Characterization and functional evidence for Orf2 of Streptomyces sp. 139 as a novel dipeptidase E.

Aspartyl dipeptidase (dipeptidase E) can hydrolyze Asp-X dipeptides (where X is any amino acid), and the enzyme plays a key role in the degradation of peptides as nutrient sources. Dipeptidase E remains uncharacterized in Streptomyces. Orf2 from Streptomyces sp. 139 is located in the exopolysaccharide biosynthesis gene cluster, which may be a novel dipeptidase E with "S134-H170-D198" catalytic triad by sequence and structure comparison. Herein, recombinant Orf2 was expressed in E. coli and characterized dipeptidase E activity using the Asp-ρNA substrate. The optimal pH and temperature for Orf2 are 7.5 and 40 ℃; Vmax and Km of Orf2 are 0.0787 mM·min-1 and 1.709 mM, respectively. Orf2 exhibits significant degradation activities to Asp-Gly-Gly, Asp-Leu, Asp-His, and isoAsp-Leu and minimal activities to Asp-Pro and Asp-Ala. Orf2 contains a Ser-His-Asp catalytic triad characterized by point mutation. In addition, the Asp147 residue of Orf2 is also proven to be critical for the enzyme's activity through molecular docking and point mutation. Transcriptome analysis reveals the upregulation of genes associated with ribosomes, amino acid biosynthesis, and aminoacyl-tRNA biosynthesis in the orf2 mutant strain. Compared with the orf2 mutant strain and WT, the yield of crude polysaccharide does not change significantly. However, crude polysaccharides from the orf2 mutant strain exhibit a wider range of molecular weight distribution. The results indicate that the Orf2 links nutrient stress to secondary metabolism as a novel dipeptidase E. KEY POINTS: • A novel dipeptidase E with a Ser-His-Asp catalytic triad was characterized from Streptomyces sp. 139. • Orf2 was involved in peptide metabolism both in vitro and in vivo. • Orf2 linked nutrient stress to mycelia formation and secondary metabolism in Streptomyces.

Effects of metformin, saxagliptin and repaglinide on gut microbiota in high-fat diet/streptozocin-induced type 2 diabetic mice.

INTRODUCTION: There has been increasing evidence that the gut microbiota is closely related to type 2 diabetes (T2D). Metformin (Met) is often used in combination with saxagliptin (Sax) and repaglinide (Rep) for the treatment of T2D. However, little is known about the effects of these combination agents on gut microbiota in T2D. RESEARCH DESIGN AND METHODS: A T2D mouse model induced by a high-fat diet (HFD) and streptozotocin (STZ) was employed. The T2D mice were randomly divided into six groups, including sham, Met, Sax, Rep, Met+Sax and Met+Rep, for 4 weeks. Fasting blood glucose level, serum biochemical index, H&E staining of liver, Oil red O staining of liver and microbiota analysis by 16s sequencing were used to access the microbiota in the fecal samples. RESULTS: These antidiabetics effectively prevented the development of HFD/STZ-induced high blood glucose, and the combination treatment had a better effect in inhibiting lipid accumulation. All these dosing regimens restored the decreasing ratio of the phylum Bacteroidetes: Firmicutes, and increasing abundance of phylum Desulfobacterota, expect for Met. At the genus level, the antidiabetics restored the decreasing abundance of Muribaculaceae in T2D mice, but when Met was combined with Rep or Sax, the abundance of Muribaculaceae was decreased. The combined treatment could restore the reduced abundance of Prevotellaceae_UCG-001, while Met monotherapy had no such effect. In addition, the reduced Lachnospiraceae_NK4A136_group was well restored in the combination treatment groups, and the effect was much greater than that in the corresponding monotherapy group. Therefore, these dosing regimens exerted different effects on the composition of gut microbiota, which might be associated with the effect on T2D. CONCLUSIONS: Supplementation with specific probiotics may further improve the hypoglycemic effects of antidiabetics and be helpful for the development of new therapeutic drugs for T2D.

Deep learning path-like collective variable for enhanced sampling molecular dynamics.

Several enhanced sampling techniques rely on the definition of collective variables to effectively explore free energy landscapes. The existing variables that describe the progression along a reactive pathway offer an elegant solution but face a number of limitations. In this paper, we address these challenges by introducing a new path-like collective variable called the "deep-locally non-linear-embedding," which is inspired by principles of the locally linear embedding technique and is trained on a reactive trajectory. The variable mimics the ideal reaction coordinate by automatically generating a non-linear combination of features through a differentiable generalized autoencoder that combines a neural network with a continuous k-nearest neighbor selection. Among the key advantages of this method is its capability to automatically choose the metric for searching neighbors and to learn the path from state A to state B without the need to handpick landmarks a priori. We demonstrate the effectiveness of DeepLNE by showing that the progression along the path variable closely approximates the ideal reaction coordinate in toy models, such as the Müller-Brown potential and alanine dipeptide. Then, we use it in the molecular dynamics simulations of an RNA tetraloop, where we highlight its capability to accelerate transitions and estimate the free energy of folding.

Comparison of quantitative whole body PET parameters on [68Ga]Ga-PSMA-11 PET/CT using ordered Subset Expectation Maximization (OSEM) vs. bayesian penalized likelihood (BPL) reconstruction algorithms in men with metastatic castration-resistant prostate cancer.

BACKGROUND: PSMA PET/CT is a predictive and prognostic biomarker for determining response to [177Lu]Lu-PSMA-617 in patients with metastatic castration resistant prostate cancer (mCRPC). Thresholds defined to date may not be generalizable to newer image reconstruction algorithms. Bayesian penalized likelihood (BPL) reconstruction algorithm is a novel reconstruction algorithm that may improve contrast whilst preventing introduction of image noise. The aim of this study is to compare the quantitative parameters obtained using BPL and the Ordered Subset Expectation Maximization (OSEM) reconstruction algorithms. METHODS: Fifty consecutive patients with mCRPC who underwent [68Ga]Ga-PSMA-11 PET/CT using OSEM reconstruction to assess suitability for [177Lu]Lu-PSMA-617 therapy were selected. BPL algorithm was then used retrospectively to reconstruct the same PET raw data. Quantitative and volumetric measurements such as tumour standardised uptake value (SUV)max, SUVmean and Molecular Tumour Volume (MTV-PSMA) were calculated on both reconstruction methods. Results were compared (Bland-Altman, Pearson correlation coefficient) including subgroups with low and high-volume disease burdens (MTV-PSMA cut-off 40 mL). RESULTS: The SUVmax and SUVmean were higher, and MTV-PSMA was lower in the BPL reconstructed images compared to the OSEM group, with a mean difference of 8.4 (17.5%), 0.7 (8.2%) and - 21.5 mL (-3.4%), respectively. There was a strong correlation between the calculated SUVmax, SUVmean, and MTV-PSMA values in the OSEM and BPL reconstructed images (Pearson r values of 0.98, 0.99, and 1.0, respectively). No patients were reclassified from low to high volume disease or vice versa when switching from OSEM to BPL reconstruction. CONCLUSIONS: [68Ga]Ga-PSMA-11 PET/CT quantitative and volumetric parameters produced by BPL and OSEM reconstruction methods are strongly correlated. Differences are proportional and small for SUVmean, which is used as a predictive biomarker. Our study suggests that both reconstruction methods are acceptable without clinical impact on quantitative or volumetric findings. For longitudinal comparison, committing to the same reconstruction method would be preferred to ensure consistency.

Hematological and renal toxicity in mice after three cycles of high activity [177Lu]Lu-PSMA-617 with or without human α1-microglobulin.

Radioligand therapy with [177Lu]Lu-PSMA-617 can be used to prolong life and reduce tumor burden in terminally ill castration resistant prostate cancer patients. Still, accumulation in healthy tissue limits the activity that can be administered. Therefore, fractionated therapy is used to lower toxicity. However, there might be a need to reduce toxicity even further with e.g. radioprotectors. The aim of this study was to (i). establish a preclinical mouse model with fractionated high activity therapy of three consecutive doses of 200 MBq [177Lu]Lu-PSMA-617 in which we aimed to (ii). achieve measurable hematotoxicity and nephrotoxicity and to (iii). analyze the potential protective effect of co-injecting recombinant α1-microglobulin (rA1M), a human antioxidant previously shown to have radioprotective effects. In both groups, three cycles resulted in increased albuminuria for each cycle, with large individual variation. Another marker of kidney injury, serum blood urea nitrogen (BUN), was only significantly increased compared to control animals after the third cycle. The number of white and red blood cells decreased significantly and did not reach the levels of control animals during the experiment. rA1M did reduce absorbed dose to kidney but did not show significant protection here, but future studies are warranted due to the recent clinical studies showing a significant renoprotective effect in patients.

Pyroptosis inhibitors MCC950 and VX-765 mitigate myocardial injury by alleviating oxidative stress, inflammation, and apoptosis in acute myocardial hypoxia.

Acute myocardial infarction (AMI) is a prevalent cardiovascular disease with high morbidity and mortality rates worldwide. Pyroptosis is an inflammatory form of programmed cell death that has been linked to various pathological conditions. However, its exact contribution to the onset and progression of heart injury in AMI has not yet fully elucidated. Herein, we established mouse AMI model by ligating the left anterior descending artery and performed transcriptome analysis during the early phase of AMI. Mouse HL-1 and human AC-16 cardiomyocytes were subjected to hypoxia to simulate ischemic injury in vitro. Our results revealed a significant activation of the inflammatory response at 3 h post-ligation, as confirmed by RNA sequencing. We identified the occurrence of NLRP3 inflammasome-mediated pyroptosis in the cardiac tissues of human cases with AMI, as well as in mouse models of AMI and hypoxia-induced cardiomyocytes, using immunohistochemistry staining and Western blotting assays. Concurrently, pharmacological inhibition of NLRP3 inflammasome-mediated pyroptosis with MCC950 and VX-765 effectively decreased hypoxia-induced cardiomyocytes injury, while mitigating myocardial oxidative stress, apoptosis and inflammation caused by hypoxia. Moreover, the circulating levels of gasdermin D (GSDMD), the pyroptosis executor, were remarkably elevated in the plasma of mice with early AMI and in the supernatant of hypoxia-exposed cardiomyocytes in a time-dependent manner using ELISA and Western blotting. Furthermore, the change in circulating GSDMD positively correlated with Creatine Kinase-MB (CK-MB) in the plasma of early-stage AMI mouse. In summary, these findings indicated a critical role for NLRP3 inflammasome-mediated pyroptosis in the progression of AMI, the administration of MCC950 and VX-765 may be attractive candidate therapeutic approaches for cardiac injury caused by acute hypoxia or even AMI. Additionally, the circulating GSDMD exhibits potential as a newly diagnostic biomarker for AMI.

Metal Cross-Linked Supramolecular Gel Noodles: Structural Insights and Antibacterial Assessment.

Achieving precise control over gelator alignment and morphology is crucial for crafting tailored materials and supramolecular structures with distinct properties. We successfully aligned the self-assembled micelles formed by a functionalized dipeptide 2NapFF into long 1-D "gel noodles" by cross-linking with divalent metal chlorides. We identify the most effective cross-linker for alignment, enhancing mechanical stability, and imparting functional properties. Our study shows that Group 2 metal ions are particularly suited for creating mechanically robust yet flexible gel noodles because of their ionic and nondirectional bonding with carboxylate groups. In contrast, the covalent nature and high directional bonds of d-block metal ions with carboxylates tend to disrupt the self-assembly of 2NapFF. Furthermore, the 2NapFF-Cu noodles demonstrated selective antibacterial activity, indicating that the potent antibacterial property of the copper(II) ion is preserved within the cross-linked system. By merging insights into molecular alignment, gel extrusion processing, and integrating specific functionalities, we illustrate how the versatility of dipeptide-based gels can be utilized in creating next-generation soft materials.

Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Birinapant Reshapes the Tumor Immunopeptidome and Enhances Antigen Presentation.

Birinapant, an antagonist of the inhibitor of apoptosis proteins, upregulates MHCs in tumor cells and displays a better tumoricidal effect when used in combination with immune checkpoint inhibitors, indicating that Birinapant may affect the antigen presentation pathway; however, the mechanism remains elusive. Based on high-resolution mass spectrometry and in vitro and in vivo models, we adopted integrated genomics, proteomics, and immunopeptidomics strategies to study the mechanism underlying the regulation of tumor immunity by Birinapant from the perspective of antigen presentation. Firstly, in HT29 and MCF7 cells, Birinapant increased the number and abundance of immunopeptides and source proteins. Secondly, a greater number of cancer/testis antigen peptides with increased abundance and more neoantigens were identified following Birinapant treatment. Moreover, we demonstrate the existence and immunogenicity of a neoantigen derived from insertion/deletion mutation. Thirdly, in HT29 cell-derived xenograft models, Birinapant administration also reshaped the immunopeptidome, and the tumor exhibited better immunogenicity. These data suggest that Birinapant can reshape the tumor immunopeptidome with respect to quality and quantity, which improves the presentation of CTA peptides and neoantigens, thus enhancing the immunogenicity of tumor cells. Such changes may be vital to the effectiveness of combination therapy, which can be further transferred to the clinic or aid in the development of new immunotherapeutic strategies to improve the anti-tumor immune response.

Gold and Silver Nanoparticles as Biosensors: Characterization of Surface and Changes in the Adsorption of Leucine Dipeptide under the Influence of Substituent Changes.

Early detection of diseases can increase the chances of successful treatment and survival. Therefore, it is necessary to develop a method for detecting or sensing biomolecules that cause trouble in living organisms. Disease sensors should possess specific properties, such as selectivity, reproducibility, stability, sensitivity, and morphology, for their routine application in medical diagnosis and treatment. This work focuses on biosensors in the form of surface-functionalized gold (AuNPs) and silver nanoparticles (AgNPs) prepared using a less-time-consuming, inexpensive, and efficient synthesis route. This allows for the production of highly pure and stable (non-aggregating without stabilizers) nanoparticles with a well-defined spherical shape, a desired diameter, and a monodisperse distribution in an aqueous environment, as confirmed by transmission electron microscopy with energy-dispersive X-ray spectroscopy (TEM-EDS), X-ray diffraction (XRD), photoelectron spectroscopy (XPS), ultraviolet-visible (UV-VIS) spectroscopy, and dynamic light scattering (DLS). Thus, these nanoparticles can be used routinely as biomarker sensors and drug-delivery platforms for precision medicine treatment. The NPs' surface was coated with phosphonate dipeptides of L-leucine (Leu; l-Leu-C(R1)(R2)PO3H2), and their adsorption was monitored using SERS. Reproducible spectra were analyzed to determine the orientation of the dipeptides (coating layers) on the nanoparticles' surface. The appropriate R2 side chain of the dipeptide can be selected to control the arrangement of these dipeptides. This allows for the proper formation of a layer covering the nanoparticles while also simultaneously interacting with the surrounding biological environment, such as cells, tissues, and biological fluids.

A Novel Tetrapeptide Ala-Phe-Phe-Pro (AFFP) Derived from Antarctic Krill Prevents Scopolamine-Induced Memory Disorder by Balancing Lipid Metabolism of Mice Hippocampus.

Memory impairment is a serious problem with organismal aging and increased social pressure. The tetrapeptide Ala-Phe-Phe-Pro (AFFP) is a synthetic analogue of Antarctic krill derived from the memory-improving Antarctic krill peptide Ser-Ser-Asp-Ala-Phe-Phe-Pro-Phe-Arg (SSDAFFPFR) after digestion and absorption. The objective of this research was to assess the neuroprotective effects of AFFP by reducing oxidative stress and controlling lipid metabolism in the brains of mice with memory impairment caused by scopolamine. The 1H Nuclear magnetic resonance spectroscopy results showed that AFFP had three active hydrogen sites that could contribute to its antioxidant properties. The findings from in vivo tests demonstrated that AFFP greatly enhanced the mice's behavioral performance in the passive avoidance, novel object recognition, and eight-arm maze experiments. AFFP reduced oxidative stress by enhancing superoxide dismutase activity and malondialdehyde levels in mice serum, thereby decreasing reactive oxygen species level in the mice hippocampus. In addition, AFFP increased the unsaturated lipid content to balance the unsaturated lipid level against the neurotoxicity of the mice hippocampus. Our findings suggest that AFFP emerges as a potential dietary intervention for the prevention of memory impairment disorders.

Exploring the role of combined external beam radiotherapy and targeted radioligand therapy with [177Lu]Lu-PSMA-617 for prostate cancer - from bench to bedside.

Management of prostate cancer (PC) might be improved by combining external beam radiotherapy (EBRT) and prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) with lutetium-177 (177Lu)-labeled PSMA inhibitors. We hypothesized a higher efficacy of the combination due to augmentation of the radiation dose to the tumor and interactions of EBRT with PSMA expression potentially increasing radiopharmaceutical uptake. Therefore, this study analyzed the influence of radiation on PSMA expression levels in vitro. The results were translated to evaluate the efficacy of the combination of photon EBRT and [177Lu]Lu-PSMA-617 in a murine PC xenograft model. Finally, a clinical case report on a combined elective field EBRT with RLT dose escalation illustrates a proof-of-concept. Methods: PSMA gene and protein expression were assessed in human PSMA-overexpressing LNCaP cells after irradiation using reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry and On-Cell Western assays. In the in vivo therapy study, LNCaP tumor-bearing BALB/c nu/nu mice were irradiated once with 2 Gy X-ray EBRT and injected with 40 MBq [177Lu]Lu-PSMA-617 after 4 h or received single or no treatment (n = 10 each). Tumor-absorbed doses by [177Lu]Lu-PSMA-617 were calculated according to the Medical Internal Radiation Dosimetry (MIRD) formalism after deriving time-activity curves using a gamma probe. An exemplified patient case is demonstrated where fractionated EBRT (54 Gy to prostate; 45 Gy to pelvic lymphatics) and three cycles of [177Lu]Lu-PSMA-617 (3.4-6.0 GBq per cycle) were sequentially combined under concurrent androgen deprivation for treating locally advanced PC. Results: At 4 h following irradiation with 2-8 Gy, LNCaP cells displayed a PSMA protein upregulation by around 18% relative to non-irradiated cells, and a stronger upregulation on mRNA level (up to 2.6-fold). This effect was reversed by 24 h when PSMA protein levels were downregulated by up to 22%. Mice treated with the combination therapy showed significantly improved outcomes regarding tumor control and median survival (p < 0.0001) as compared to single or no treatment. Relative to monotherapy with PSMA-RLT or EBRT, the tumor doubling time was prolonged 1.7- or 2.7-fold and the median survival was extended by 24% or 60% with the combination, respectively. Additionally, tumors treated with EBRT exhibited a 14% higher uptake of the radiopharmaceutical as evident from the calculated tumor-absorbed dose, albeit with high variability in the data. Concerning the patient case, the tri-modality treatment was well tolerated and the patient responded with a long-lasting complete biochemical remission for five years following end of PSMA-RLT. The patient then developed a biochemical relapse with oligo-recurrent disease on follow-up imaging. Conclusion: The present preclinical and clinical data demonstrate that the combination of EBRT with dose escalation by PSMA-RLT improves tumor control and potentially prolongs survival. This may pave the way for further clinical investigations of this approach to explore the curative potential of the combination therapy.

pH-sensitive release of nitric oxide gas using peptide-graphene co-assembled hybrid nanosheets.

Nitric oxide (NO) donating drugs such as organic nitrates have been used to treat cardiovascular diseases for more than a century. These donors primarily produce NO systemically. It is however sometimes desirable to control the amount, location, and time of NO delivery. We present the design of a novel pH-sensitive NO release system that is achieved by the synthesis of dipeptide diphenylalanine (FF) and graphene oxide (GO) co-assembled hybrid nanosheets (termed as FF@GO) through weak molecular interactions. These hybrid nanosheets were characterised by using X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, zeta potential measurements, X-ray photoelectron spectroscopy, scanning and transmission electron microscopies. The weak molecular interactions, which include electrostatic, hydrogen bonding and π-π stacking, are pH sensitive due to the presence of carboxylic acid and amine functionalities on GO and the dipeptide building blocks. Herein, we demonstrate that this formulation can be loaded with NO gas with the dipeptide acting as an arresting agent to inhibit NO burst release at neutral pH; however, at acidic pH it is capable of releasing NO at the rate of up to 0.6 µM per minute, comparable to the amount of NO produced by healthy endothelium. In conclusion, the innovative conjugation of dipeptide with graphene can store and release NO gas under physiologically relevant concentrations in a pH-responsive manner. pH responsive NO-releasing organic-inorganic nanohybrids may prove useful for the treatment of cardiovascular diseases and other pathologies.

Contribution of amino acids in the active site of dipeptidyl peptidase 4 to the catalytic action of the enzyme.

Dipeptidyl peptidase 4 (DP4)/CD26 regulates the biological function of various peptide hormones by releasing dipeptides from their N-terminus. The enzyme is a prominent target for the treatment of type-2 diabetes and various DP4 inhibitors have been developed in recent years, but their efficacy and side effects are still an issue. Many available crystal structures of the enzyme give a static picture about enzyme-ligand interactions, but the influence of amino acids in the active centre on binding and single catalysis steps can only be judged by mutagenesis studies. In order to elucidate their contribution to inhibitor binding and substrate catalysis, especially in discriminating the P1 amino acid of substrates, the amino acids R125, N710, E205 and E206 were investigated by mutagenesis studies. Our studies demonstrated, that N710 is essential for the catalysis of dipeptide substrates. We found that R125 is not important for dipeptide binding but interacts in the P1`position of the peptide backbone. In contrast to dipeptide substrates both amino acids play an essential role in the binding and arrangement of long natural substrates, particularly if lacking proline in the P1 position. Thus, it can be assumed that the amino acids R125 and N710 are important in the DP4 catalysed substrate hydrolysis by interacting with the peptide backbone of substrates up- and downstream of the cleavage site. Furthermore, we confirmed the important role of the amino acids E205 and E206. However, NP Y, displaying proline in P1 position, is still processed without the participation of E205 or E206.

[Advances on cyclopeptides of plants].

Cyclopeptides isolated from a variety of plants are a class of cyclic nitrogen-containing compounds, and they are primarily formed by peptide bonds between amino acids, generally containing 2 to 37 L-configuration encoded or non-encoded amino acid residues. Cyclopeptides have significant values in scientific research as natural small-molecule metabolites produced by plants. The available studies have revealed that such natural products are ubiquitous in plants, which mainly include cyclic dipeptides, cyclic tetrapeptides, cyclic pentapeptides, cyclic hexapeptides, cyclic heptapeptides, cyclic octapeptides, cyclic nonapeptides, and cyclic decapeptides. Among them, cyclic dipeptides, cyclic hexapeptides, and cyclic octapeptides are the major active compounds. It has been reported that plant cyclopeptides have novel and unique chemical structures. They possess diverse pharmacological activities, such as antineoplastic, antimicrobial, antimalarial, anti-inflammatory, and antiviral activities. This paper summarizes the research achievements of plant cyclopeptides since 2006, aiming to provide theoretical reference for the research and application of plant cyclopeptides in medicine, health, and agriculture fields.

GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells.

Bladder cancer aggressiveness is correlated with abnormal N-cadherin transmembrane glycoprotein expression. This protein is cleaved by the metalloprotease ADAM10 and the γ-secretase complex releasing a pro-angiogenic N-terminal fragment (NTF) and a proliferation-activating soluble C-terminal fragment (CTF2). Tetraspanin 15 (Tspan15) is identified as an ADAM10-interacting protein to induce selective N-cadherin cleavage. We first demonstrated, in invasive T24 bladder cancer cells, that N-cadherin was cleaved by ADAM10 generating NTF in the extracellular environment and leaving a membrane-anchored CTF1 fragment and that Tspan15 is required for ADAM10 to induce the selective N-cadherin cleavage. Targeting N-cadherin function in cancer is relevant to preventing tumor progression and metastases. For antitumor molecules to inhibit N-cadherin function, they should be complete and not cleaved. We first showed that the GW501516, an agonist of the nuclear receptor PPARß/δ, decreased Tspan15 and prevented N-cadherin cleavage thus decreasing NTF. Interestingly, the drug did not modify ADAM10 expression, which was important because it could limit side effects since ADAM10 cleaves numerous substrates. By targeting Tspan15 to block ADAM10 activity on N-cadherin, GW501516 could prevent NTF pro-tumoral effects and be a promising molecule to treat bladder cancer. More interestingly, it could optimize the effects of the N-cadherin antagonists those such as ADH-1 that target the N-cadherin ectodomain.

Stabilization of Prebiotic Vesicles by Peptides Depends on Sequence and Chirality: A Mechanism for Selection of Protocell-Associated Peptides.

Cells require oligonucleotides and polypeptides with specific, homochiral sequences to perform essential functions, but it is unclear how such oligomers were selected from random sequences at the origin of life. Cells were probably preceded by simple compartments such as fatty acid vesicles, and oligomers that increased the stability, growth, or division of vesicles could have thereby increased in frequency. We therefore tested whether prebiotic peptides alter the stability or growth of vesicles composed of a prebiotic fatty acid. We find that three of 15 dipeptides tested reduce salt-induced flocculation of vesicles. All three contain leucine, and increasing their length increases the efficacy. Also, leucine-leucine but not alanine-alanine increases the size of vesicles grown by multiple additions of micelles. In a molecular simulation, leucine-leucine docks to the membrane, with the side chains inserted into the hydrophobic core of the bilayer, while alanine-alanine fails to dock. Finally, the heterochiral forms of leucine-leucine, at a high concentration, rapidly shrink the vesicles and make them leakier and less stable to high pH than the homochiral forms do. Thus, prebiotic peptide-membrane interactions influence the flocculation, growth, size, leakiness, and pH stability of prebiotic vesicles, with differential effects due to sequence, length, and chirality. These differences could lead to a population of vesicles enriched for peptides with beneficial sequence and chirality, beginning selection for the functional oligomers that underpin life.

Inhibition of Notch1 signal promotes brain recovery by modulating glial activity after stroke.

BACKGROUND: Notch1 signaling inhibiton with N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butylester] (DAPT) treatment could promote brain recovery and the intervention effect is different between striatum (STR) and cortex (CTX), which might be accounted for different changes of glial activities, but the in-depth mechanism is still unknown. The purpose of this study was to identify whether DAPT could modulate microglial subtype shifts and astroglial-endfeet aquaporin-4 (AQP4) mediated waste solute drainage. METHODS: Sprague-Dawley rats (n=10) were subjected to 90min of middle cerebral artery occlusion (MCAO) and were treated with DAPT (n=5) or act as control with no treatment (n=5). Two groups of rats underwent MRI scans at 24h and 4 week, and sacrificed at 4 week after stroke for immunofluorescence (IF). RESULTS: Compared with control rats, MRI data showed structural recovery in ipsilateral STR but not CTX. And IF showed decreased pro-inflammatory M1 microglia and increased anti-inflammatory M2 microglia in striatal lesion core and peri-lesions of STR, CTX. Meanwhile, IF showed decreased AQP4 polarity in ischemic brain tissue, however, AQP4 polarity in striatal peri-lesions of DAPT treated rats was higher than that in control rats but shows no difference in cortical peri-lesions between control and treated rats. CONCLUSIONS: The present study indicated that DAPT could promote protective microglia subtype shift and striatal astrocyte mediated waste solute drainage, that the later might be the major contributor of waste solute metabolism and one of the accounts for discrepant recovery of STR and CTX.

Genomic Correlates of Prostate-Specific Membrane Antigen Expression and Response to 177Lu-PSMA-617: A Retrospective Multicenter Cohort Study.

PURPOSE: While 177Lu-PSMA-617 (LuPSMA) is an effective therapy for many patients with metastatic castration-resistant prostate cancer (mCRPC), biomarkers associated with outcomes are not well defined. We hypothesized that prostate cancer mutational profile may associate with clinical activity of LuPSMA. We devised a study to evaluate associations between mCRPC mutational profile with LuPSMA clinical outcomes. METHODS: This was a multicenter retrospective analysis of patients with mCRPC with next-generation sequencing (NGS) who received LuPSMA. PSA50 response (ie, ≥50% decline in prostate-specific antigen [PSA]) rate, PSA progression free survival (PSA PFS), and overall survival (OS) were compared between genetically defined subgroups. RESULTS: One hundred twenty-six patients with NGS results who received at least one cycle of LuPSMA were identified. The median age was 73 (IQR, 68-78) years, 124 (98.4%) received ≥1 prior androgen receptor-signaling inhibitor, and 121 (96%) received ≥1 taxane-based chemotherapy regimen. Fifty-eight (46%) patients with a DNA damage repair gene mutation (DNA damage response group) and 59 (46.8%) with a mutation in TP53, RB1, or PTEN tumor suppressor genes (TSG group) were identified. After adjusting for relevant confounders, the presence of ≥1 TSG mutation was associated with shorter PSA PFS (hazard ratio [HR], 1.93 [95% CI, 1.05 to 3.54]; P = .034) and OS (HR, 2.65 [95% CI, 1.15 to 6.11]; P = .023). There was improved OS favoring the DNA damage response group (HR, 0.37 [95% CI, 0.14 to 0.97]; P = .044) on multivariable analysis. Univariate analysis of patients with ATM mutations had significantly higher rates of PSA50 response, PSA PFS, and OS. CONCLUSION: Outcomes on LuPSMA varied on the basis of mutational profile. Prospective studies to define the clinical activity of LuPSMA in predefined genomic subgroups are justified.

Cyclodipeptide oxidase is an enzyme filament.

Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization of the Streptomyces noursei enzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,ß-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that other CDO-like enzymes are likely enzyme filaments. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.