Circular and L50-like leaderless enterocins share a common ABC-transporter immunity gene

Microbes live within complex communities of interacting populations, either free-living in waters and soils or symbionts of animals and plants. Their interactions include the production of antimicrobial peptides (bacteriocins) to antagonize competitors, and these producers must carry their own immunity gene for self-protection. Whether other coexisting populations are sensitive or resistant to the bacteriocin producer will be key for the population dynamics within the microbial community. The immunity gene frequently consists of an ABC transporter to repel its own bacteriocin but rarely protects against a nonrelated bacteriocin. A case where this cross-resistance occurs mediated by a shared ABC transporter has been shown between enterocins MR10A/B and AS-48. The first is an L50-like leaderless enterocin, while AS-48 is a circular enterocin. In addition, L50-like enterocins such as MR10A/B have been found in E. faecalis and E. faecium, but AS-48 appears only in E. faecalis. Thus, using the ABC transporter of the enterocin MR10A/B gene cluster of Enterococcus faecalis MRR10-3 as a cross-resistance model, we aimed to unravel to what extent a particular ABC transporter can be shared across multiple bacteriocinogenic bacterial populations. To this end, we screened the MR10A/B-ABC transporters in available microbial genomes and analyzed their sequence homologies and distribution. Overall, our main findings are as follows: (i) the MR10A/B-ABC transporter is associated with multiple enterocin gene clusters; (ii) the different enterocins associated with this transporter have a saposin-like fold in common; (iii) the Mr10E component of the transporter is more conserved within its associated enterocin, while the Mr10FGH components are more conserved within the carrying species. This is the least known component of the transporter, but it has shown the greatest specificity to its corresponding enterocin. Bacteriocins are now being investigated as an alternative to antibiotics; hence, the wider or narrower distribution of the particular immunity gene should be taken into account for clinical applications to avoid the selection of resistant strains. Further research will be needed to investigate the mechanistic interactions between the Mr10E transporter component and the bacteriocin as well as the specific ecological and evolutionary mechanisms involved in the spread of the immunity transporter across multiple bacteriocins.Junta de Andalucía A-BIO-083-UGR18, BIO 309Ministerio de Ciencia e Innovación PEJ2018-003019-

Unveiling the Binding between the Armadillo-Repeat Domain of Plakophilin 1 and the Intrinsically Disordered Transcriptional Repressor RYBP

Plakophilin 1 (PKP1), a member of the p120ctn subfamily of the armadillo (ARM)-repeat-containing proteins, is an important structural component of cell–cell adhesion scaffolds although it can also be ubiquitously found in the cytoplasm and the nucleus. RYBP (RING 1A and YY1 binding protein) is a multifunctional intrinsically disordered protein (IDP) best described as a transcriptional regulator. Both proteins are involved in the development and metastasis of several types of tumors. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with RYBP by using in cellulo methods, namely immunofluorescence (IF) and proximity ligation assay (PLA), and in vitro biophysical techniques, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), and isothermal titration calorimetry (ITC). We also characterized the binding of the two proteins by using in silico experiments. Our results showed that there was binding in tumor and non-tumoral cell lines. Binding in vitro between the two proteins was also monitored and found to occur with a dissociation constant in the low micromolar range (~10 μM). Finally, in silico experiments provided additional information on the possible structure of the binding complex, especially on the binding ARM-PKP1 hot-spot. Our findings suggest that RYBP might be a rescuer of the high expression of PKP1 in tumors, where it could decrease the epithelial–mesenchymal transition in some cancer cells

The armadillo-repeat domain of plakophilin 1 binds the C-terminal sterile alpha motif (SAM) of p73

12 pags, 7 figs. -- Supplementary material includes five figures containing: (i) the fluorescence spectrum of the mixture containing prothymosin α and ARM-PKP1 (Fig. S1); (ii) the fluorescence titration curve of SAMp73 and ARM-PKP1 in the presence of 100 mM NaCl (Fig. S2); (iii) the 2D 15N, 1H- HSQC-NMR spectra of isolated SAMp73 and in the presence of ARM-PKP1 in 100 mM NaCl; (iv) binding of ARM-PKP1 and SAMp73 as monitored by ITC in 100 mM of NaCl (Fig. S4); and (v) the fluorescence titrations of the fragments α1α2 and α4α5 with ARM-PKP1 (Fig. S5). Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbagen.2021.129914Plakophilin 1 (PKP1) is a component of desmosomes, which are key structural components for cell-cell adhesion, and can also be found in other cell locations. The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing crucial roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); such domain, SAMp73, is involved in the interaction with other macromolecules.This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN; BFU2016-78232-P to AVC; BES-2017-080739 to DOA]; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, Investing in your future') [PI15/00663 and PI18/00349 to OA]; PAIDI program, Group BIO309 (Junta de Andalucia) to MEF-V; Diputacion General de Aragon [Protein Targets and Bioactive Compounds Group E45_20R to AVC, and Digestive Pathology Group B25_20R to OA]; and Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd).Peer reviewe

Progressive changes in composition of lymphocytes in lung tissues from patients with non-small-cell lung cancer.

Immune cell infiltration is a common feature of many human solid tumors. Innate and adaptative immune systems contribute to tumor immunosurveillance. We investigated whether tumors evade immune surveillance by inducing states of tolerance and/or through the inability of some immune subpopulations to effectively penetrate tumor nests. Immunohistochemistry and flow cytometry analysis were used to study the composition and distribution of immune subpopulations in samples of peripheral blood, tumor tissue (TT), adjacent tumor tissue (ATT), distant non-tumor tissue (DNTT), cancer nests, cancer stroma, and invasive margin in 61 non-small-cell lung cancer (NSCLC) patients. A significantly higher percentage of T and B cells and significantly lower percentage of NK cells were detected in TT than in DNTT. Memory T cells (CD4+CD45RO+, CD8+CD45RO+) and activated T cells (CD8+DR+) were more prevalent in TT. Alongside this immune activation, the percentage of T cells with immunosuppressive activity was higher in TT than in DNTT. B- cells were practically non-existent in tumor nests and were preferentially located in the invasive margin. The dominant NK cell phenotype in peripheral blood and DNTT was the cytotoxic phenotype (CD56+ CD16+), while the presence of these cells was significantly decreased in ATT and further decreased in TT. Finally, the immunologic response differed between adenocarcinoma and squamous cell carcinoma and according to the tumor differentiation grade. These findings on the infiltration of innate and adaptative immune cells into tumors contribute to a more complete picture of the immune reaction in NSCLC

The isolated armadillo-repeat domain of Plakophilin 1 is a monomer in solution with a low conformational stability.

Plakophilin 1 (PKP1) is a member of the armadillo repeat family of proteins. It serves as a scaffold component of desmosomes, which are key structural components for cell-cell adhesion. We have embarked on the biophysical and conformational characterization of the ARM domain of PKP1 (ARM-PKP1) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC)). ARM-PKP1 was a monomer in solution at physiological pH, with a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by fluorescence and CD. The presence or absence of disulphide bridges did not affect its low stability. The protein unfolded through an intermediate which has lost native-like secondary structure. ARM-PKP1 acquired a native-like structure in a narrow pH range (between pH 6.0 and 8.0), indicating that its adherent properties might only work in a very narrow pH range

PKP1 and MYC create a feedforward loop linking transcription and translation in squamous cell lung cancer

[Purpose] Plakophilin 1 (PKP1) is well-known as an important component of the desmosome, a cell structure specialized in spot-like cell-to-cell adhesion. Although desmosomes have generally been associated with tumor suppressor functions, we recently found that PKP1 is recurrently overexpressed in squamous cell lung cancer (SqCLC) to exert an oncogenic role by enhancing the translation of MYC (c-Myc), a major oncogene. In this study, we aim to further characterize the functional relationship between PKP1 and MYC.[Methods] To determine the functional relationship between PKP1 and MYC, we performed correlation analyses between PKP1 and MYC mRNA expression levels, gain/loss of function models, chromatin immunoprecipitation (ChIP) and promoter mutagenesis followed by luciferase assays.[Results] We found a significant correlation between the mRNA levels of MYC and PKP1 in SqCLC primary tumor samples. In addition, we found that MYC is a direct transcription factor of PKP1 and binds to specific sequences within its promoter. In agreement with this, we found that MYC knockdown reduced PKP1 protein expression in different SqCLC models, which may explain the PKP1-MYC correlation that we found. Conversely, we found that PKP1 knockdown reduced MYC protein expression, while PKP1 overexpression enhanced MYC expression in these models.[Conclusions] Based on these results, we propose a feedforward functional relationship in which PKP1 enhances MYC translation in conjunction with the translation initiation complex by binding to the 5’-UTR of MYC mRNA, whereas MYC promotes PKP1 transcription by binding to its promoter. These results suggest that PKP1 may serve as a therapeutic target for SqCLC.The CTS-993 group is funded by the Ministry of Economy of Spain (SAF2015-67919-R), the Junta de Andalucía (Pl-0245-2017, PIGE-0440-2019, PI-0135-2020, PIGE-0213-2020, P20-00688), the Plan propio de la Universidad de Granada (PPJIA2019-06, and B‐CTS‐126‐UGR18), an International Association for the Study of Lung Cancer (IASLC) Young Investigator Award 2017, and the Spanish Association for Cancer Research (LAB-AECC-2018). L.B, J.M-P, and D.J.G. were supported by a “Fundación Benéfica Anticáncer Santa Cándida y San Francisco Javier” predoctoral fellowship. L.B. is currently funded by the Ministry of Health and Social Welfare of Junta de Andalucía (RH-0051-2020). J.C.A.-P is a Marie Curie Postdoctoral Researcher (European Commission. H2020-MSCA-IF-2018 #837897). P.P. was supported by a PhD “La Caixa Foundation” LCF/BQ/DE15/10360019 Fellowship. A.A, A.M.A. and F.R-S were supported by a Spanish Ministry of Education, Culture and Sports FPU fellowship: FPU17/00067, FPU17/01258, and FPU19/05124.Peer reviewe