Integrative lactylation and tumor microenvironment signature as prognostic and therapeutic biomarkers in skin cutaneous melanoma.

BACKGROUND: The incidence of skin cutaneous melanoma (SKCM), one of the most aggressive and lethal skin tumors, is increasing worldwide. However, for advanced SKCM, we still lack an accurate and valid way to predict its prognosis, as well as novel theories to guide the planning of treatment options for SKCM patients. Lactylation (LAC), a novel post-translational modification of histones, has been shown to promote tumor growth and inhibit the antitumor response of the tumor microenvironment (TME) in a variety of ways. We hope that this study will provide new ideas for treatment options for SKCM patients, as well as research on the molecular mechanisms of SKCM pathogenesis and development. METHODS: At the level of the RNA sequencing set (TCGA, GTEx), we used differential expression analysis, LASSO regression analysis, and multifactor Cox regression analysis to screen for prognosis-related genes and calculate the corresponding LAC scores. The content of TME cells in the tumor tissue was calculated using the CIBERSORT algorithm, and the TME score was calculated based on its results. Finally, the LAC-TME classifier was established and further analyzed based on the two scores, including the construction of a prognostic model, analysis of clinicopathological characteristics, and correlation analysis of tumor mutation burden (TMB) and immunotherapy. Based on single-cell RNA sequencing data, this study analyzed the cellular composition in SKCM tissues and explored the role of LAC scores in intercellular communication. To validate the functionality of the pivotal gene CLPB in the model, cellular experiments were ultimately executed. RESULTS: We screened a total of six prognosis-related genes (NDUFA10, NDUFA13, CLPB, RRM2B, HPDL, NARS2) and 7 TME cells with good prognosis. According to Kaplan-Meier survival analysis, we found that the LAClow/TMEhigh group had the highest overall survival (OS) and the LAChigh/TMElow group had the lowest OS (p value < 0.05). In further analysis of immune infiltration, tumor microenvironment (TME), functional enrichment, tumor mutational load and immunotherapy, we found that immunotherapy was more appropriate in the LAClow/TMEhigh group. Moreover, the cellular assays exhibited substantial reductions in proliferation, migration, and invasive potentials of melanoma cells in both A375 and A2058 cell lines upon CLPB knockdown. CONCLUSIONS: The prognostic model using the combined LAC score and TME score was able to predict the prognosis of SKCM patients more consistently, and the LAC-TME classifier was able to significantly differentiate the prognosis of SKCM patients across multiple clinicopathological features. The LAC-TME classifier has an important role in the development of immunotherapy regimens for SKCM patients.

Identification and characterization of aspartyl-tRNA synthetase inhibitors against Mycobacterium tuberculosis by an integrated whole-cell target-based approach.

Mycobacterium tuberculosis, the causative agent of tuberculosis, has surpassed HIV as the leading cause of death due to an infectious disease worldwide, being responsible for more than 1.5 million deaths in low-income countries. In response to a pandemic threat by drug resistant strains, the tuberculosis research community is searching for new chemical entities with novel mechanisms of action to avoid drug resistance and shorten treatment regimens using combinatorial chemotherapy. Herein, we have identified several novel chemical scaffolds, GSK97C (spiro-oxazolidin-2-one), GSK93A (2-amino-1,3-thiazole, GSK85A and GSK92A (enamides), which target M. tuberculosis aspartyl-tRNA synthetase (Mt-AspRS), an essential component of the protein synthesis machinery of tuberculosis, using a whole-cell target-based screening strategy against a genetically modified Mycobacterium bovis BCG strain. We also provide further evidence of protein inhibition and inhibitor profiling through a classical aminoacylation reaction and a tRNA-independent assay, respectively. Altogether, our results have identified a number of hit new molecules with novel mechanism of action for further development through medicinal chemistry as hits and leads.

Virtual screening of traditional Chinese medicine (TCM) database: identification of fragment-like lead molecules for filariasis target asparaginyl-tRNA synthetase.

Lymphatic filariasis (LF) is a vector borne infectious disease caused by the nematode Wuchereria bancrofti, Brugia malayi, and Brugia timori. Over 120 million people are affected by LF in the world, of which two-thirds are in Asia. The infection restricts the normal flow of lymph from the infected area resulting in swelling of the extremities and causing permanent disability. As the available drugs for the treatment of LF are becoming ineffective due to the development of resistance, there is an urgent need to find new leads for drug development. In this study, asparaginyl-tRNA synthetase (AsnRS; PDB ID: 2XGT) essential for the protein bio-synthesis in the filarial nematode was used to carry out virtual screening (VS) of plant constituents from traditional Chinese medicine (TCM) database. Docking as well as E-pharmacophore based VS were carried out to identify the hits. The top scoring hits, Agri 1 (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one-3-O-beta-D-glucopyranoside) and Agri 2 (5,7-dihydroxy-2-propylchromone 7-O-beta-D-glucopyranoside), constituents of Agrimonia pilosa, were selected for molecular dynamics (MD) simulation study for 10 ns. MD simulation showed that both the glycosides Agri 1 and Agri 2 were forming stable interactions with the target protein. Moreover, docking and MD simulation of the lead A (1,3,8-trihydroxy-4,5-dimethoxyxanthen-9-one; Mol. Wt.: 304.25; CLogP: 3.07) and lead B (5,7-dihydroxy-2-propylchromone; Mol. Wt.: 220.22; CLogP: 3.02), the aglycones of Agri 1 and Agri 2, respectively, were carried out with the target AsnRS. The in silico investigations of the aglycones suggest that the lead B could be a suitable fragment-like lead molecule for anti-filarial drug discovery.

Asparaginyl-tRNA synthetase pre-transfer editing assay.

Aminoacyl-tRNA synthetases (AARSs) are a structurally heterogeneous family of enzymes present in prokaryotes, archaea and eukaryotes. They catalyze the attachment of tRNA to its corresponding amino acid via an aminoacyl adenylate intermediate. Errors in protein synthesis will occur if an incorrect amino acid is attached to the tRNA. To prevent such errors, AARSs have evolved editing mechanisms that eliminate incorrect aminoacyl adenylates (pre-transfer editing) or misacylated tRNAs (post-transfer editing). Various AARSs are the targets of natural antibiotics and are considered validated targets for chemotherapy. We have developed a high-throughput screening (HTS) assay measuring the pre-transfer editing activity of pathogen-derived asparaginyl-tRNA synthetase (AsnRS). This was achieved by monitoring the formation of pyrophosphate via cleavage to phosphate, which was quantified by reaction with Malachite Green. L-Aspartate-ß-hydroxamate, an asparagine analogue, was most effective in promoting the editing activity of AsnRS from Brugia malayi (BmAsnRS) and Staphylococcus epidermidis (SeAsnRS) with KM values close to 100 mM. The assay sensitivity was enhanced by the thiol agents, DTT and L-Cysteine, which significantly increased the turn-over of aminoacyl adenylate by BmAsnRS, but not SeAsnRS. The HTS assay was used to screen a library of 37,120 natural-product extracts for inhibitors of BmAsnRS. A small number of extracts that inhibited the pre-transfer editing by BmAsnRS was identified for future isolation of the active component(s). The principle of this assay can be applied to all enzymes having a pre- or post-editing activity.

Calcium regulates the expression of a Dictyostelium discoideum asparaginyl tRNA synthetase gene.

In a screen for calcium-regulated gene expression during growth and development of Dictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. The ddAsnRS gene shows many unique features. One, it is repressed by lowering cellular calcium, making it the first known calcium-regulated tRNA synthetase. Two, despite the calcium-dependence, its expression is unaltered during the cell cycle, making this the first D. discoideum gene to show a calcium-dependent but cell cycle phase-independent expression. Finally, the N-terminal domain of the predicted ddAsnRS protein shows higher sequence similarity to Glutaminyl tRNA synthetases than to other Asn tRNA synthetases. These unique features of the AsnRS from this primitive eukaryote not only point to a novel mechanism regulating the components of translation machinery and gene expression by calcium, but also hint at a link between the evolution of GlnRS and AsnRS in eukaryotes.

Anomalous X-ray diffraction with soft X-ray synchrotron radiation.

Anomalous diffraction with soft X-ray synchrotron radiation opens new possibilities in protein crystallography and materials science. Low-Z elements like silicon, phosphorus, sulfur and chlorine become accessible as new labels in structural studies. Some of the heavy elements like uranium exhibit an unusually strong dispersion at their M(V) absorption edge (lambdaMV = 3.497 A, E(MV) = 3545 eV) and so does thorium. Two different test experiments are reported here showing the feasibility of anomalous X-ray diffraction at long wavelengths with a protein containing uranium and with a salt containing chlorine atoms. With 110 electrons the anomalous scattering amplitude of uranium exceeds by a factor of 4 the resonance scattering of other strong anomalous scatterers like that of the lanthanides at their L(III) edge. The resulting exceptional phasing power of uranium is most attractive in protein crystallography using the multi-wavelength anomalous diffraction (MAD) method. The anomalous dispersion of an uranium derivative of asparaginyl-tRNA synthetase (hexagonal unit cell; a = 123.4 A, c = 124.4 A) has been measured for the first time at 4 wavelengths near the M(V) edge using the beamline ID1 of ESRF (Grenoble, France). The present set up allowed to measure only 30% of the possible reflections at a resolution of 4 A, mainly because of the low sensitivity of the CCD detector. In the second experiment, the dispersion of the intensity of 5 X-ray diffraction peaks from pentakismethylammonium undecachlorodibismuthate (PMACB, orthorhombic unit cell; a = 13.003 A, b = 14.038 A, c = 15.450 A) has been measured at 30 wavelengths near the K absorption edge of chlorine (lambdaK = 4.397 A, EK= 2819.6 eV). All reflections within the resolution range from 6.4 A to 3.4 A expected in the 20 degree scan were observed. The chemical state varies between different chlorine atoms of PMACB, and so does the dispersion of different Bragg peaks near the K-edge of chlorine. The results reflect the performance of the beamline ID1 of ESRF at wavelengths beyond 3 A at the end of 1998. A gain by a factor 100 for diffraction experiments with 4.4 A photons was achieved in Autumn 1999 when two focusing mirrors had been added to the X-ray optics. Further progress is expected from area detectors more sensitive to soft X-rays. Both CCD detectors and image plates would provide a gain of two orders of measured intensity. Image plates would have the additional advantage that they can be bent cylindrically and thus cover a larger solid angle in reciprocal space. In many cases, samples need to be cooled: closed and open systems are presented. A comparison with the state of art of soft X-ray diffraction, as it had been reached at HASYLAB (Hamburg, Germany), and as it is developing at the Brookhaven National Laboratory (USA), is given.