Transient interactions of a slow-folding protein with the Hsp70 chaperone machinery

Most known proteins have at least one local Hsp70 chaperone binding site. Does this mean that all proteins interact with Hsp70 as they fold? This study makes an initial step to address the above question by examining the interaction of the E.coli Hsp70 chaperone (known as DnaK) and its co-chaperones DnaJ and GrpE with a slow-folding E.coli substrate, RNase H^D. Importantly, this protein is a nonobligatory client, and it is able to fold in vitro even in the absence of chaperones. We employ stopped-flow mixing, chromatography, and activity assays to analyze the kinetic perturbations induced by DnaK/DnaJ/GrpE (K/J/E) on the folding of RNase H^D. We find that K/J/E slows down RNase H^D''s apparent folding, consistent with the presence of transient chaperone-substrate interactions. However, kinetic retardation is moderate for this slow-folding client and it is expected to be even smaller for faster-folding substrates. Given that the interaction of folding-competent substrates such as RNase H^D with the K/J/E chaperones is relatively short-lived, it does not significantly interfere with the timely production of folded biologically active substrate. The above mode of action is important because it preserves K/J/E bioavailability, enabling this chaperone system to act primarily by assisting the folding of other misfolded and (or) aggregation-prone cellular proteins that are unable to fold independently. When refolding is carried out in the presence of K/J and absence of the nucleotide exchange factor GrpE, some of the substrate population becomes trapped as a chaperone-bound partially unfolded state.     

Mining the gastric cancer secretome: identification of GRN as a potential diagnostic marker for early gastric cancer

Gastric cancer is the second leading cause of cancer deaths worldwide, and currently, there are no clinically relevant biomarkers for gastric cancer diagnosis or prognosis. In this study, we applied a 2D-LC-MS/MS based approach, in combination with iTRAQ labeling, to study the secretomes of the gastric cancer cell lines AGS and MKN7. By performing a comparative analysis between the conditioned media and the whole cell lysates, our workflow allowed us to differentiate the bona fide secreted proteins from the intracellular contaminants within the conditioned media. Ninety proteins were found to have higher abundance in the conditioned media as compared to the whole cell lysates of AGS and MKN7 cells. Using a signal peptide and nonclassical secretion prediction tool and an online exosome database, we demonstrated that up to 92.2% of these 90 proteins can be exported out of the cells by classical or nonclassical secretory pathways. We then performed quantitative comparisons of the secretomes between AGS and MKN7, identifying 43 differentially expressed secreted proteins. Among them, GRN was found to be frequently expressed in gastric tumor tissues, but not in normal gastric epithelia by immunohistochemistry. Sandwich ELISA assay also showed elevation of serum GRN levels in gastric cancer patients, particularly those with early gastric cancer. Receiver operating characteristic (ROC) curves analysis confirmed that serum GRN can provide diagnostic discriminations for gastric cancer patients.     

Identification of bartonella trw host-specific receptor on erythrocytes

Each Bartonella species appears to be highly adapted to one or a limited number of reservoir hosts, in which it establishes long-lasting intraerythrocytic bacteremia as the hallmark of infection. Recently, we identified Trw as the bacterial system involved in recognition of erythrocytes according to their animal origin. The T4SS Trw is characterized by a multiprotein complex that spans the inner and outer bacterial membranes, and possesses a hypothetical pilus structure. TrwJ, I, H and trwL are present in variable copy numbers in different species and the multiple copies of trwL and trwJ in the Bartonella trw locus are considered to encode variant forms of surface-exposed pilus components. We therefore aimed to identify which of the candidate Trw pilus components were located on the bacterial surface and involved in adhesion to erythrocytes, together with their erythrocytic receptor. Using different technologies (electron microscopy, phage display, invasion inhibition assay, far western blot), we found that only TrwJ1 and TrwJ2 were expressed and localized at the cell surface of B. birtlesii and had the ability to bind to mouse erythrocytes, and that their receptor was band3, one of the major outer-membrane glycoproteins of erythrocytes, (anion exchanger). According to these results, we propose that the interaction between TrwJ1, TrwJ2 and band 3 leads to the critical host-specific adherence of Bartonella to its host cells, erythrocytes.     

Antibody repertoires in humanized NOD-scid-IL2Rγ(null) mice and human B cells reveals human-like diversification and tolerance checkpoints in the mouse

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγ^null engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγ^null mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential D_H-J_H pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.     

BDNF‐mediated migration of cardiac microvascular endothelial cells is impaired during ageing

This study indicates that brain‐derived neurotrophic factor (BDNF) can promote young cardiac microvascular endothelial cells (CMECs) to migrate via the activation of the BDNF‐TrkB‐FL‐PI3K/Akt pathway, which may benefit angiogenesis after myocardial infarction (MI). However, the ageing of CMECs led to changes in the expression of receptor Trk isoforms in that among the three isoforms (TrkB‐FL, TrkB‐T1 and TrkB‐T2), only one of its truncated isoforms, TrkB‐T1, continued to be expressed, which leads to the dysfunction of its ligand, a decrease in the migration of CMECs and increased injury in ageing hearts. This shift in receptor isoforms in aged CMECs, together with changes in the ageing microenvironment, might predispose ageing hearts to decreased angiogenic potential and increased cardiac pathology.     

Impaired hepatitis C virus (HCV)-specific effector CD8+ T cells undergo massive apoptosis in the peripheral blood during acute HCV infection and in the liver during the chronic phase of infection

A majority of patients infected with hepatitis C virus (HCV) do not sustain an effective T-cell response, and viremia persists. The mechanism leading to failure of the HCV-specific CD8⁺ T-cell response in patients developing chronic infection is unclear. We investigated apoptosis susceptibility of HCV-specific CD8⁺ T cells during the acute and chronic stages of infection. Although HCV-specific CD8⁺ T cells in the blood during the acute phase of infection and in the liver during the chronic phase were highly activated and expressed an effector phenotype, the majority was undergoing apoptosis. In contrast, peripheral blood HCV-specific CD8⁺ T cells during the chronic phase expressed a resting memory phenotype. Apoptosis susceptibility of HCV-specific CD8⁺ T cells was associated with very high levels of programmed death-1 (PD-1) and low CD127 expression and with significant functional T-cell deficits. Further evaluation of the “death phase” of HCV-specific CD8⁺ T cells during acute HCV infection showed that the majority of cells were dying by a process of cytokine withdrawal, mediated by activated caspase 9. Contraction during the acute phase occurred rapidly via this process despite the persistence of the virus. Remarkably, in the chronic phase of HCV infection, at the site of infection in the liver, a substantial frequency of caspase 9-mediated T-cell death was also present. This study highlights the importance of cytokine deprivation-mediated apoptosis with consequent down-modulation of the immune response to HCV during acute and chronic infections.     

Charting epilepsy by searching for intelligence in network space with the help of evolving autonomous agents

The problem of demarcating neural network space is formidable. A simple fully connected recurrent network of five units (binary activations, synaptic weight resolution of 10) has 3.2 *10(26) possible initial states. The problem increases drastically with scaling. Here we consider three complementary approaches to help direct the exploration to distinguish epileptic from healthy networks. [1] First, we perform a gross mapping of the space of five-unit continuous recurrent networks using randomized weights and initial activations. The majority of weight patterns (>70%) were found to result in neural assemblies exhibiting periodic limit-cycle oscillatory behavior. [2] Next we examine the activation space of non-periodic networks demonstrating that the emergence of paroxysmal activity does not require changes in connectivity. [3] The next challenge is to focus the search of network space to identify networks with more complex dynamics. Here we rely on a major available indicator critical to clinical assessment but largely ignored by epilepsy modelers, namely: behavioral states. To this end, we connected the above network layout to an external robot in which interactive states were evolved. The first random generation showed a distribution in line with approach [1]. That is, the predominate phenotypes were fixed-point or oscillatory with seizure-like motor output. As evolution progressed the profile changed markedly. Within 20 generations the entire population was able to navigate a simple environment with all individuals exhibiting multiply-stable behaviors with no cases of default locked limit-cycle oscillatory motor behavior. The resultant population may thus afford us a view of the architectural principles demarcating healthy biological networks from the pathological. The approach has an advantage over other epilepsy modeling techniques in providing a way to clarify whether observed dynamics or suggested therapies are pointing to computational viability or dead space.     

Salmonella enters a dormant state within human epithelial cells for persistent infection

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. S. Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinct S. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella INtracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella-containing vacuoles (SCV) as well as the previously reported niche of dormant S. Typhimurium in macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche and provides an alternative strategy for S. Typhimurium pathogenicity and its persistence.