ABSTRACT
Introduction: Colorectal cancer (CRC) ranks as the third most prevalent malignancy globally, with a concerning rise in incidence among young adults. Despite progress in understanding genetic predispositions and lifestyle risk factors, the intricate molecular mechanisms of CRC demand exploration. MicroRNAs (miRNAs) emerge as key regulators of gene expression and their deregulation in tumor cells play pivotal roles in cancer progression. Methods: NanoString's nCounter technology was utilized to measure the expression of 827 cancer-related miRNAs in tumor tissue and adjacent non-involved normal colon tissue from five patients with locoregional CRC progression. These expression profiles were then compared to those from the primary colon adenocarcinoma (COAD) cohort in The Cancer Genome Atlas (TCGA). Results and discussion: Intriguingly, 156 miRNAs showed a contrasting dysregulation pattern in reccurent tumor compared to their expression in the TCGA COAD cohort. This observation implies dynamic alterations in miRNA expression patterns throughout disease progression. Our exploratory study contributes to understanding the regulatory landscape of recurrent CRC, emphasizing the role of miRNAs in disease relapse. Notable findings include the prominence of let-7 miRNA family, dysregulation of key target genes, and dynamic changes in miRNA expression patterns during progression. Univariate Cox proportional hazard models highlighted miRNAs associated with adverse outcomes and potential protective factors. The study underscores the need for more extensive investigations into miRNA dynamics during tumor progression and the value of stage specific biomarkers for prognosis.
ABSTRACT
Bacterial plasmids and chromosomes widely contain toxin-antitoxin (TA) loci, which are implicated in stress response, growth regulation and even tolerance to antibiotics and environmental stress. Type I TA systems consist of a stable toxin-expressing mRNA, which is counteracted by an unstable RNA antitoxin. The Long Direct Repeat (LDR-) D locus, a type I TA system of Escherichia Coli (E. coli) K12, encodes a 35 amino acid toxic peptide, LdrD. Despite being characterized as a bacterial toxin, causing rapid killing and nucleoid condensation, little was known about its function and its mechanism of toxicity. Here, we show that LdrD specifically interacts with ribosomes which potentially blocks translation. Indeed, in vitro translation of LdrD-coding mRNA greatly reduces translation efficiency. The structure of LdrD in a hydrophobic environment, similar to the one found in the interior of ribosomes was determined by NMR spectroscopy in 100% trifluoroethanol solution. A single compact α-helix was found which would fit nicely into the ribosomal exit tunnel. Therefore, we conclude that rather than destroying bacterial membranes, LdrD exerts its toxic activity by inhibiting protein synthesis through binding to the ribosomes.
Subject(s)
Antitoxins , Bacterial Toxins , Escherichia coli/genetics , Escherichia coli/metabolism , Bacterial Toxins/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repetitive Sequences, Nucleic Acid , Protein Biosynthesis , Antitoxins/chemistry , Antitoxins/genetics , Antitoxins/metabolism , Bacterial Proteins/chemistryABSTRACT
Tumor-related death is primarily caused by metastasis; consequently, understanding, preventing, and treating metastasis is essential to improving clinical outcomes. Metastasis is mainly governed by the dissemination of tumor cells in the systemic circulation: so-called circulating tumor cells (CTCs). CTCs typically arise from epithelial tumor cells that undergo epithelial-to-mesenchymal transition (EMT), resulting in the loss of cell-cell adhesions and polarity, and the reorganization of the cytoskeleton. Various oncogenic factors can induce EMT, among them the transforming growth factor (TGF)-ß, as well as Wnt and Notch signaling pathways. This entails the activation of numerous transcription factors, including ZEB, TWIST, and Snail proteins, acting as transcriptional repressors of epithelial markers, such as E-cadherin and inducers of mesenchymal markers such as vimentin. These genetic and phenotypic changes ultimately facilitate cancer cell migration. However, to successfully form distant metastases, CTCs must primarily withstand the hostile environment of circulation. This includes adaption to shear stress, avoiding being trapped by coagulation and surviving attacks of the immune system. Several applications of CTCs, from cancer diagnosis and screening to monitoring and even guided therapy, seek their way into clinical practice. This review describes the process leading to tumor metastasis, from the generation of CTCs in primary tumors to their dissemination into distant organs, as well as the importance of subtyping CTCs to improve personalized and targeted cancer therapy.
ABSTRACT
Pancreatic cancer (PC) has a complex and unique tumor microenvironment (TME). Due to the physical barrier formed by the desmoplastic stroma, the delivery of drugs to the tumor tissue is limited. The TME also contributes to resistance to various immunotherapies such as cancer vaccines, chimeric antigen receptor T cell therapy and immune checkpoint inhibitors. Overcoming and/or modulating the TME is therefore one of the greatest challenges in developing new therapeutic strategies for PC. Nanoparticles have been successfully used as drug carriers and delivery systems in cancer therapy. Recent experimental and engineering developments in nanotechnology have resulted in increased drug delivery and improved immunotherapy for PC. In this review we discuss and analyze the current nanoparticle-based immunotherapy approaches that are at the verge of clinical application. Particularly, we focus on nanoparticle-based delivery systems that improve the effectiveness of PC immunotherapy. We also highlight current clinical research that will help to develop new therapeutic strategies for PC and especially targeted immunotherapies based on immune checkpoint inhibitors.
ABSTRACT
Recently, the V-domain immunoglobulin suppressor of T-cell activation (VISTA) was identified as a negative immune checkpoint regulator (NCR) that is mainly expressed in hematopoietic cells. Preclinical studies have shown that VISTA blockade results in impeded tumor growth and improved survival. Nevertheless, little is known about the physiological role of VISTA expression in macrophages. This study focused on the differential expression of VISTA in human monocytes and macrophages in order to elucidate a putative role of VISTA regulation upon macrophage polarization and activation. We observed that human peripheral monocytes constitutively release soluble VISTA, which was regulated via matrix metalloproteinases. However, monocyte stimulation with cytokines that induce macrophage differentiation, such as granulocyte-macrophage colony-stimulating (GM-CSF) and macrophage colony-stimulating factor (M-CSF), substantially reduced soluble VISTA release. VISTA release was further affected by various pro- and anti-inflammatory stimuli that led to macrophage polarization, where activated M1 macrophages generally released more VISTA than M2 macrophages. Additionally, we observed that stimulation of activated macrophages with the toll-like receptor 4 ligand lipopolysaccharide (LPS) led to a further decrease of soluble VISTA release. Moreover, we found that soluble VISTA impairs T cell cytotoxic activity but did not induce their programmed death. Our results suggest that VISTA is constantly produced and released in the peripheral blood where it may contribute to peripheral tolerance.
Subject(s)
Immune Checkpoint Proteins , Lymphocyte Activation , B7-H1 Antigen/metabolism , Cytokines/metabolism , Humans , MacrophagesABSTRACT
Immune checkpoint proteins play crucial roles in human embryonic development but are also used by cancer cells to escape immune surveillance. These proteins and biochemical pathways associated with them form a complex machinery capable of blocking the ability of cytotoxic immune lymphoid cells to attack cancer cells and, ultimately, to fully suppress anti-tumor immunity. One of the more recently discovered immune checkpoint proteins is V-domain Ig-containing suppressor of T cell activation (VISTA), which plays a crucial role in anti-cancer immune evasion pathways. The biochemical mechanisms underlying regulation of VISTA expression remain unknown. Here, we report for the first time that VISTA expression is controlled by the transforming growth factor beta type 1 (TGF-ß)-Smad3 signaling pathway. However, in T lymphocytes, we found that VISTA expression was differentially regulated by TGF-ß depending on their immune profile. Taken together, our results demonstrate the differential biochemical control of VISTA expression in human T cells and various types of rapidly proliferating cells, including cancer cells, fetal cells and keratinocytes.
ABSTRACT
ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a "DNA-catcher" to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.
Subject(s)
Bacterial Proteins/chemistry , DNA-Binding Proteins/chemistry , Transcription Factors/chemistry , Vibrio cholerae/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Helix-Turn-Helix Motifs , Protein Domains , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Vibrio cholerae/genetics , Vibrio cholerae/metabolismABSTRACT
Galectin-9 is a member of the galectin family of proteins, which were first identified to specifically bind to carbohydrates containing ß-galactosides. Galectin-9 is conserved through evolution and recent evidence demonstrated its involvement in innate immune reactions to bacterial infections as well as the suppression of cytotoxic immune responses of T and natural killer cells. However, the molecular mechanisms underlying such differential immunological functions of galectin-9 remain largely unknown. In this work we confirmed that soluble galectin-9 derived from macrophages binds to Gram-negative bacteria by interacting with lipopolysaccharide (LPS), which forms their cell wall. This opsonisation effect most likely interferes with the mobility of bacteria leading to their phagocytosis by innate immune cells. Galectin-9-dependent opsonisation also promotes the innate immune reactions of macrophages to these bacteria and significantly enhances the production of pro-inflammatory cytokines - interleukin (IL) 6, IL-1ß and tumour necrosis factor alpha (TNF-α). In contrast, galectin-9 did not bind peptidoglycan (PGN), which forms the cell wall of Gram-positive bacteria. Moreover, galectin-9 associated with cellular surfaces (studied in primary human embryonic cells) was not involved in the interaction with bacteria or bacterial colonisation. However, galectin-9 expressed on the surface of primary human embryonic cells, as well as soluble forms of galectin-9, were able to target T lymphocytes and caused apoptosis in T cells expressing granzyme B. Furthermore, "opsonisation" of T cells by galectin-9 led to the translocation of phosphatidylserine onto the cell surface and subsequent phagocytosis by macrophages through Tim-3, the receptor, which recognises both galectin-9 and phosphatidylserine as ligands.
Subject(s)
Apoptosis , Escherichia coli/metabolism , Galectins/metabolism , Immunity, Innate , Macrophages/metabolism , Opsonization , T-Lymphocytes/metabolism , Cytokines/metabolism , Escherichia coli/immunology , Escherichia coli/pathogenicity , Granzymes/metabolism , Host-Pathogen Interactions , Humans , Inflammation Mediators/metabolism , Jurkat Cells , Macrophages/immunology , Macrophages/microbiology , Macrophages/pathology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , THP-1 CellsABSTRACT
Acute myeloid leukemia (AML), a blood/bone marrow cancer, is a severe and often fatal malignancy. AML cells are capable of impairing the anti-cancer activities of cytotoxic lymphoid cells. This includes the inactivation of natural killer (NK) cells and killing of T lymphocytes. Here we report for the first time that V-domain Ig-containing suppressor of T cell activation (VISTA), a protein expressed by T cells, recognizes galectin-9 secreted by AML cells as a ligand. Importantly, we found that soluble VISTA released by AML cells enhances the effect of galectin-9, most likely by forming multiprotein complexes on the surface of T cells and possibly creating a molecular barrier. These events cause changes in the plasma membrane potential of T cells leading to activation of granzyme B inside cytotoxic T cells, resulting in apoptosis.
Subject(s)
B7 Antigens/metabolism , Galectins/metabolism , T-Lymphocytes, Cytotoxic/immunology , Antigens, Neoplasm , Apoptosis , Cytotoxicity, Immunologic , Granzymes/metabolism , Humans , Immunosuppression Therapy , Ligands , Membrane Potentials , Protein Binding , Protein Multimerization , THP-1 Cells , Tumor EscapeABSTRACT
The mechanisms by which protein complexes convert from functional to pathogenic are the subject of intensive research. Here, we report how functionally unfavorable protein interactions can be induced by structural fuzziness, i.e., by persisting conformational disorder in protein complexes. We show that extreme disorder in the bound state transforms the intrinsically disordered protein SERF1a from an RNA-organizing factor into a pathogenic enhancer of alpha-synuclein (aSyn) amyloid toxicity. We demonstrate that SERF1a promotes the incorporation of RNA into nucleoli and liquid-like artificial RNA-organelles by retaining an unusually high degree of conformational disorder in the RNA-bound state. However, this type of structural fuzziness also determines an undifferentiated interaction with aSyn. RNA and aSyn both bind to one identical, positively charged site of SERF1a by an analogous electrostatic binding mode, with similar binding affinities, and without any observable disorder-to-order transition. The absence of primary or secondary structure discriminants results in SERF1a being unable to select between nucleic acid and amyloidogenic protein, leading the pro-amyloid aSyn:SERF1a interaction to prevail in the cytosol under conditions of cellular stress. We suggest that fuzzy disorder in SERF1a complexes accounts for an adverse gain-of-interaction which favors toxic binding to aSyn at the expense of nontoxic RNA binding, thereby leading to a functionally distorted and pathogenic process. Thus, structural fuzziness constitutes a direct link between extreme conformational flexibility, amyloid aggregation, and the malfunctioning of RNA-associated cellular processes, three signatures of neurodegenerative proteinopathies.
Subject(s)
Nerve Tissue Proteins/metabolism , RNA/chemistry , alpha-Synuclein/metabolism , Animals , Cytosol/metabolism , Humans , Intrinsically Disordered Proteins/chemistry , Intrinsically Disordered Proteins/metabolism , Mice , Nerve Tissue Proteins/chemistry , Nucleic Acids/chemistry , Protein Binding , RNA/metabolism , Static Electricity , alpha-Synuclein/chemistryABSTRACT
The recent discovery of biologically active fully disordered, so called random fuzzy protein-protein interactions leads to the question of how the high flexibility of these protein complexes correlates to aggregation and pathologic misfolding. We identify the structural mechanism by which a random fuzzy protein complex composed of the intrinsically disordered proteins alpha-Synuclein and SERF1a is able to potentiate cytotoxic aggregation. A structural model derived from an integrated NMR/SAXS analysis of the reconstituted aSyn:SERF1a complex enabled us to observe the partial deprotection of one precise aSyn amyloid nucleation element in the fully unstructured ensemble. This minimal exposure was sufficient to increase the amyloidogenic tendency of SERF1a-bound aSyn. Our findings provide a structural explanation of the previously observed pro-amyloid activity of SERF1a. They further demonstrate that random fuzziness can trigger a structurally organized disease-associated reaction such as amyloid polymerization.
Subject(s)
Amyloid/chemistry , Brain/metabolism , Intrinsically Disordered Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neuroblastoma/metabolism , alpha-Synuclein/chemistry , alpha-Synuclein/metabolism , Amino Acid Sequence , Animals , Brain/cytology , Humans , Intrinsically Disordered Proteins/chemistry , Mice , Mice, Inbred C57BL , Models, Molecular , Nerve Tissue Proteins/chemistry , Neuroblastoma/pathology , Protein Binding , Protein Conformation , Protein Multimerization , Sequence HomologyABSTRACT
Bullous pemphigoid (BP) is characterized by substantial skin and blood eosinophilia as well as intensive pruritus. Since the pruritogenic cytokine interleukin (IL)-31 is increased in inflammatory skin diseases the aim of this study was to determine whether IL-31 plays a role in BP. Using immunofluorescence, IL-31 expression was analysed in eosinophils derived from blister fluids and skin from patients with BP and IL-31 levels in blister fluids, serum and culture supernatants were determined by enzyme-linked immunoassay (ELISA). High levels of IL-31 expression were observed in BP blister fluids, but they were only marginally elevated in BP serum compared with healthy controls. Eosinophils from either BP blister fluids or skin biopsies showed strong expression of IL-31. Furthermore, peripheral blood eosinophils from patients with BP, but not healthy controls, released high levels of IL-31, reflecting those in blister fluids. In conclusion, eosinophils are a major source of IL-31 in BP and this cytokine may contribute to itch in patients with BP.
Subject(s)
Eosinophilia/immunology , Eosinophils/immunology , Interleukins/immunology , Skin Diseases, Vesiculobullous/immunology , Skin/immunology , Urticaria/immunology , Aged , Aged, 80 and over , Case-Control Studies , Cells, Cultured , Eosinophilia/blood , Eosinophilia/diagnosis , Eosinophils/metabolism , Female , Fluorescent Antibody Technique , Humans , Interleukins/blood , Male , Middle Aged , Pruritus/immunology , Skin/metabolism , Skin Diseases, Vesiculobullous/blood , Skin Diseases, Vesiculobullous/diagnosis , Up-Regulation , Urticaria/blood , Urticaria/diagnosisABSTRACT
Nanoparticles have been recognized as promising tools for targeted drug-delivery and protein therapeutics. However, the mechanisms of protein-nanoparticle interaction and the dynamics underlying the binding process are poorly understood. Here, we present a general methodology for the characterization of protein-nanoparticle interaction on a molecular level. To this end we combined biophysical techniques including nuclear magnetic resonance (NMR), circular dichroism (CD), resonance energy transfer (RET) and surface plasmon resonance (SPR). Particularly, we analyzed molecular mechanisms and dynamics of the interaction of CaF2 nanoparticles with the prototypical calcium sensor calmodulin (CaM). We observed the transient formation of an intermediate encounter complex involving the structural region linking the two domains. Specific interaction of CaM with CaF2 NPs is driven by the N-terminal EF-hands, which seem to recognize Ca2+ on the surface of the nanoparticle. We conclude that CaF2 NP-CaM interaction is fully compatible with potential applications in nanomedicine. Overall, the methods presented in this work can be extended to other systems and may be useful to quantitatively characterize structural and dynamic features of protein-NP interactions with important implications for nanomedicine and nano-biotechnology.
Subject(s)
Calcium Fluoride/metabolism , Calmodulin/metabolism , Nanoparticles/metabolism , Calcium Fluoride/chemistry , Calmodulin/chemistry , Circular Dichroism , EF Hand Motifs , Humans , Luminescence , Models, Molecular , Nanoparticles/chemistry , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Conformation , Protein Stability , Surface Plasmon ResonanceABSTRACT
Untreatable bacterial infections caused by a perpetual increase of antibiotic resistant strains represent a serious threat to human healthcare in the 21(st) century. Conjugative DNA transfer is the most important mechanism for antibiotic resistance and virulence gene dissemination among bacteria and is mediated by a protein complex, known as type IV secretion system (T4SS). The core of the T4SS is a multiprotein complex that spans the bacterial envelope as a channel for macromolecular secretion. We report the NMR structure and functional characterization of the transfer protein TraH encoded by the conjugative Gram-positive broad-host range plasmid pIP501. The structure exhibits a striking similarity to VirB8 proteins of Gram-negative secretion systems where they play an essential role in the scaffold of the secretion machinery. Considering TraM as the first VirB8-like protein discovered in pIP501, TraH represents the second protein affiliated with this family in the respective transfer operon. A markerless traH deletion in pIP501 resulted in a total loss of transfer in Enterococcus faecalis as compared with the pIP501 wild type (wt) plasmid, demonstrating that TraH is essential for pIP501 mediated conjugation. Moreover, oligomerization state and topology of TraH in the native membrane were determined providing insights in molecular organization of a Gram-positive T4SS.
Subject(s)
Bacterial Proteins/metabolism , Biological Transport , Conjugation, Genetic , DNA/metabolism , Enterococcus faecalis/metabolism , Gene Transfer, Horizontal , Nuclear Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Enterococcus faecalis/genetics , Gene Deletion , Humans , Magnetic Resonance Spectroscopy , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Plasmids , Protein ConformationABSTRACT
Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Alternative Splicing , DNA-Binding Proteins/metabolism , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Animals , Dimerization , HEK293 Cells , Humans , Male , Mice , Molecular Sequence Data , Nucleotide Motifs , Protein Structure, Tertiary , RNA/metabolism , Structure-Activity RelationshipABSTRACT
Conjugative transfer of DNA represents the most important transmission pathway in terms of antibiotic resistance and virulence gene dissemination among bacteria. TraH is a putative transfer protein of the type IV secretion system (T4SS) encoded by the Gram-positive (G+) conjugative plasmid pIP501. This molecular machine involves a multi-protein core complex spanning the bacterial envelope thereby serving as a macromolecular secretion channel. Here, we report the near complete (1)H, (13)C and (15)N resonance assignment of a soluble TraH variant comprising the C-terminal domain.
Subject(s)
Bacterial Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Enterococcus faecalisABSTRACT
The IL-4-inducing principle from Schistosoma mansoni eggs (IPSE/α-1), the major secretory product of eggs from the parasitic worm S. mansoni, efficiently triggers basophils to release the immunomodulatory key cytokine interleukin-4. Activation by IPSE/α-1 requires the presence of IgE on the basophils, but the detailed molecular mechanism underlying activation is unknown. NMR and crystallographic analysis of IPSEΔNLS, a monomeric IPSE/α-1 mutant, revealed that IPSE/α-1 is a new member of the ßγ-crystallin superfamily. We demonstrate that this molecule is a general immunoglobulin-binding factor with highest affinity for IgE. NMR binding studies of IPSEΔNLS with the 180-kDa molecule IgE identified a large positively charged binding surface that includes a flexible loop, which is unique to the IPSE/α-1 crystallin fold. Mutational analysis of amino acids in the binding interface showed that residues contributing to IgE binding are important for IgE-dependent activation of basophils. As IPSE/α-1 is unable to cross-link IgE, we propose that this molecule, by taking advantage of its unique IgE-binding crystallin fold, activates basophils by a novel, cross-linking-independent mechanism.