Host cell deformability is linked to transmission in the human malaria parasite Plasmodium falciparum

Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over 2 weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modelling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together, we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long-term survival of the parasite.     

The crystal structure of Escherichia coli spermidine synthase SpeE reveals a unique substrate-binding pocket

Polyamines are essential in all branches of life. Biosynthesis of spermidine, one of the most ubiquitous polyamines, is catalyzed by spermidine synthase (SpeE). Although the function of this enzyme from Escherichia coli has been thoroughly characterised, its structural details remain unknown. Here, we report the crystal structure of E. coli SpeE and study its interaction with the ligands by isothermal titration calorimetry and computational modelling. SpeE consists of two domains – a small N-terminal β-strand domain, and a C-terminal catalytic domain that adopts a canonical methyltransferase (MTase) Rossmann fold. The protein forms a dimer in the crystal and in solution. Structural comparison of E. coli SpeE to its homologs reveals that it has a large and unique substrate-binding cleft that may account for its lower amine substrate specificity.     

Stretching and Relaxation of Malaria-Infected Red Blood Cells

The invasion of red blood cells (RBCs) by malaria parasites is a complex dynamic process, in which the infected RBCs gradually lose their deformability and their ability to recover their original shape is greatly reduced with the maturation of the parasites. In this work, we developed two types of cell model, one with an included parasite, and the other without an included parasite. The former is a representation of real malaria-infected RBCs, in which the parasite is treated as a rigid body. In the latter, where the parasite is absent, the membrane modulus and viscosity are elevated so as to produce the same features present in the parasite model. In both cases, the cell membrane is modeled as a viscoelastic triangular network connected by wormlike chains. We studied the transient behaviors of stretching deformation and shape relaxation of malaria-infected RBCs based on these two models and found that both models can generate results in agreement with those of previously published studies. With the parasite maturation, the shape deformation becomes smaller and smaller due to increasing cell rigidity, whereas the shape relaxation time becomes longer and longer due to the cell’s reduced ability to recover its original shape.     

Unexpected X chromosome skewing during culture and reprogramming of human somatic cells can be alleviated by exogenous telomerase

Somatic tissues in female eutherian mammals are mosaic due to random X inactivation. In contrast to mice, X chromosome reactivation does not occur during the reprogramming of human female somatic cells to induced pluripotent stem cells (iPSCs), although this view is contested. Using balanced populations of female Rett patient and control fibroblasts, we confirm that all cells in iPSC colonies contain an inactive X, and additionally find that all colonies made from the same donor fibroblasts contain the same inactive X chromosome. Notably, this extreme "skewing" toward a particular dominant, active X is also a general feature of primary female fibroblasts during proliferation, and the skewing seen in reprogramming and fibroblast culture can be alleviated by overexpression of telomerase. These results have important implications for in?vitro modeling of X-linked diseases and the interpretation of long-term culture studies in cancer and senescence using primary female fibroblast cell lines.     

IL-17A expression is localised to both mononuclear and polymorphonuclear synovial cell infiltrates

Introduction

This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.

Methods

IL-17A expression was quantified in synovial tissue (ST), serum and synovial fluid (SF) by immunohistochemistry and MSD-plex assays. IL-6 SF and serum levels were measured by MSD-plex assays. Dual immunofluorescence for IL-17A was quantified in ST CD15+ cells (neutrophils), Tryptase+ (mast cells) and CD4+ (T cells). Synovial tissue oxygen (tpO₂) levels were measured under direct visualisation at arthroscopy. Synovial infiltration was assessed using immunohistochemistry for cell specific markers. Peripheral blood mononuclear and polymorphonuclear cells were isolated and exposed to normoxic or 3% hypoxic conditions. IL-17A and IL-6 were quantified as above in culture supernatants.

Results

IL-17A expression was localised to mononuclear and polymorphonuclear (PMN) cells in inflamed ST. Dual immunoflourescent staining co-localised IL-17A expression with CD15+ neutrophils Tryptase+ mast cells and CD4+T cells. % IL-17A positivity was highest on CD15+ neutrophils, followed by mast cells and then CD4+T-cells. The number of IL-17A-secreting PMN cells significantly correlated with sublining CD68 expression (r = 0.618, p<0.01). IL-17A SF levels correlated with IL-6 SF levels (r = 0.675, p<0.01). Patients categorized according to tp0₂< or >20mmHg, showed those with low tp0₂<20mmHg had significantly higher IL-17A+ mononuclear cells with no difference observed for PMNs. Exposure of mononuclear and polymorphonuclear cells to 3% hypoxia, significantly induced IL-6 in mononuclear cells, but had no effect on IL-17A expression in mononuclear and polymorphonuclear cells.

Conclusion

This study demonstrates IL-17A expression is localised to several immune cell subtypes within the inflamed synovial tissue, further supporting the concept that IL-17A is a key mediator in inflammatory arthritis. The association of hypoxia with Il-17A expression appears to be indirect, probably through hypoxia-induced pro-inflammatory pathways and leukocyte influx within the joint microenvironment.     

A Quantitative Chemical Proteomics Approach to Profile the Specific Cellular Targets of Andrographolide,a Promising Anticancer Agent That Suppresses Tumor Metastasis

Drug target identification is a critical step toward understanding the mechanism of action of a drug, which can help one improve the drug''s current therapeutic regime and expand the drug''s therapeutic potential. However, current in vitro affinity-chromatography-based and in vivo activity-based protein profiling approaches generally face difficulties in discriminating specific drug targets from nonspecific ones. Here we describe a novel approach combining isobaric tags for relative and absolute quantitation with clickable activity-based protein profiling to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. Our findings, validated through cell migration and invasion assays, showed that Andro has a potential novel application as a tumor metastasis inhibitor. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analog synthesis, protein engineering, and mass-spectrometry-based approaches and determined the drug-binding sites of two protein targets, NF-κB and actin.As most drugs exert pharmacological effects by interacting with their target proteins, the identification of these target proteins is a critical step in unraveling the mechanisms of drug action. It is also imperative for our understanding of the pharmacodynamics of a known drug, suggesting potentially unrevealed actions and thus refining future clinical applications of the substance. Traditional approaches used to identify protein targets of a drug typically utilize immobilized drug affinity chromatography coupled with mass spectrometry (MS)¹ (1, 2). These methods can be applied to cell lysates, but not in an in vivo setting, because of the requirement of a solid support. In vitro target profiling might not accurately reflect the drug''s actions in the in vivo physiological environment. To overcome this limitation, several groups have used activity-based protein profiling (ABPP) combined with bio-orthogonal click chemistry to identify drug targets both in vitro and in vivo (supplemental Fig. S1) (3–15). ABPP probes exert their functions via covalent reactions with the target proteins or photoaffinity-based labeling via the incorporation of photoreactive groups. With the increasing sensitivity of modern MS platforms, low-abundance protein targets can be successfully identified. Although both conventional affinity chromatography and recent ABPP-based methods allow us to detect a set of candidate protein targets for a drug, it remains difficult to discriminate specific interactions from nonspecific ones. Thus, more time and effort are needed for subsequent validation because of the presence of a large number of nonspecific binders. Therefore, there is an urgent need to develop comprehensive unbiased methods for specific target identification. Quantitative proteomics has been used to profile enriched kinases using cell-lysate-based kinobead pull-down. However, these types of experiments are mainly suitable for studying kinase inhibitors (16). Recently, proteomics methods based on stable isotope labeling of amino acids in cell culture (SILAC) have been applied to determine the specific binders of small molecules or proteins with certain post-translational modifications (17–19). These studies have shed light on how quantitative proteomics can improve the specificity of target protein identification. Nevertheless, because of the inherent limitations of SILAC, the complete incorporation of isotopic amino acids via such an approach takes a long time. Furthermore, it is also extremely difficult to apply the SILAC approach to tissue and body fluid samples, which are of particular relevance to biomedical research.Here we introduce a clickable activity-based protein profiling (ICABPP) approach based on the use of isobaric tags for relative and absolute quantitation (iTRAQ) for the unbiased specific and comprehensive identification of target proteins in live cells. iTRAQ is a stable isotope labeling approach for multiplexed quantitative proteome profiling (20). An overview of the technique is illustrated in Fig. 1A. In this assay, cells are first incubated with a clickable probe or with DMSO, which serves as a negative control. After the probe permeates the cell, and covalently binds to its dedicated in situ targets, the washed cells are lysed, clicked with biotin-N₃ tag, and enriched through avidin pull-down in parallel. The beads are washed thoroughly, and the bond proteins are directly digested on the beads with trypsin. The resulting peptides are labeled with their respective iTRAQ reagents, pooled together for further identification and quantification via LC-MS/MS. This technique enabled us to discriminate specific protein targets from nonspecific, and endogenously biotinylated proteins. Biological replicates of probe- or DMSO-treated samples are included to overcome experimental variations. As shown in Fig. 1B, nonspecific binding proteins'' iTRAQ reporters have equal or similar intensities, whereas specific target proteins enriched by the probe show highly differential intensities relative to the DMSO-treated control samples (as illustrated by the significantly higher reporter intensities of 116 and 117 versus 113 and 114 shown in Fig. 1B). The multiplexing nature of the iTRAQ-based chemical proteomics method allows replicated enrichments to be compared within a single LC-MS/MS analysis, thereby increasing the accuracy of specific target identifications, and minimizing experimental errors.Open in a separate windowFig. 1.Identifying specific drug targets using ICABPP approach in live cells. A, live cells were treated with DMSO, and clickable probe in duplicate. Cells were lysed and tagged with biotin-N3 using click chemistry in parallel. The biotin-bearing target proteins were enriched through avidin pull-down, and directly digested on beads. The resulting peptides of the two biological replicates of control pulled-down samples were labeled with 113 and 114, respectively, and two probe pulled-down samples were labeled with 116 and 117, respectively. The labeled peptides were combined in order to be identified, and quantified via LC-MS/MS. B, for the nonspecific targets, the iTRAQ reporters showed similar intensities, whereas for the specific targets, the reporters showed highly differential intensities.In this context, the ICABPP approach was applied to identify protein targets of andrographolide (Andro) (Fig. 2), a natural product with known anti-inflammation, and anti-cancer effects (21–25), in live cancer cells. A spectrum of 75 potential Andro targets was identified with high confidence, which suggested that Andro may exert anti-cancer effects by acting on multiple targets to interfere with several cellular signaling pathways. Two targets, NF-κB and β-actin, were validated by in vitro binding assay, and direct binding site mapping. Furthermore, our data revealed a novel mechanism of Andro in suppressing tumor metastasis.Open in a separate windowFig. 2.Chemical structures of Andro, reduced Andro analog RA, and Andro-based clickable ABPP probes P1 and P2.     

Atomic force microscope imaging of chromatin assembled in <Emphasis Type="Italic">Xenopus laevis</Emphasis> egg extract

Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.     

Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

N⁶-Methyladenosine (m⁶A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m⁶A in single-stranded RNA. Here we report crystal structures of ALKBH5 in the presence of either its cofactors or the ALKBH5 inhibitor citrate. Catalytic assays demonstrate that the ALKBH5 catalytic domain can demethylate both single-stranded RNA and single-stranded DNA. We identify the TCA cycle intermediate citrate as a modest inhibitor of ALKHB5 (IC₅₀, ∼488 μm). The structural analysis reveals that a loop region of ALKBH5 is immobilized by a disulfide bond that apparently excludes the binding of dsDNA to ALKBH5. We identify the m⁶A binding pocket of ALKBH5 and the key residues involved in m⁶A recognition using mutagenesis and ITC binding experiments.