A current view of mitochondria damage and the diversity of lipopigment inclusions in neuronal ceroid lipofuscinose type 2 from rectal biopsy.

Neuronal ceroid lipofuscinoses (NCLs) are a growing group of neurodegenerative storage diseases, in which specific features are sought to facilitate the creation of a universal diagnostic algorithm in the future. In our ultrastructural studies, the group of NCLs was represented by the CLN2 disease caused by a defect in the TPP1 gene encoding the enzyme tripeptidyl-peptidase 1. A 3.5-year-old girl was affected by this disease. Due to diagnostic difficulties, the spectrum of clinical, enzymatic, and genetic tests was extended to include analysis of the ultrastructure of cells from a rectal biopsy. The aim of our research was to search for pathognomonic features of CLN2 and to analyse the mitochondrial damage accompanying the disease. In the examined cells of the rectal mucosa, as expected, filamentous deposits of the curvilinear profile (CVP) type were found, which dominated quantitatively. Mixed deposits of the CVP/fingerprint profile (FPP) type were observed less frequently in the examined cells. A form of inclusions of unknown origin, not described so far in CLN2 disease, were wads of osmophilic material (WOMs). They occurred alone or co-formed mixed deposits. In addition, atypically damaged mitochondria were observed in muscularis mucosae. Their deformed cristae had contact with inclusions that looked like CVPs. Considering the confirmed role of the c subunit of the mitochondrial ATP synthase in the formation of filamentous lipopigment deposits in the group of NCLs, we suggest the possible significance of other mitochondrial proteins, such as mitochondrial contact site and cristae organizing system (MICOS), in the formation of these deposits. The presence of WOMs in the context of searching for ultrastructural pathognomonic features in CLN2 disease also requires further research.

A study of the association between single nucleotide polymorphisms of the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene and the risk of ankylosing spondylitis in Egyptians.

BACKGROUND: Ankylosing spondylitis (AS) is often regarded as the prototypical manifestation of spondylo-arthropathies that prevalently involves the axial skeleton with the potential attribution of ERAP2 polymorphisms to AS predisposition. The purpose of this study was to determine the genetic association between ERAP2 gene rs2910686, and rs2248374 single nucleotide polymorphisms (SNPs) and the risk of ankylosing spondylitis in the Egyptian population. METHODS AND RESULTS: A cross-sectional work involved 200 individuals: 100 AS individuals diagnosed based on modified New York criteria in 1984 with 100 healthy controls matched in age and gender. The study included a comprehensive evaluation of historical data, clinical examinations, and evaluation of the activity of the disease using the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). A comprehensive laboratory and radiological evaluation were conducted, accompanied by an assessment and genotyping of the ERAP2 gene variants rs2248374 and rs2910686. This genotyping was performed utilizing a real-time allelic discrimination methodology.Highly statistically substantial variations existed among the AS patients and the healthy control group regarding rs2910686 and rs2248374 alleles. There was a statistically significant difference between rs2910686 and rs2248374 regarding BASDAI, BASFI, mSASSS, ASQoL, V.A.S, E.S.R, and BASMI in the active AS group. CONCLUSIONS: ERAP2 gene SNPs have been identified as valuable diagnostic biomarkers for AS patients in the Egyptian population being a sensitive and non-invasive approach for AS diagnosis especially rs2910686. Highly statistically significant variations existed among the AS patients and the healthy control group regarding rs2910686 alleles and genotypes.Further research is recommended to explore the potential therapeutic implications of these SNPs.

A needle in a haystack? The impact of a targeted epilepsy gene panel in the identification of a treatable but rapidly progressive metabolic epilepsy: CLN2 disease.

BACKGROUND: Neuronal ceroid lipofuscinoses (NCL) are a group of autosomal recessive, inherited, lysosomal, and neurodegenerative diseases that causes progressive dementia, seizures, movement disorders, language delay/regression, progressive visual failure, and early death. Neuronal ceroid lipofuscinosis type 2 (CLN2), caused by biallelic pathogenic variants of the TPP1 gene, is the only NCL with an approved targeted therapy. The laboratory diagnosis of CLN2 is established through highly specific tests, leading to diagnostic delays and eventually hampering the provision of specific treatment for patients with CLN2. Epilepsy is a common and clinically-identifiable feature among NCLs, and seizure onset is the main driver for families to seek medical care. OBJECTIVE: To evaluate the results of the Latin America Epilepsy and Genetics Program, an epilepsy gene panel, as a comprehensive tool for the investigation of CLN2 among other genetic causes of epilepsy. METHODS: A total of 1,284 patients with epilepsy without a specific cause who had at least 1 symptom associated with CLN2 were screened for variants in 160 genes associated with epilepsy or metabolic disorders presenting with epilepsy through an epilepsy gene panel. RESULTS: Variants of the TPP1 gene were identified in 25 individuals (1.9%), 21 of them with 2 variants. The 2 most frequently reported variants were p.Arg208* and p.Asp276Val, and 2 novel variants were detected in the present study: p.Leu308Pro and c.89 + 3G > C Intron 2. CONCLUSION: The results suggest that these genetic panels can be very useful tools to confirm or exclude CLN2 diagnosis and, if confirmed, provide disease-specific treatment for the patients.

ANTECEDENTES: As lipofuscinoses ceroides neuronais (neuronal ceroid lipofuscinoses, NCLs, em inglês) são um grupo de doenças autossômicas recessivas, hereditárias, lisossomais e neurodegenerativas que causam demência progressiva, crises epiléticas, distúrbios de movimento, atraso/regressão da linguagem, deficiência visual progressiva e morte precoce. A lipofuscinose ceroide neuronal tipo 2 (neuronal ceroid lipofuscinosis type 2, CLN2, em inglês), causada por variantes patogênicas bialélicas do gene TPP1, é a única com terapia-alvo aprovada. O diagnóstico laboratorial é realizado por testes específicos, o que leva a atrasos diagnósticos e, consequentemente, prejudica a disponibilização de tratamento. A epilepsia é uma característica comum e clinicamente identificável entre as NCLs, e o início das convulsões é o principal motivo para as famílias buscarem atendimento médico. OBJETIVO: Avaliar os resultados do Programa de Epilepsia e Genética da América Latina, um painel genético, como uma ferramenta abrangente para a investigação de CLN2 entre outras causas genéticas de epilepsia. MéTODOS: Um total de 1.284 pacientes com epilepsia sem uma causa específica e que tinham pelo menos 1 sintoma associado à CLN2 foram rastreados em busca de variantes em 160 genes associados à epilepsia ou a distúrbios metabólicos que apresentam epilepsia, por meio de um painel genético. RESULTADOS: Variantes do gene TPP1 foram identificadas em 25 indivíduos (1,9%), sendo que ; 21 apresentavam duas variantes. As duas variantes mais frequentes foram p.Arg208* e p.Asp276Val, e duas variantes novas foram detectadas neste: p.Leu308Pro e c.89 + 3G > C Intron 2. CONCLUSãO: Os resultados sugerem que os painéis genéticos de epilepsia podem ser uma ferramenta útil para confirmar ou excluir o diagnóstico de CLN2 e, se confirmado, fornecer tratamento específico para os pacientes.

Novel genetic variants of HLA gene associated with Thai Behcet's disease (BD) patients using next generation sequencing technology.

Behçet's disease (BD) manifests as an autoimmune disorder featuring recurrent ulcers and multi-organ involvement, influenced by genetic factors associated with both HLA and non-HLA genes, including TNF-α and ERAP1. The study investigated the susceptible alleles of both Class I and II molecules of the HLA gene in 56 Thai BD patients and 192 healthy controls through next-generation sequencing using a PacBio kit. The study assessed 56 BD patients, primarily females (58.9%), revealing diverse manifestations including ocular (41.1%), vascular (35.7%), skin (55.4%), CNS (5.4%), and GI system (10.7%) involvement. This study found associations between BD and HLA-A*26:01:01 (OR 3.285, 95% CI 1.135-9.504, P-value 0.028), HLA-B*39:01:01 (OR 6.176, 95% CI 1.428-26.712, P-value 0.015), HLA-B*51:01:01 (OR 3.033, 95% CI 1.135-8.103, P-value 0.027), HLA-B*51:01:02 (OR 6.176, 95% CI 1.428-26.712, P-value 0.015), HLA-C*14:02:01 (OR 3.485, 95% CI 1.339-9.065, P-value 0.01), HLA-DRB1*14:54:01 (OR 1.924, 95% CI 1.051-3.522, P-value 0.034), and HLA-DQB1*05:03:01 (OR 3.00, 95% CI 1.323-6.798, P-value 0.008). However, after Bonferroni correction none of these alleles were found to be associated with BD. In haplotype analysis, we found a strong linkage disequilibrium in HLA-B*51:01:01, HLA-C*14:02:01 (P-value 0.0, Pc-value 0.02). Regarding the phenotype, a significant association was found between HLA-DRB1*14:54:01 (OR 11.67, 95% CI 2.86-47.57, P-value 0.001) and BD with ocular involvement, apart from this, no distinct phenotype-HLA association was documented. In summary, our study identifies specific HLA associations in BD. Although limited by a small sample size, we acknowledge the need for further investigation into HLA relationships with CNS, GI, and neurological phenotypes in the Thai population.

The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds.

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

Inhibition of Insulin-Regulated Aminopeptidase by Imidazo [1,5-α]pyridines-Synthesis and Evaluation.

Inhibition of insulin-regulated aminopeptidase (IRAP) has been shown to improve cognitive functions in several animal models. Recently, we performed a screening campaign of approximately 10,000 compounds, identifying novel small-molecule-based compounds acting as inhibitors of the enzymatic activity of IRAP. Here we report on the chemical synthesis, structure-activity relationships (SAR) and initial characterization of physicochemical properties of a series of 48 imidazo [1,5-α]pyridine-based inhibitors, including delineation of their mode of action as non-competitive inhibitors with a small L-leucine-based IRAP substrate. The best compound displays an IC50 value of 1.0 µM. We elucidate the importance of two chiral sites in these molecules and find they have little impact on the compound's metabolic stability or physicochemical properties. The carbonyl group of a central urea moiety was initially believed to mimic substrate binding to a catalytically important Zn2+ ion in the active site, although the plausibility of this binding hypothesis is challenged by observation of excellent selectivity versus the closely related aminopeptidase N (APN). Taken together with the non-competitive inhibition pattern, we also consider an alternative model of allosteric binding.

Identification of immunological patterns characterizing immune-related psoriasis reactions in oncological patients in therapy with anti-PD-1 checkpoint inhibitors.

Introduction: Immunotherapy with biologics targeting programmed cell death protein-1 (PD-1) is highly effective in the treatment of various malignancies. Nevertheless, it is frequently responsible for unexpected cutaneous manifestations, including psoriasis-like dermatitis. The pathogenesis of anti-PD-1-induced psoriasis has yet to be clarified, even though it is plausible that some innate and adaptive immunity processes are in common with canonical psoriasis. The genetic predisposition to psoriasis of patients could also be a contributing factor. Here, we investigated the immunological and genetic profiles of two patients with metastatic melanoma and one patient affected by lung cancer, who developed severe psoriasis after receiving anti-PD-1 nivolumab therapy. Methods: The immune patterns of the three patients were compared with those detectable in classical, chronic plaque-type psoriasis or paradoxical psoriasis induced by anti-TNF-α therapy, mostly sustained by adaptive and innate immunity processes, respectively. Therefore, immunohistochemistry and mRNA analyses of innate and adaptive immunity molecules were conducted on skin biopsy of patients. Genetic analysis of polymorphisms predisposing to psoriasis was carried out by NGS technology. Results: We found that anti-PD-1-induced psoriasis showed immunological features similar to chronic psoriasis, characterized by the presence of cellular players of adaptive immunity, with abundant CD3+, CD8+ T cells and CD11c+ dendritic cells infiltrating skin lesions, and producing IL-23, IL-6, TNF-α, IFN-γ and IL-17. On the contrary, a lower number of innate immunity cells (BDCA2+ plasmacytoid dendritic cells, CD15+ neutrophils, CD117+ mast cells) and reduced IFN-α/ß, lymphotoxin (LT)-α/ß, were observed in anti-PD-1-induced psoriasis lesions, as compared with anti-TNF-α-induced paradoxical psoriasis. Importantly, the disintegrin and metalloprotease domain containing thrombospondin type 1 motif-like 5 (ADAMTSL5) psoriasis autoantigen was significantly upregulated in psoriasis lesions of anti-PD-1-treated patients, at levels comparable with chronic plaque-type psoriasis. Finally, NGS analysis revealed that all patients carried several allelic variants in psoriasis susceptibility genes, such as HLA-C, ERAP1 and other genes of the major psoriasis susceptibility PSORS1 locus. Discussion: Our study showed that adaptive immunity predominates over innate immunity in anti-PD-1-induced psoriasis lesions, consistently with the local ADAMTSL5 overexpression. The presence of numerous SNPs in psoriasis susceptibility genes of the three patients also suggested their strong predisposition to the disease.

Characterization, Semirational Design for pH Robustness, and the Application in Bioactive Peptide Production of a X-Prolyl Dipeptidyl Aminopeptidase from Lactococcus lactis MY-3.

PepXLcMY-3, an X-prolyl dipeptidyl aminopeptidase derived from Lactobacillus lactis MY-3, was screened and recombinantly expressed in Escherichia coli. The enzyme could exhibit about 40% activity within the pH range of 6.0-10. To further improve the pH robustness, site E396 located in the active pocket was discovered through alanine scanning. The mutant E396I displayed both developed activity and kcat/Km. The optimal pH of E396I shifted from 6.0 to 10 compared to WT, with the relative activity within the pH range of 6.0-10 significantly increased. The site K648 was then proposed by semirational design. The activity of mutant E396I/K648D reached 4.03 U/mg. The optimal pH was restored to 6.0, and the pH stability was further improved. E396I/K648D could totally hydrolyze ß-casomorphin 7 within 30 min. The hydrolysate showed 64.5% inhibition on angiotensin I converting enzyme, which was more efficient than those produced by E396I and WT, 23.2 and 44.7%, respectively.

Role of endoplasmic reticulum aminopeptidase-1 gene polymorphism (rs13167972) in occurrence susceptibility of ankylosing spondylitis in a sample of Iraqi male patients.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic and systemic seronegative inflammatory spondyloarthropathy, an autoimmune disease that has been associated with impaired Endoplasmic Reticulum Aminopeptidase (ERAP)-1 activity, which is involved in priming antigenic peptides. The purpose of this study is to investigate the association of 3-UTR of ERAP1 gene polymorphism (rs13167972) with the AS occurrence susceptibility in a sample of Iraqi male patients. METHODS: The AS patients were diagnosed clinically and by magnetic resonance imaging (MRI) and other clinical and laboratory criteria like symptoms, increased C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). The blood grouping and Body Mass Index (BMI) were also investigated to be associated with AS occurrence. The genotyping of the 3-UTR region of the ERAP1 gene (rs13167972) was done by Sanger sequencing. RESULTS: The results revealed that the AS occurred significantly in the age group of 20-35 years (p = 0.013). The BMI shows that the AS patients were overweighted males (p = 0.013) and the most predominant blood group in AS patients was O- (p = 0.002). The ESR and serum level of CRP were significantly raised in AS patient sera (< 0.001). The results of the receiver-operating characteristics curve analysis (ROC) revealed that the CRP (AUC: 0.995, cut-off: 2.48 mg/L, had 95% %sensitivity, 100% specificity, p < 0.001) is more discriminative than BMI (AUC: 0.300, cut-off: 46.91 kg, had 0% sensitivity, 100% specificity, p = 0.001), and ESR (AUC: 0.808, cut-off: 7.50 mm/hr, had 60% sensitivity, 88% specificity, p < 0.001) in distinguishing between AS patients and control group. The genotyping of the 3-UTR region of ERAP1 gene (rs13167972) result shows that the AG and GG genotypes are significantly occurring in AS patients (70%, OR: 2.33, 95%CI: 1.02-5.36, p = 0.04). The G allele is significantly occurring in AS patients (47%, OR: 2.07, 95CI%: 1.15-3.71, p = 0.01). CONCLUSION: The AS occurred in young overweight males with blood group O-. The AG and GG genotypes are risk factors for AS development while the G allele is a risk factor that increases the chances for disease incidence.

Crystal structure and solution scattering of Geobacillus stearothermophilus S9 peptidase reveal structural adaptations for carboxypeptidase activity.

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase.

Innovate therapeutic targets for autoimmune diseases: insights from proteome-wide mendelian randomization and Bayesian colocalization.

BACKGROUND: Despite growing knowledge regarding the pathogenesis of autoimmune diseases (ADs) onset, the current treatment remains unsatisfactory. This study aimed to identify innovative therapeutic targets for ADs through various analytical approaches. RESEARCH DESIGN AND METHODS: Utilizing Mendelian randomization, Bayesian co-localization, phenotype scanning, and protein-protein interaction network, we explored potential therapeutic targets for 14 ADs and externally validated our preliminary findings. RESULTS: This study identified 12 circulating proteins as potential therapeutic targets for six ADs. Specifically, IL12B was judged to be a risk factor for ankylosing spondylitis (p = 1.61E - 07). TYMP (p = 6.28E - 06) was identified as a protective factor for ulcerative colitis. For Crohn's disease, ERAP2 (p = 4.47E - 14), HP (p = 2.08E - 05), and RSPO3 (p = 6.52E - 07), were identified as facilitators, whereas FLRT3 (p = 3.42E - 07) had a protective effect. In rheumatoid arthritis, SWAP70 (p = 3.26E - 10), SIGLEC6 (p = 2.47E - 05), ISG15 (p = 3.69E - 05), and FCRL3 (p = 1.10E - 10) were identified as risk factors. B4GALT1 (p = 6.59E - 05) was associated with a lower risk of Type 1 diabetes (T1D). Interestingly, CTSH was identified as a protective factor for narcolepsy (p = 1.58E - 09) but a risk factor for T1D (p = 7.36E - 11), respectively. External validation supported the associations of eight of these proteins with three ADs. CONCLUSIONS: Our integrated study identified 12 potential therapeutic targets for ADs and provided novel insights into future drug development for ADs.

NPEPPS Is a Druggable Driver of Platinum Resistance.

There is an unmet need to improve the efficacy of platinum-based cancer chemotherapy, which is used in primary and metastatic settings in many cancer types. In bladder cancer, platinum-based chemotherapy leads to better outcomes in a subset of patients when used in the neoadjuvant setting or in combination with immunotherapy for advanced disease. Despite such promising results, extending the benefits of platinum drugs to a greater number of patients is highly desirable. Using the multiomic assessment of cisplatin-responsive and -resistant human bladder cancer cell lines and whole-genome CRISPR screens, we identified puromycin-sensitive aminopeptidase (NPEPPS) as a driver of cisplatin resistance. NPEPPS depletion sensitized resistant bladder cancer cells to cisplatin in vitro and in vivo. Conversely, overexpression of NPEPPS in sensitive cells increased cisplatin resistance. NPEPPS affected treatment response by regulating intracellular cisplatin concentrations. Patient-derived organoids (PDO) generated from bladder cancer samples before and after cisplatin-based treatment, and from patients who did not receive cisplatin, were evaluated for sensitivity to cisplatin, which was concordant with clinical response. In the PDOs, depletion or pharmacologic inhibition of NPEPPS increased cisplatin sensitivity, while NPEPPS overexpression conferred resistance. Our data present NPEPPS as a druggable driver of cisplatin resistance by regulating intracellular cisplatin concentrations. SIGNIFICANCE: Targeting NPEPPS, which induces cisplatin resistance by controlling intracellular drug concentrations, is a potential strategy to improve patient responses to platinum-based therapies and lower treatment-associated toxicities.

Recent Advances of Aminopeptidases-Responsive Small-Molecular Probes for Bioimaging.

Aminopeptidases, enzymes with critical roles in human body, are emerging as vital biomarkers for metabolic processes and diseases. Aberrant aminopeptidase levels are often associated with diseases, particularly cancer. Small-molecule probes, such as fluorescent, fluorescent/photoacoustics, bioluminescent, and chemiluminescent probes, are essential tools in the study of aminopeptidases-related diseases. The fluorescent probes provide real-time insights into protein activities, offering high sensitivity in specific locations, and precise spatiotemporal results. Additionally, photoacoustic probes offer signals that are able to penetrate deeper tissues. Bioluminescent and chemiluminescent probes can enhance in vivo imaging abilities by reducing the background. This comprehensive review is focused on small-molecule probes that respond to four key aminopeptidases: aminopeptidase N, leucine aminopeptidase, Pyroglutamate aminopeptidase 1, and Prolyl Aminopeptidase, and their utilization in imaging tumors and afflicted regions. In this review, the design strategy of small-molecule probes, the variety of designs from previous studies, and the opportunities of future bioimaging applications are discussed, serving as a roadmap for future research, sparking innovations in aminopeptidase-responsive probe development, and enhancing our understanding of these enzymes in disease diagnostics and treatment.

Pharmacological Characterization of SDX-7320/Evexomostat: A Novel Methionine Aminopeptidase Type 2 Inhibitor with Anti-tumor and Anti-metastatic Activity.

Methionine aminopeptidase type 2 (METAP2) is a ubiquitous, evolutionarily conserved metalloprotease fundamental to protein biosynthesis which catalyzes removal of the N-terminal methionine residue from nascent polypeptides. METAP2 is an attractive target for cancer therapeutics based upon its over-expression in multiple human cancers, the importance of METAP2-specific substrates whose biological activity may be altered following METAP2 inhibition, and additionally, that METAP2 was identified as the target for the anti-angiogenic natural product, fumagillin. Irreversible inhibition of METAP2 using fumagillin analogues has established the anti-angiogenic and anti-tumor characteristics of these derivatives; however, their full clinical potential has not been realized due to a combination of poor drug-like properties and dose-limiting central nervous system (CNS) toxicity. This report describes the physicochemical and pharmacological characterization of SDX-7320 (evexomostat), a polymer-drug conjugate of the novel METAP2 inhibitor (METAP2i) SDX-7539. In vitro binding, enzyme, and cell-based assays demonstrated that SDX-7539 is a potent and selective METAP2 inhibitor. In utilizing a high molecular weight, water-soluble polymer to conjugate the novel fumagillol-derived, cathepsin-released, METAP2i SDX-7539, limitations observed with prior generation, small molecule fumagillol derivatives were ameliorated including reduced CNS exposure of the METAP2i, and prolonged half-life enabling convenient administration. Multiple xenograft and syngeneic cancer models were utilized to demonstrate the anti-tumor and anti-metastatic profile of SDX-7320. Unlike polymer-drug conjugates in general, reductions in small molecule-equivalent efficacious doses following polymer conjugation were observed. SDX-7320 has completed a phase I clinical safety study in patients with late-stage cancer and is currently being evaluated in multiple phase Ib/II clinical studies in patients with advanced solid tumors.

Characterization of a secreted aminopeptidase of M28 family from B. fragilis and its possible role in protein metabolism in the gut.

Products of microbial protein metabolism in the gut can influence the health of the host in many ways. Members of the Bacteriodales, major commensals of the human colon have been associated with long-term intake of high-protein diets. Undigested proteins or peptides that reach the colon can be hydrolyzed by extra-cellular proteases found in some Bacteroides species into amino acids and peptides which can be further catabolized. In this communication, we have characterized one such secreted aminopeptidase (BfAP) from Bacteroides fragilis belonging to the M28 family which is capable of degrading peptides released from soybean protein after predigestion in the small intestine. The BfAP enzyme was cloned, expressed in E. coli, and purified to homogeneity. It is a metallopeptidase requiring Co2+ ion for optimum activity at 55 °C and pH 8 and preferentially cleaves neutral aliphatic (Met/Leu) and positively charged (Arg/Lys) amino acids from the N-terminus of peptides. It showed high specificity for long peptides as well as proteins like ß-casein. Structural analysis of BfAP and its orthologues using AlphaFold2 reveal a shared highly conserved M28 domain, but vary with respect to their N-terminal region with some of them possessing an additional cap domain which may be important for regulation of substrate binding. Although BfAP lacks the typical cap domain, it shows small extensions that can form a loop adjacent to the proposed active site and may affect substrate binding. We suggest that this secreted enzyme may play an important role in protein metabolism in the colon where Bacteroides species are abundant.

Tripeptidyl peptidase II coordinates the homeostasis of calcium and lipids in the central nervous system and its depletion causes presenile dementia in female mice through calcium/lipid dyshomeostasis-induced autophagic degradation of CYP19A1.

Rationale: Tripeptidyl peptidase II (TPP2) has been proven to be related to human immune and neurological diseases. It is generally considered as a cytosolic protein which forms the largest known protease complex in eukaryotic cells to operate mostly downstream of proteasomes for degradation of longer peptides. However, this canonical function of TPP2 cannot explain its role in a wide variety of biological and pathogenic processes. The mechanistic interrelationships and hierarchical order of these processes have yet to be clarified. Methods: Animals, cells, plasmids, and viruses established and/or used in this study include: TPP2 knockout mouse line, TPP2 conditional knockout mouse lines (different neural cell type oriented), TRE-TPP2 knockin mouse line on the C57BL/6 background; 293T cells with depletion of TPP2, ATF6, IRE1, PERK, SYVN1, UCHL1, ATG5, CEPT1, or CCTα, respectively; 293T cells stably expressing TPP2, TPP2 S449A, TPP2 S449T, or CCTα-KDEL proteins on the TPP2-depleted background; Plasmids for eukaryotic transient expression of rat CYP19A1-Flag, CYP19A1 S118A-Flag, CYP19A1 S118D-Flag, Sac I ML GFP Strand 11 Long, OMMGFP 1-10, G-CEPIA1er, GCAMP2, CEPIA3mt, ACC-GFP, or SERCA1-GFP; AAV2 carrying the expression cassette of mouse CYP19A1-3 X Flag-T2A-ZsGreen. Techniques used in this study include: Flow cytometry, Immunofluorescence (IF) staining, Immunohistochemical (IHC) staining, Luxol fast blue (LFB) staining, ß-galactosidase staining, Lipid droplet (LD) staining, Calcium (Ca2+) staining, Stimulated emission depletion (STED) imaging, Transmission electron microscopic imaging, Two-photon imaging, Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end Labeling (TUNEL) assay, Bromodeoxyuridine (BrdU) assay, Enzymatic activity assay, Proximity ligation assay (PLA), In vivo electrophysiological recording, Long-term potentiation (LTP) recording, Split-GFP-based mitochondria-associated membrane (MAM) detection, Immunoprecipitation (IP), Cellular fractionation, In situ hybridization, Semi-quantitative RT-PCR, Immunoblot, Mass spectrometry-based lipidomics, metabolomics, proteomics, Primary hippocampal neuron culture and Morris water maze (MWM) test. Results: We found that TPP2, independent of its enzymatic activity, plays a crucial role in maintaining the homeostasis of intracellular Ca2+ and phosphatidylcholine (PC) in the central nervous system (CNS) of mice. In consistence with the critical importance of Ca2+ and PC in the CNS, TPP2 gene ablation causes presenile dementia in female mice, which is closely associated with Ca2+/PC dysregulation-induced endoplasmic reticulum (ER) stress, abnormal autophagic degradation of CYP19A1 (aromatase), and estrogen depletion. This work therefore uncovers a new role of TPP2 in lipogenesis and neurosteroidogenesis which is tightly related to cognitive function of adult female mice. Conclusion: Our study reveals a crucial role of TPP2 in controlling homeostasis of Ca2+ and lipids in CNS, and its deficiency causes sexual dimorphism in dementia. Thus, this study is not only of great significance for elucidating the pathogenesis of dementia and its futural treatment, but also for interpreting the role of TPP2 in other systems and their related disorders.

"Zero" Intrinsic Fluorescence Sensing-Platforms Enable Ultrasensitive Whole Blood Diagnosis and In Vivo Imaging.

Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re-engineered far-red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of "zero" PIF sensing-platforms were created by systematically regulating the open-loop/spirocyclic forms. Leveraging these advancements, we devised various ultra-sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300-fold). Among these indicators, 8-LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8-LAP with an endoplasmic reticulum-targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases.

Peripheral retinal finding on fluorescein angiography in neuronal ceroid lipofuscinosis type 2 (CLN2).

Classically, peripheral vascular changes in the retina in patients with neuronal ceroid lipofuscinosis type 2 (CLN2) are described as vascular attenuation seen in the late stages of disease on the Weill Connell Ophthalmic Severity Score (WCOSS) staging system. We describe isolated, mild, peripheral vasculitis with peripheral arteriolar dropout identified by fluorescein angiography in patients with a WCOSS grade of stage 2. We believe this vasculitis represents an early vasodegenerative phase of disease that leads to the vascular attenuation seen in later stages of the disease.

Association of a single nucleotide polymorphism (rs27434) in the ERAP1 gene with plural tissue weight.

Our study was designed to examine the correlation between single nucleotide polymorphism (SNP) in the endoplasmic reticulum aminopeptidase 1 (ERAP1) gene, specifically focusing on rs27434, and plural tissue weight. We conducted this investigation using autopsy samples from the Japanese population. Blood samples were collected from 178 Japanese subjects who had undergone autopsies in Shimane Prefecture. Genomic DNA was subsequently extracted from these samples. SNP (rs27434, G＞A substitution) was analyzed by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. In the present study, rs27434 exhibited a statistically significant association with brain weight (g) in both female and male individuals. Among males, rs27434 displayed significant relationships with liver weight (g), and body surface area (m2). In females, rs27434 was significantly related to the length of the appendix. Across both genders, individuals with GA and AA genotypes tended to exhibit higher levels in these respective measurements compared to those with the GG genotype. These results suggest that genetic variant of ERAP1 gene may influence the weight of the organs. To the best of our knowledge, this is the first study investigating the interaction between the association of rs27434 in the ERAP1 gene and data routinely measured at autopsy, such as tissue weight. However, conducting further investigations with larger population samples could provide more comprehensive insights to clarify this issue.