Insights into the dual nature of αB-crystallin chaperone activity from the p.P39L mutant at the N-terminal region.

The substitution of leucine to proline at position 39 (p.P39L) in human αB-crystallin (αB-Cry) has been associated with conflicting interpretations of pathogenicity in cataracts and cardiomyopathy. This study aimed to investigate the effects of the p.P39L mutation on the structural and functional features of human αB-Cry. The mutant protein was expressed in Escherichia coli (E. coli) and purified using anion exchange chromatography. We employed a wide range of spectroscopic analyses, gel electrophoresis, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques to investigate the structure, function, stability, and fibrillation propensity of the mutant protein. The p.P39L mutation caused significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry and increased the thermal stability of the protein. The mutant αB-Cry exhibited an increased chaperone activity and an altered oligomeric size distribution, along with an increased propensity to form amyloid aggregates. It is worth mentioning, increased chaperone activity has important positive and negative effects on damaged cells related to cataracts and cardiomyopathy, particularly by interfering in the process of apoptosis. Despite the apparent positive nature of the increased chaperone activity, it is also linked to adverse consequences. This study provides important insights into the effect of proline substitution by leucine at the N-terminal region on the dual nature of chaperone activity in human αB-Cry, which can act as a double-edged sword.

Systematic analysis and expression of Gossypium ATG8 family reveals the roles of GhATG8f responding to salt stress in cotton.

KEY MESSAGE: Comprehensive analysis of Gossypium ATG8 family indicates that GhATG8f could improve salt tolerance of cotton by increasing SOD, POD and CAT activity and proline accumulation. In plants, autophagy is regulated by several genes that play important roles in initiating and controlling the process. ATG8, functioning as a protein similar to ubiquitin, is involved in crucial tasks throughout the autophagosome formation process. In this research, we conducted an extensive and all-encompassing investigation of 64 ATG8 genes across four varieties of cotton. According to the subcellular localization prediction results, 49 genes were found in the cytoplasm, 6 genes in the chloroplast, 1 gene in the peroxisome, 5 genes in the nucleus, and 3 genes in the extracellular region. Phylogenetic analysis categorized a total of 5 subfamilies containing sixty-four ATG8 genes. The expression of the majority of GhATG8 genes was induced by salt, drought, cold, and heat stresses, as revealed by RNA-seq and real-time PCR. Analysis of cis-elements in the promoters of GhATG8 genes revealed the predominant presence of responsive elements for plant hormones and abiotic stress, suggesting that GhATG8 genes might have significant functions in abiotic stress response. Furthermore, we additionally performed a gene interaction network analysis for the GhATG8 proteins. The salt stress resistance of cotton was reduced due to the downregulation of GhATG8f expression, resulting in decreased activity of CAT, SOD, and POD enzymes, as well as decreased fresh weight and proline accumulation. In summary, our research is the initial exploration of ATG8 gene components in cotton, providing a basis for future investigations into the regulatory mechanisms of ATG8 genes in autophagy and their response to abiotic stress.

Glutamate-dependent arginine biosynthesis requires the inactivation of spoVG, sarA, and ahrC in Staphylococcus aureus.

Genome sequencing has demonstrated that Staphylococcus aureus encodes arginine biosynthetic genes argDCJBFGH synthesizing proteins that mediate arginine biosynthesis using glutamate as a substrate. Paradoxically, however, S. aureus does not grow in a defined, glutamate-replete medium lacking arginine and glucose (CDM-R). Studies from our laboratory have found that specific mutations are selected by S. aureus that facilitate growth in CDM-R. However, these selected mutants synthesize arginine utilizing proline as a substrate rather than glutamate. In this study, we demonstrate that the ectopic expression of the argDCJB operon supports the growth of S. aureus in CDM-R, thus documenting the functionality of this pathway. Furthermore, suppressor mutants of S. aureus JE2 putA::Tn, which is defective in synthesizing arginine from proline, were selected on CDM-R agar. Genome sequencing revealed that these mutants had compensatory mutations within both spoVG, encoding an ortholog of the Bacillus subtilis stage V sporulation protein, and sarA, encoding the staphylococcal accessory regulator. Transcriptional studies document that argD expression is significantly increased when JE2 spoVG sarA was grown in CDM-R. Lastly, we found that a mutation in ahrC was required to induce argD expression in JE2 spoVG sarA when grown in an arginine-replete medium (CDM), suggesting that AhrC also functions to repress argDCJB in an arginine-dependent manner. In conclusion, these data indicate that the argDCJB operon is functional when transcribed in vitro and that SNPs within potential putative regulatory proteins are required to alleviate the repression.IMPORTANCEAlthough Staphylococcus aureus has the capability to synthesize all 20 amino acids, it is phenotypically auxotrophic for several amino acids including arginine. This work identifies putative regulatory proteins, including SpoVG, SarA, and AhrC, that function to inhibit the arginine biosynthetic pathways using glutamate as a substrate. Understanding the ultimate mechanisms of why S. aureus is selected to repress arginine biosynthetic pathways even in the absence of arginine will add to the growing body of work assessing the interactions between metabolism and S. aureus pathogenesis.

Transcriptional responses of Crassostrea hongkongensis under high and low salinity stress.

Salinity, a key limiting factor, affects the distribution and survival of marine species. The Hong Kong oyster (Crassostrea hongkongensis), a euryhaline species found along the coast of the South China Sea, has become a major aquaculture bivalve species. To determine the molecular mechanism by which oysters respond to coastal waters with varying salinity levels, we used RNA-seq to sequence the gill samples of oysters exposed to normal (25 ‰, S25), low (5 ‰, S5) and high (35 ‰, S35) salinity conditions for one month. The results revealed different expression transcriptome levels among oysters living under low and high salinity conditions. Using high-throughput sequencing, we identified 811 up-regulated genes and 769 down-regulated genes. As determined by KEGG pathway mapping, the differentially expressed genes (DEGs) were significantly enriched in the prion diseases, histidine metabolism, arginine and proline metabolism, and beta-alanine metabolism pathways in both the S5 vs. S25 and S35 vs. S25 group comparison. Several DEGs including heat shock 70 kDa protein 12B-like, poly (ADP-ribose) polymerase (PARP), and tripartite motif-containing protein 2 (TRIM2), and low-density lipoprotein receptor-like, as well as KEGG pathways, including arginine and proline metabolism, apoptosis, PPAR signaling pathway, the thyroid hormone signaling pathway, were concerning response to salinity stress. Additionally, eight DEGs involved in salinity adaptation were selected for RT-qPCR validation, and the results confirmed the credibility of the transcriptome sequencing data. Overall, we designed a one-month, medium-term experiment to examine the responses of C. hongkongensis exposed to different levels of salinity stress and performed transcriptome analysis using high-throughput sequencing. Our results enhance current understanding of the molecular mechanisms of salinity stress responses in C. hongkongensis and provided insights into the osmotic biology of oysters.

Virulence evaluation of Israeli Marek's disease virus isolates from commercial poultry using their meq gene sequence.

Fifty-seven Gallid alphaherpesvirus 2 (GaHV-2) isolates, collected during a 30-year period (1990-2019) from commercial poultry flocks affected by Marek's disease (MD), were molecularly characterised. The GaHV-2 meq gene was amplified and sequenced to evaluate the virus virulence, based on the number of PPPPs within the proline-rich repeats (PRRs) of its transactivation domain. The present illustration of virus virulence evaluation on a large scale of field virus isolates by molecular analysis exemplifies the practical benefit and usefulness of the molecular marker in commercial GaVH-2 isolates. The alternative assay of GaVH-2 virulence pathotyping is the classical Gold Standard ADOL method, which is difficult and impossible to employ on a large scale using the Specific Pathogen Free (SPF) chicks of the ADOL strains kept in isolators for two months. The phylogenetic analysis performed in the present study showed that the meq gene amino acid sequences of the 57 Israeli strains divide into 16 phylogenetic branches. The virulence evaluation was performed in comparison with 36 GaHV-2 prototype strains, previously characterised by the in vivo Gold Standard ADOL assay. The results obtained revealed that the GaHV-2 strains circulating in Israel have evolved into a higher virulence potential during the years, as the four-proline stretches number in the meq gene decreased over the investigated period, typically of very virulent virus prototypes. The present study supports the meq gene molecular markers for the assessment of field GaVH-2 strains virulence.

Homozygous substitution of threonine 191 by proline in polymerase η causes Xeroderma pigmentosum variant.

DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη function is associated with the human disease Xeroderma pigmentosum variant (XPV). We hereby report the case of a 60-year-old woman known for XPV and carrying a Polη Thr191Pro variant in homozygosity. We further characterize the variant in vitro and in vivo, providing molecular evidence that the substitution abrogates polymerase activity and results in UV sensitivity through deficient damage bypass. This is the first functional molecular characterization of a missense variant of Polη, whose reported pathogenic variants have thus far been loss of function truncation or frameshift mutations. Our work allows the upgrading of Polη Thr191Pro from 'variant of uncertain significance' to 'likely pathogenic mutant', bearing direct impact on molecular diagnosis and genetic counseling. Furthermore, we have established a robust experimental approach that will allow a precise molecular analysis of further missense mutations possibly linked to XPV. Finally, it provides insight into critical Polη residues that may be targeted to develop small molecule inhibitors for cancer therapeutics.

Pseudomonas mosselii improves cold tolerance of Asian rice (Oryza sativa L.) in a genotype-dependent manner by increasing proline in japonica and reduced glutathione in indica varieties.

Cold stress is an important factor limiting rice production and distribution. Identifying factors that contribute to cold tolerance in rice is of primary importance. While some plant specific genetic factors involved in cold tolerance have been identified, the role of the rice microbiome remains unexplored. In this study, we evaluated the influence of plant growth promoting bacteria (PGPB) with the ability of phosphate solubilization on rice cold tolerance and survival. To reach this goal, inoculated and uninoculated 2-week-old seedlings were cold stressed and evaluated for survival and other phenotypes such as electrolyte leakage (EL) and necessary elements for cold tolerance. The results of this study showed that of the five bacteria, Pseudomonas mosselii, improved both indica and japonica varietal plants' survival and decreased EL, indicating increased membrane integrity. We observed different possible cold tolerance mechanisms in japonica and indica plants such as increases in proline and reduced glutathione levels, respectively. This bacterium also improved the shoot growth of cold exposed indica plants during the recovery period. This study confirmed the host genotype dependent activity of P. mosselii and indicated that there is an interaction between specific plant genes and bacterial genes that causes different plant responses to cold stress.

Integrative analysis of transcriptomic and metabolomic profiles reveals enhanced arginine metabolism in androgen-independent prostate cancer cells.

BACKGROUND: Prostate cancer is a common solid tumor that affects a significant number of men worldwide. Conventional androgen deprivation therapy (ADT) increases the risk of developing castration-resistant prostate cancer (CRPC). Effective clinical management of patients with CRPC is challenging due to the limited understanding. METHODS: In this study, transcriptomic and metabolomic profiles of androgen-dependent prostate cancer cell line LNCaP and the androgen-independent cells developed from LNCaP cells (LNCaP-ADR) were investigated using RNA-sequencing and LC-MS/MS, respectively. The differentially expressed genes and metabolites were analyzed, and integrative analysis of transcriptomic and metabolomic data was further conducted to obtain a comprehensive understanding of the metabolic characteristics in LNCaP-ADR cells. Quantitative real-time PCR (QPCR) was employed to ascertain the mRNA expression levels of the selected differentially expressed genes. RESULTS: The arginine and proline metabolism pathway was identified as a commonly altered pathway at both the transcriptional and metabolic levels. In the LNCaP-ADR cells, significant upregulation was observed for metabolites including 5-Aminopentanoic acid, L-Arginine, L-Glutamic acid, N-Acetyl-L-alanine, and Pyrrole-2-carboxylic acid at the metabolic level. At the transcriptional level, MAOA, ALDH3A2, ALDH2, ARG1, CKMT2, and CNDP1 were found to be significantly upregulated in the LNCaP-ADR cells. Gene set enrichment analysis (GSEA) identified various enriched gene sets in the LNCaP-ADR cells, encompassing inflammatory response, 9plus2 motile cilium, motile cilium, ciliary plasm, cilium or flagellum-dependent cell motility, cilium movement, cilium, response to endoplasmic reticulum stress, PTEN DN.V1 DN, SRC UP.V1 UP, IL15 UP.V1 DN, RB DN.V1 DN, AKT UP MTOR DN.V1 UP, VEGF A UP.V1 UP, and KRAS.LUNG.BREAST UP.V1 UP. CONCLUSIONS: These findings highlight the substantial association between the arginine and proline metabolism pathway and CRPC, emphasizing the need to prioritize strategies that target dysregulated metabolites and differentially expressed genes as essential interventions in the clinical management of CRPC.

[Type I Hyperprolinemia - What about the Kidney?]

Hyperprolinemia is a rare genetic condition due to mutations in proline metabolic pathway. Type I Hyperprolinemia (HPI) typically causes neuropsychiatric disorders, and diagnosis is usually confirmed in pediatric population with suggestive neuropsychiatric involvement by elevated serum proline levels and elevated urinary proline, hydroxyproline, and glycine levels. The possible coexistence of nephropathy in patients with HPI, often specified as malformative urinary disease, is often mentioned. However, reports of HPI diagnosis due to kidney impairment do not exist in scientific literature yet. Here we present the case of a patient presenting with chronic kidney disease secondary to obstructive nephropathy who received a HPI diagnosis in adulthood. Interestingly, the family study showed the same 22q11.21 deletion and elevated blood proline levels in the father, who had no clinical anomalies. We therefore suggest, in light of the high frequency of mutations involving 22q11 and PRODH in the general population, to consider these rare alterations in patients with congenital urinary malformations, even in the presence of nuanced neurological symptoms and negative family history.

Assessment of intra- and inter-genetic diversity in tetraploid and hexaploid wheat genotypes based on omega, gamma and alpha-gliadin profiles.

Durum and bread wheat are well adapted to the Mediterranean Basin. Twenty-three genotypes of each species were grown to evaluate the intra- and inter-genetic diversity based on omega (ω), gamma (γ) and alpha (α)-gliadin profiles. To achieve this purpose, the endosperm storage proteins (both gliadins and glutenins) were extracted from wheat grains and electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The results of SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) revealed nine polymorphic loci out of 16 loci with durum wheat genotypes and nine polymorphic loci out of 18 loci with bead wheat genotypes. The polymorphisms revealed by the SDS-PAGE were 56% and 50% in durum and bread wheat genotypes, respectively. Using the cluster analysis, the durum wheat genotypes were clustered into five groups, while the bread wheat genotypes were grouped into six clusters using un-weighed pair group mean analyses based on ω, γ, and α-gliadins profiles. The 46 durum and bread wheat genotypes were grouped into seven clusters based on the combined ω, γ, and α-gliadins profiles revealed by the SDS-PAGE. The in silico analysis determined the intra-genetic diversity between bread and durum wheat based on the sequences of ω, γ, and α-gliadins. The alignment of ω-gliadin revealed the highest polymorphism (52.1%) between bread and durum wheat, meanwhile, the alignment of γ and α-gliadins revealed very low polymorphism 6.6% and 15.4%, respectively. According to computational studies, all gliadins contain a lot of glutamine and proline residues. The analysis revealed that the bread wheat possessed ω and γ -gliadins with a lower content of proline and a higher content of glutamine than durum wheat. In contrast, durum wheat possessed α-gliadin with a lower content of proline and a higher content of glutamine than bread wheat. In conclusion, the SDS-PAGE, in silico and computational analyses are effective tools to determine the intra- and inter-genetic diversity in tetraploid and hexaploid wheat genotypes based on ω, γ, and α-gliadins profiles.

Comparative genomics of light harvesting chlorophyll (LHC) gene family and impact of chlorophyll-A contents under drought stress in Helianthus annuus.

Drought is one of the main environmental stressors that can alter the water status of plants; negatively affect growth, assimilation, and photosynthesis; and eventually reduce crop yield. We explored the dependence of drought tolerance traits on chlorophyll-A content. Local sunflower cultivars (FH-01, FH-628, FH-633, FH-572, and FH-653) were grown in pots and subjected to drought by withholding water for 10, 15, or 20 d. One month after germination, the leaves of the treated and non-treated plants were collected and subjected to biochemical analyses. Under different water stress levels, the levels of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and proline increased, whereas those of chlorophyll-A decreased. Regression analysis clearly found that proline (-0.442), POD (-0.528), SOD (-0.532), and CAT (-0.814) have negative beta coefficient values. Phylogenetic analysis revealed that the LHC gene family is divided into six clades. Subcellular locations indicated that most LHC genes were located in the chloroplast; however, only few genes were present in the peroxisomes and endoplasmic reticulum. Our research found that Arabidopsis thaliana LHC genes were highly homologous to the LHC genes of Helianthus annuus. Furthermore, the LHC genes of both species are located in the chloroplasts; therefore, they play a role in photosynthesis and renewable energy production. This study opens a new horizon for discussing the role of chlorophyll-A in the drought-related traits of sunflowers.

C9orf72 proline-arginine dipeptide repeats disrupt the proteasome and perturb proteolytic activities.

The hexanucleotide G4C2 repeat expansion in C9orf72 is the most frequent genetic cause of familial amyotrophic lateral sclerosis (ALS). Aberrant translation of this hexanucleotide sequence leads to production of 5 dipeptide repeats (DPRs). One of these DPRs is proline-arginine (polyPR), which is found in C9orf72-expanded ALS (C9ALS) patient brain tissue and is neurotoxic across multiple model systems. PolyPR was previously reported to bind and impair proteasomes in vitro. Nevertheless, the clinical relevance of the polyPR-proteasome interaction and its functional consequences in vivo are yet to be established. Here, we aim to confirm and functionally characterize polyPR-induced impairment of proteolysis in C9ALS patient tissue and an in vivo model system. Confocal microscopy and immunofluorescence studies on both human and Drosophila melanogaster brain tissues revealed sequestration of proteasomes by polyPR into inclusion-like bodies. Co-immunoprecipitation in D. melanogaster showed that polyPR strongly binds to the proteasome. In vivo, functional evidence for proteasome impairment is further shown by the accumulation of ubiquitinated proteins along with lysosomal accumulation and hyper-acidification, which can be rescued by a small-molecule proteasomal enhancer. Together, we provide the first clinical report of polyPR-proteasome interactions and offer in vivo evidence proposing polyPR-induced proteolytic dysfunction as a pathogenic mechanism in C9ALS.

Molecular characterization and phylogenetic analysis of Marek's disease virus in chickens from Ogun State, Nigeria.

Marek's disease (MD) is caused by oncogenic MD virus serotype 1 (MDV1) and is characterized by lymphoproliferative lesions resulting in high morbidity and mortality in chickens. Despite being ubiquitous on poultry farms, there is a dearth of information on its molecular characteristics in Nigeria. This study aimed at characterizing three virulence genes (Meq, pp38, and vIL-8) of MDV1 from chickens in Ogun state, Nigeria. Blood, feather quill, and tumour samples of chickens from different commercial poultry farms in Ogun State were pooled, spotted on 107 FTA cards, and screened for MDV1 by polymerase chain reaction (PCR). Phylogenetic analysis was carried out to compare Nigerian MDV1 Meq, pp38, and vIL-8 genes sequences with the published references. Thirteen samples were MDV1-positive and the Meq, as well as pp38, and vIL-8 genes from the different samples were 100% identical. The Meq genes contained 339 amino acids (aa) with three PPPP motifs in the transactivation domain and two interruptions of the PPPP motifs due to proline-to-arginine substitutions at positions 176 and 217 resulting in a 20.88% proline composition. Phylogenetic analysis revealed that the Meq gene clustered with strains from Egypt and very virulent ATE2539 strain from Hungary. Mutations were observed in the pp38 protein (at positions 107 and 109) and vIL-8 protein (at positions 4 and 31). Based on the molecular analysis of the three genes, the results indicate the presence of MDV1 with virulence signatures; therefore, further studies on in vivo pathotyping of Nigerian MDV1 from all states should be performed.RESEARCH HIGHLIGHTS Meq, pp38 and vIL-8 genes were 100% identical between Nigerian MDV strains.Proline content in Nigerian meq gene was 20.88% with two PPPP motifs interruptions.Meq, pp38 and vIL-8 genes of Nigerian MDV were similar to Egyptian and Indian strains.

Point mutations including a novel Pro-197-Phe mutation confer cross-resistance to acetolactate synthase (ALS) inhibiting herbicides in Lactuca serriola in Australia.

BACKGROUND: Control of prickly lettuce has become increasingly difficult for lentil growers in southern Australia because of widespread resistance to common herbicides, a lack of alternative herbicide options and the prolific production of highly mobile seed. This study aimed to quantify acetolactate synthase (ALS)-inhibiting herbicide resistance in the Mid North (MN) and Yorke Peninsula (YP) of South Australia, characterize the resistance mutations present and investigate population structure and gene flow in this species. RESULTS: Resistance was identified in all populations tested, with average survival of 92% to chlorsulfuron and 95% to imazamox + imazapyr. Five different amino acid substitutions were identified at proline 197 of the ALS gene. There was no significant difference in the median lethal dose (LD50 ) between plants with these five different substitutions when treated with metsulfuron-methyl; however, the imidazolinone resistance level was higher in plants with a phenylalanine substitution and lower in plants with a serine. Population structure based on 701 single nucleotide polymorphisms and 271 individuals provided evidence for both independent evolution of the same mutation in different populations, as well as frequent short- to medium-distance dispersal accompanied by occasional long-distance dispersal events. The overall inbreeding coefficient (FIS ) was calculated at 0.5174, indicating an intermediate level of outcrossing despite the cross-pollination experiment showing only low outcrossing. In the structure analyses, most individuals from YP were assigned to a single cluster, whereas most individuals from MN were assigned 50% to each of two clusters, indicating some genetic differences between these two regions, but also evidence for dispersal between them. CONCLUSIONS: Use of imidazolinone herbicides has selected for mutations conferring higher levels of resistance, such as the Pro-197-Phe mutation, and resulted in further spread of resistance in this species. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

ALS Variants of Annexin A11's Proline-Rich Domain Impair Its S100A6-Mediated Fibril Dissolution.

Mutations in the proline-rich domain (PRD) of annexin A11 are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, and generate abundant neuronal A11 inclusions by an unknown mechanism. Here, we demonstrate that recombinant A11-PRD and its ALS-associated variants form liquidlike condensates that transform into ß-sheet-rich amyloid fibrils. Surprisingly, these fibrils dissolved in the presence of S100A6, an A11 binding partner overexpressed in ALS. The ALS variants of A11-PRD showed longer fibrillization half-times and slower dissolution, even though their binding affinities for S100A6 were not significantly affected. These findings indicate a slower fibril-to-monomer exchange for these ALS variants, resulting in a decreased level of S100A6-mediated fibril dissolution. These ALS-A11 variants are thus more likely to remain aggregated despite their slower fibrillization.

Analysis of thermostability for seven Phe to Ala and six Pro to Gly mutants in the Fab constant region of adalimumab.

To identify amino acids that play important roles in the structural stability of Fab, seven phenylalanine residues in the Fab constant region of the therapeutic antibody adalimumab were subjected to alanine mutagenesis. Six Fab mutants, H:F130A, H:F154A, H:F174A, L:F118A, L:F139A and L:F209A, showed decreased thermostability compared with wild-type Fab. In contrast, the Tm for the L:F116A mutant was 1.7°C higher than that of wild-type Fab, indicating that the F116 residue was unfavorable for Fab thermostability. Six proline mutants, H:P131G, H:P155G, H:P175G, L:P119G, L:P120G and L:P141G, were also prepared to investigate the effect of proline residues adjacent to mutated phenylalanine residues. The thermostability of the H:P155G and L:P141G mutants in particular was significantly reduced, with decreases in Tm of 5.0 and 3.0°C, respectively, compared with wild-type Fab. The H:P155 and L:P141 residues have a cis conformation, whereas the other mutated proline residues have a trans conformation. H:P155 and L:P141 had stacking interactions with the H:F154 and L:Y140, respectively, at the interface between the variable and constant regions. It is suggested that the interactions of the aromatic ring with a cis-form proline at the interface between the variable and constant regions is important for stability of Fab.

Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl-tRNA synthetase 1.

Prolyl-tRNA synthetase 1 (PARS1) has attracted much interest in controlling pathologic accumulation of collagen containing high amounts of proline in fibrotic diseases. However, there are concerns about its catalytic inhibition for potential adverse effects on global protein synthesis. We developed a novel compound, DWN12088, whose safety was validated by clinical phase 1 studies, and therapeutic efficacy was shown in idiopathic pulmonary fibrosis model. Structural and kinetic analyses revealed that DWN12088 binds to catalytic site of each protomer of PARS1 dimer in an asymmetric mode with different affinity, resulting in decreased responsiveness at higher doses, thereby expanding safety window. The mutations disrupting PARS1 homodimerization restored the sensitivity to DWN12088, validating negative communication between PARS1 promoters for the DWN12088 binding. Thus, this work suggests that DWN12088, an asymmetric catalytic inhibitor of PARS1 as a novel therapeutic agent against fibrosis with enhanced safety.

Role of the proline-rich disordered domain of DROSHA in intronic microRNA processing.

DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD's enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.

Antisense oligonucleotide therapy for proline-23-histidine autosomal dominant retinitis pigmentosa.

PURPOSE OF REVIEW: To discuss antisense oligonucleotide (ASON) therapy for autosomal dominant retinitis pigmentosa (adRP) caused by the proline-23-histidine (P23H) mutation in the rhodopsin gene. RECENT FINDINGS: Viral and nonviral therapies to treat adRP are currently under investigation. A promising therapeutic option is a nonviral approach using ASONs. This form of genetic therapy has demonstrated a dose-dependent and highly selective reduction of P23H mutant rhodopsin mRNA in animal models, and it is currently being investigated as a human phase 1/2 clinical trial. SUMMARY: There are promising new therapies to treat adRP. ASON has shown encouraging results in animal models and has undergone a phase 1 clinical trial. ASON does not use a viral vector, is delivered with standard intravitreal injection, and its effects are reversible.

Prolidase deficiency: A novel PEPD missense variant in exon 2.

Prolidase deficiency is an autosomal recessive disease that causes impaired collagen degradation. Altered collagen homeostasis results in the intracellular accumulation of imidodipeptides, which contain proline and hydroxyproline. The many clinical manifestations of prolidase deficiency include dysmorphic facial features, skeletal deformities, hepatosplenomegaly, necrotizing skin ulcers, and recurrent infections. Current clinical knowledge of this genetic disease relies upon few case reports due to its extreme rarity. Diagnosis is dependent on the detection of a pathologic gene variant. Additional diagnostic confirmation may be provided by urine amino acid quantification or reduced in vitro prolidase activity. We present a case of prolidase deficiency caused by a novel variant manifested by skeletal malformations and lifelong multisystemic infections. Genetic testing revealed a homozygous missense variant in the PEPD gene at nucleotide position 200, whereby adenine was replaced by guanine (c.200A > G). The corresponding amino acid change replaced glutamine with arginine at codon 67 (p.Gln67Arg). After boiling the urine sample for hydrolysis, quantitative urine amino acids demonstrated a markedly elevated proline level, confirming the diagnosis. We also provide a discussion of the pathophysiology, clinical manifestations, diagnostic testing, and clinical management of this disease.