structure, biological and biophysical properties of the amyloid fibrils formed by the peptides. We also explored how a position specific and type of amino acid substitution hugely contributes to the amyloidogenic property of the peptides. The third was a drug discovery approach to identify small fragments or compounds that can bind to the TGFBIp and modulate the protein, thereby delaying the aggregation process or changing the proteolytic processing of the mutant protein and preventing the formation of highly amyloidogenic peptide seeds. The lead compound identified will be tested to see if it can alter the thermodynamic stability of the protein and thereby stabilize the protein or delay the formation of toxic oligomers or fibrils. The compounds may possibly play a role in altering the proteolytic processing of the mutant TGFBIp. Thus the screening of 2500 compounds from the Maybridge library using initial Weak affinity chromatography and secondary 15N-HSQC-based assays have identified 3 lead compounds that bind to the mutant protein near a predicted binding pocket. The lead compounds were added to the proteins, and limited proteolysis by trypsin showed that the addition of the compounds stabilised the secondary structure of the protein and prevented the formation of highly amyloidogenic peptide seeds. The compounds, when bound to the mutant proteins, may alter the proteolytic processing for efficient clearance or to prevent the formation of smaller peptides. The lead compounds are to be taken to the next stage of the drug discovery process. A combination of computational, proteomics, structural and biophysical studies along with the clinical data provided an insight into the mechanism of cornea-specific protein aggregation and identification of lead compounds that may play a role in protein processing and proteolysis. Even the smallest progress made in each of the different approaches will provide additional information to the basic understanding of the mechanism of protein aggregation, disease progression, and development of novel therapeutic strategies to treat TGFBI-associated Corneal Dystrophies.​Doctor of Philosophy (SBS

Pharmaceutical modulation of the proteolytic profile of Transforming Growth Factor Beta induced protein (TGFBIp) offers a new avenue for treatment of TGFBI-corneal dystrophy

Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman's membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.Published versio

Matrix-assisted laser desorption ionization mass spectrometry imaging of key proteins in corneal samples from lattice dystrophy patients with TGFBI-H626R and TGFBI-R124C mutations

Scope: The purpose of this study is to identify and visualize the spatial distribution of proteins present in amyloid corneal deposits of TGFBI‐CD patients using Mass Spectrometry Imaging (MSI) and compare it with healthy control cornea. Corneal Dystrophies (CD) constitute a group of genetically inherited protein aggregation disorders that affects different layers of the cornea. With accumulated protein deposition, the cornea becomes opaque with decreased visual acuity. CD affecting the stroma and Bowman's membrane, is associated with mutations in transforming growth factor β‐induced (TGFBI ) gene. Methods: MALDI‐Mass Spectrometry Imaging (MSI) is performed on 2 patient corneas and is compared with 1 healthy control cornea using a 7T‐MALDI‐FTICR. Molecular images obtained are overlaid with congo‐red stained sections to visualize the proteins associated with the corneal amyloid aggregates. Results: MALDI‐MSI provides a relative abundance and two dimensional spatial protein signature of key proteins (TGFBIp, Apolipoprotein A‐I, Apolipoprotein A‐IV, Apolipoprotein E, Kaliocin‐1, Pyruvate Kinase and Ras related protein Rab‐10) in the patient deposits compared to the control. This is the first report of the anatomical localization of key proteins on corneal tissue section from CD patients. This may provide insight in understanding the mechanism of amyloid fibril formation in TGFBI ‐corneal dystrophy

In vivo liquid–liquid phase separation protects amyloidogenic and aggregation-prone peptides during overexpression in Escherichia coli

Funding Information: This research was funded by the Singapore Ministry of Education (MOE) through an Academic Research Fund (AcRF) Tier 3 grant (grant # MOE 2019‐T3‐1‐012) and the Academy of Finland project 315140. Publisher Copyright: © 2022 The Protein Society.Studying pathogenic effects of amyloids requires homogeneous amyloidogenic peptide samples. Recombinant production of these peptides is challenging due to their susceptibility to aggregation and chemical modifications. Thus, chemical synthesis is primarily used to produce amyloidogenic peptides suitable for high-resolution structural studies. Here, we exploited the shielded environment of protein condensates formed via liquid–liquid phase separation (LLPS) as a protective mechanism against premature aggregation. We designed a fusion protein tag undergoing LLPS in Escherichia coli and linked it to highly amyloidogenic peptides, including β amyloids. We find that the fusion proteins form membraneless organelles during overexpression and remain fluidic-like. We also developed a facile purification method of functional Aβ peptides free of chromatography steps. The strategy exploiting LLPS can be applied to other amyloidogenic, hydrophobic, and repetitive peptides that are otherwise difficult to produce.Peer reviewe

Effects of Rho-Associated Kinase (Rock) Inhibitors (Alternative to Y-27632) on Primary Human Corneal Endothelial Cells

(1) Rho-associated coiled-coil protein kinase (ROCK) signaling cascade impacts a wide array of cellular events. For cellular therapeutics, scalable expansion of primary human corneal endothelial cells (CECs) is crucial, and the inhibition of ROCK signaling using a well characterized ROCK inhibitor (ROCKi) Y-27632 had been shown to enhance overall endothelial cell yield. (2) In this study, we compared several classes of ROCK inhibitors to both ROCK-I and ROCK-II, using in silico binding simulation. We then evaluated nine ROCK inhibitors for their effects on primary CECs, before narrowing it down to the two most efficacious compounds—AR-13324 (Netarsudil) and its active metabolite, AR-13503—and assessed their impact on cellular proliferation in vitro. Finally, we evaluated the use of AR-13324 on the regenerative capacity of donor cornea with an ex vivo corneal wound closure model. Donor-matched control groups supplemented with Y-27632 were used for comparative analyses. (3) Our in silico simulation revealed that most of the compounds had stronger binding strength than Y-27632. Most of the nine ROCK inhibitors assessed worked within the concentrations of between 100 nM to 30 µM, with comparable adherence to that of Y-27632. Of note, both AR-13324 and AR-13503 showed better cellular adherence when compared to Y-27632. Similarly, the proliferation rates of CECs exposed to AR-13324 were comparable to those of Y-27632. Interestingly, CECs expanded in a medium supplemented with AR-13503 were significantly more proliferative in (i) untreated vs. AR-13503 (1 μM; * p p p p p < 0.05). Lastly, an ex vivo corneal wound healing study showed a comparable wound healing rate for the final healed area in corneas exposed to Y-27632 or AR-13324. (4) In conclusion, we were able to demonstrate that various classes of ROCKi compounds other than Y-27632 were able to exert positive effects on primary CECs, and systematic donor-match controlled comparisons revealed that the FDA-approved ROCK inhibitor, AR-13324, is a potential candidate for cellular therapeutics or as an adjunct drug in regenerative treatment for corneal endothelial diseases in humans

pH Induced Conformational Transitions in the Transforming Growth Factor β-Induced Protein (TGFβIp) Associated Corneal Dystrophy Mutants

Most stromal corneal dystrophies are associated with aggregation and deposition of the mutated transforming growth factor-β induced protein (TGFβIp). The 4th_FAS1 domain of TGFβIp harbors ~80% of the mutations that forms amyloidogenic and non-amyloidogenic aggregates. To understand the mechanism of aggregation and the differences between the amyloidogenic and non-amyloidogenic phenotypes, we expressed the 4th_FAS1 domains of TGFβIp carrying the mutations R555W (non-amyloidogenic) and H572R (amyloidogenic) along with the wild-type (WT). R555W was more susceptible to acidic pH compared to H572R and displayed varying chemical stabilities with decreasing pH. Thermal denaturation studies at acidic pH showed that while WT did not undergo any conformational transition, the mutants exhibited a clear pH-dependent irreversible conversion from αβ conformation to β-sheet oligomers. The β-oligomers of both mutants were stable at physiological temperature and pH. Electron microscopy and dynamic light scattering studies showed that β-oligomers of H572R were larger compared to R555W. The β-oligomers of both mutants were cytotoxic to primary human corneal stromal fibroblast (pHCSF) cells. The β-oligomers of both mutants exhibit variations in their morphologies, sizes, thermal and chemical stabilities, aggregation patterns and cytotoxicities

Proteomic Analysis of Amyloid Corneal Aggregates from <i>TGFBI</i>-H626R Lattice Corneal Dystrophy Patient Implicates Serine-Protease HTRA1 in Mutation-Specific Pathogenesis of TGFBIp

<i>TGFBI</i>-associated corneal dystrophies are inherited disorders caused by <i>TGFBI</i> gene variants that promote deposition of mutant protein (TGFBIp) as insoluble aggregates in the cornea. Depending on the type and position of amino acid substitution, the aggregates may be amyloid fibrillar, amorphous globular or both, but the molecular mechanisms that drive these different patterns of aggregation are not fully understood. In the current study, we report the protein composition of amyloid corneal aggregates from lattice corneal dystrophy patients of Asian origin with H626R and R124C mutation and compared it with healthy corneal tissues via LC–MS/MS. We identified several amyloidogenic, nonfibrillar amyloid associated proteins and TGFBIp as the major components of the deposits. Our data indicates that apolipoprotein A-IV, apolipoprotein E, and serine protease HTRA1 were significantly enriched in patient deposits compared to healthy controls. HTRA1 was also found to be 7-fold enriched in the amyloid deposits of patients compared to the controls. Peptides sequences (G⁵¹¹DNRFSM­LVAAIQS­AGLTETLNR⁵³³ and Y⁵⁷¹HIGDE­ILVSGGI­GALVR⁵⁸⁸) derived from the fourth FAS-1 domain of TGFBIp were enriched in the corneal aggregates in a mutation-specific manner. Biophysical studies of these two enriched sequences revealed high propensity to form amyloid fibrils under physiological conditions. Our data suggests a possible proteolytic processing mechanism of mutant TGFBIp by HTRA1 and peptides generated by mutant protein may form the β-amyloid core of corneal aggregates in dystrophic patients

Release of frustration drives corneal amyloid disaggregation by brain chaperone

TGFBI-related corneal dystrophy (CD) is characterized by the accumulation of insoluble protein deposits in the corneal tissues, eventually leading to progressive corneal opacity. Here we show that ATP-independent amyloid-β chaperone L-PGDS can effectively disaggregate corneal amyloids in surgically excised human cornea of TGFBI-CD patients and release trapped amyloid hallmark proteins. Since the mechanism of amyloid disassembly by ATP-independent chaperones is unknown, we reconstructed atomic models of the amyloids self-assembled from TGFBIp-derived peptides and their complex with L-PGDS using cryo-EM and NMR. We show that L-PGDS specifically recognizes structurally frustrated regions in the amyloids and releases those frustrations. The released free energy increases the chaperone's binding affinity to amyloids, resulting in local restructuring and breakage of amyloids to protofibrils. Our mechanistic model provides insights into the alternative source of energy utilized by ATP-independent disaggregases and highlights the possibility of using these chaperones as treatment strategies for different types of amyloid-related diseases.Ministry of Education (MOE)Published versionThe work is supported by the Ministry of Education, Singapore, under its Academic Research Fund Tier 1 (RG28/19) and Academic Research Fund Tier 3 grant (Project ID: MOE2019-T3-1-012). The authors would like to acknowledge the funding support from SNEC-HREF R1429/12/2017 and SERI Lee –Foundation Pilot Grant R1586/85/ 2018 awarded to JSM and VA respectively. YM acknowledges Singapore MOE Tier 1 grant (RG27/21)