Bacterial flagella hijack type IV pili proteins to control motility.

Bacterial flagella and type IV pili (TFP) are surface appendages that enable motility and mechanosensing through distinct mechanisms. These structures were previously thought to have no components in common. Here, we report that TFP and some flagella share proteins PilO, PilN, and PilM, which we identified as part of the flagellar motor.

Suppression of MR1 by human cytomegalovirus inhibits MAIT cell activation.

Introduction: The antigen presentation molecule MHC class I related protein-1 (MR1) is best characterized by its ability to present bacterially derived metabolites of vitamin B2 biosynthesis to mucosal-associated invariant T-cells (MAIT cells).

Methods: Through in vitro human cytomegalovirus (HCMV) infection in the presence of MR1 ligand we investigate the modulation of MR1 expression. Using coimmunoprecipitation, mass spectrometry, expression by recombinant adenovirus and HCMV deletion mutants we investigate HCMV gpUS9 and its family members as potential regulators of MR1 expression.

The human cytomegalovirus decathlon: Ten critical replication events provide opportunities for restriction.

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen that can cause severe disease in immunocompromised individuals, transplant recipients, and to the developing foetus during pregnancy. There is no protective vaccine currently available, and with only a limited number of antiviral drug options, resistant strains are constantly emerging. Successful completion of HCMV replication is an elegant feat from a molecular perspective, with both host and viral processes required at various stages.

UL49 is an essential subunit of the viral pre-initiation complex that regulates human cytomegalovirus gene transcription.

More than half the world's population is infected with human cytomegalovirus (HCMV), causing congenital birth defects and impacting the immuno-compromised. Many of the >170 HCMV genes remain uncharacterized, and this gap in knowledge limits the development of novel antivirals. In this study, we investigated the essential viral protein UL49 and found it displayed leaky late expression kinetics, and localized to nuclear replication compartments.

A Highly Specific Holin-Mediated Mechanism Facilitates the Secretion of Lethal Toxin TcsL in .

Protein secretion is generally mediated by a series of distinct pathways in bacteria. Recently, evidence of a novel bacterial secretion pathway involving a bacteriophage-related protein has emerged. TcdE, a holin-like protein encoded by toxigenic isolates of , mediates the release of the large clostridial glucosylating toxins (LCGTs), TcdA and TcdB, and TpeL from uses another holin-like protein, TpeE, for its secretion; however, it is not yet known if TcdE or TpeE secretion is specific to these proteins.

The host exosome pathway underpins biogenesis of the human cytomegalovirus virion.

Human Cytomegalovirus (HCMV) infects over half the world's population, is a leading cause of congenital birth defects, and poses serious risks for immuno-compromised individuals. To expand the molecular knowledge governing virion maturation, we analysed HCMV virions using proteomics, and identified a significant proportion of host exosome constituents. To validate this acquisition, we characterized exosomes released from uninfected cells, and demonstrated that over 99% of the protein cargo was subsequently incorporated into HCMV virions during infection.

Herpesviruses hijack host exosomes for viral pathogenesis.

Herpesviruses are remarkable pathogens possessing elaborate mechanisms to seize various host cellular components for immune evasion, replication, and virion egress. As viruses are dependent upon their hosts, investigating this intricate interplay has revealed that the exosome pathway is utilised by alpha (Herpes Simplex Virus 1), beta (Human Cytomegalovirus, and Human Herpesvirus 6) and gamma (Epstein-Barr Virus, and Kaposi Sarcoma-associated Herpesvirus) herpesviruses. Virions and exosomes share similar properties and functions.

Extracellular vesicles: their role in cancer biology and epithelial-mesenchymal transition.

Cell-cell communication is critical across an assortment of physiological and pathological processes. Extracellular vesicles (EVs) represent an integral facet of intercellular communication largely through the transfer of functional cargo such as proteins, messenger RNAs (mRNAs), microRNA (miRNAs), DNAs and lipids. EVs, especially exosomes and shed microvesicles, represent an important delivery medium in the tumour micro-environment through the reciprocal dissemination of signals between cancer and resident stromal cells to facilitate tumorigenesis and metastasis.

A Portrait of the Human Organelle Proteome In Space and Time during Cytomegalovirus Infection.

The organelles within a eukaryotic host are manipulated by viruses to support successful virus replication and spread of infection, yet the global impact of viral infection on host organelles is poorly understood. Integrating microscopy, subcellular fractionation, mass spectrometry, and functional analyses, we conducted a cell-wide study of organelles in primary fibroblasts throughout the time course of human cytomegalovirus (HCMV) infection. We used label-free and isobaric-labeling proteomics to characterize nearly 4,000 host and 100 viral proteins, then classified their specific subcellular locations over time using machine learning.

Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly.

Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered.

Transformed MDCK cells secrete elevated MMP1 that generates LAMA5 fragments promoting endothelial cell angiogenesis.

Epithelial-mesenchymal transition (EMT) enhances the migration and invasion of cancer cells, and is regulated by various molecular mechanisms including extracellular matrix metalloproteinase (MMP) activity. Previously, we reported transformation of epithelial Madin-Darby canine kidney (MDCK) cells with oncogenic H-Ras (21D1 cells) induces EMT, and significantly elevates MMP1 expression. To explore the biological significance, in this study we characterized 21D1 cells with knocked-down MMP1 expression (21D1(-MMP1)).

Identification of Sirtuin4 (SIRT4) Protein Interactions: Uncovering Candidate Acyl-Modified Mitochondrial Substrates and Enzymatic Regulators.

Recent studies have highlighted the three mitochondrial human sirtuins (SIRT3, SIRT4, and SIRT5) as critical regulators of a wide range of cellular metabolic pathways. A key factor to understanding their impact on metabolism has been the discovery that, in addition to their ability to deacetylate substrates, mitochondrial sirtuins can have other prominent enzymatic activities. SIRT4, one of the least characterized mitochondrial sirtuins, was shown to be the first known cellular lipoamidase, removing lipoyl modifications from lysine residues of substrates.

Oncogenic epithelial cell-derived exosomes containing Rac1 and PAK2 induce angiogenesis in recipient endothelial cells.

The metastatic cascade describes the escape of primary tumour cells to distant secondary sites. Cells at the leading tumour edge are thought to undergo epithelial-mesenchymal transition (EMT), to enhance their motility and invasion for spreading. Whether EMT cells directly promote tumour angiogenesis, and the role of exosomes (30-150 nm extracellular vesicles) remains largely unknown.

YBX1/YB-1 induces partial EMT and tumourigenicity through secretion of angiogenic factors into the extracellular microenvironment.

Epithelial-mesenchymal transition (EMT) describes a morphogenetic program which confers mesenchymal cell properties, such as reduced cell-cell contact and increased cell migration and invasion, to epithelial cells. Here we investigate the role of the pleiotropic transcription/splicing factor and RNA-binding protein nuclease-sensitive element-binding protein 1 (YBX1/YB-1) in increasing the oncogenic potential of epithelial MDCK cells. Characterization of MDCK cells expressing YBX1 (MDCKYBX1 cells) revealed a partial EMT phenotype, including cytosolic relocalization of E-cadherin, increased cell scattering, and anchorage-independent growth.

Emerging roles of exosomes during epithelial-mesenchymal transition and cancer progression.

Epithelial-mesenchymal transition (EMT) is a highly conserved process defined by the loss of epithelial characteristics, and acquisition of the mesenchymal phenotype. In addition to its central role in development, EMT has been implicated as a cellular process during tumourigenesis which facilitates tumour cell invasion and metastasis. The EMT process has been largely defined by signal transduction networks and transcriptional factors that activate mesenchymal-associated gene expression.

Post-translational modifications regulate class IIa histone deacetylase (HDAC) function in health and disease.

Class IIa histone deacetylases (HDACs4, -5, -7, and -9) modulate the physiology of the human cardiovascular, musculoskeletal, nervous, and immune systems. The regulatory capacity of this family of enzymes stems from their ability to shuttle between nuclear and cytoplasmic compartments in response to signal-driven post-translational modification. Here, we review the current knowledge of modifications that control spatial and temporal histone deacetylase functions by regulating subcellular localization, transcriptional functions, and cell cycle-dependent activity, ultimately impacting on human disease.

Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.

Sirtuins (SIRTs) are critical enzymes that govern genome regulation, metabolism, and aging. Despite conserved deacetylase domains, mitochondrial SIRT4 and SIRT5 have little to no deacetylase activity, and a robust catalytic activity for SIRT4 has been elusive. Here, we establish SIRT4 as a cellular lipoamidase that regulates the pyruvate dehydrogenase complex (PDH).

Probing phosphorylation-dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5).

Class IIa histone deacetylases (HDACs) are critical transcriptional regulators, shuttling between nuclear and cytoplasmic cellular compartments. Within the nucleus, these HDACs repress transcription as components of multiprotein complexes, such as the nuclear corepressor and beclin-6 corepressor (BCoR) complexes. Cytoplasmic relocalization relieves this transcriptional repressive function.

Oncogenic H-ras reprograms Madin-Darby canine kidney (MDCK) cell-derived exosomal proteins following epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles following EMT, we conducted a proteomic analysis of exosomes released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells).

Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids.

Exosomes are naturally occurring biological nanomembranous vesicles (∼40 to 100 nm) of endocytic origin that are released from diverse cell types into the extracellular space. They have pleiotropic functions such as antigen presentation and intercellular transfer of protein cargo, mRNA, microRNA, lipids, and oncogenic potential. Here we describe the isolation, via sequential immunocapture using anti-A33- and anti-EpCAM-coupled magnetic beads, of two distinct populations of exosomes released from organoids derived from human colon carcinoma cell line LIM1863.

Contribution of cells undergoing epithelial-mesenchymal transition to the tumour microenvironment.

Human cancer mortality is predominantly determined by the spread of tumour cells from a primary to secondary anatomical location. The metastatic cascade follows tumour cell dissemination, passage through the blood and/or lymphatic system, and colonisation at a distant site. Increased cell motility of cancer cells at the leading tumour edge has been attributed to the epithelial-mesenchymal transition (EMT) which facilitates their release and invasiveness.

Restoration of full-length APC protein in SW480 colon cancer cells induces exosome-mediated secretion of DKK-4.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are common in both inherited and sporadic forms of colorectal cancer (CRC), and are associated with dysregulated Wnt signaling. Colon carcinoma SW480 cells restored with stable expression of wild-type APC (SW480APC cells) exhibit attenuated Wnt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW480 and SW480APC cells to examine the effects of restored APC on exosome protein expression.

Proteomic profiling of the epithelial-mesenchymal transition using 2D DIGE.

Metastasis remains the primary cause of cancer patient death. Although the precise molecular mechanisms at play remain largely unknown, tumor progression is currently hypothesized to follow a series of sequential steps known as the metastatic cascade. An important component, thought to be involved early in this cascade, is the process known as epithelial-mesenchymal transition (EMT), whereby epithelial cells undergo morphogenetic alterations and acquire properties typical of mesenchymal cells.

Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes.

Exosomes are 40-100nm extracellular vesicles that are released from a multitude of cell types, and perform diverse cellular functions including intercellular communication, antigen presentation, and transfer of oncogenic proteins as well as mRNA and miRNA. Exosomes have been purified from biological fluids and in vitro cell cultures using a variety of strategies and techniques. However, all preparations invariably contain varying proportions of other membranous vesicles that co-purify with exosomes such as shed microvesicles and apoptotic blebs.