Dynamic reorganization of metabolic enzymes into intracellular bodies.

Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci--such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis--to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality--and dysfunctionality--of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable.

ZFAND1 Recruits p97 and the 26S Proteasome to Promote the Clearance of Arsenite-Induced Stress Granules.

Stress granules (SGs) are cytoplasmic assemblies of mRNPs stalled in translation initiation. They are induced by various stress conditions, including exposure to the environmental toxin and carcinogen arsenic. While perturbed SG turnover is linked to the pathogenesis of neurodegenerative diseases, the molecular mechanisms underlying SG formation and turnover are still poorly understood. Here, we show that ZFAND1 is an evolutionarily conserved regulator of SG clearance. ZFAND1 interacts with two key factors of protein degradation, the 26S proteasome and the ubiquitin-selective segregase p97, and recruits them to arsenite-induced SGs. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products, and persist after arsenite removal, indicating their transformation into aberrant, disease-linked SGs. Accordingly, ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder IBMPFD/ALS.

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase.

Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure-biosynthesis-activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

Clues to the function of bacterial microcompartments from ancillary genes.

Bacterial microcompartments (BMCs) are prokaryotic organelles. Their bounding membrane is a selectively permeable protein shell, encapsulating enzymes of specialized metabolic pathways. While the function of a BMC is dictated by the encapsulated enzymes which vary with the type of the BMC, the shell is formed by conserved protein building blocks. The genes necessary to form a BMC are typically organized in a locus; they encode the shell proteins, encapsulated enzymes as well as ancillary proteins that integrate the BMC function into the cell's metabolism. Among these are transcriptional regulators which usually found at the beginning or end of a locus, and transmembrane proteins that presumably function to conduct the BMC substrate into the cell. Here, we describe the types of transcriptional regulators and permeases found in association with BMC loci, using a recently collected data set of more than 7000 BMC loci distributed over 45 bacterial phyla, including newly discovered BMC loci. We summarize the known BMC regulation mechanisms, and highlight how much remains to be uncovered. We also show how analysis of these ancillary proteins can inform hypotheses about BMC function; by examining the ligand-binding domain of the regulator and the transporter, we propose that nucleotides are the likely substrate for an enigmatic uncharacterized BMC of unknown function.

Natural Polymorphisms in Arabidopsis Result in Wide Variation or Loss of the Amylose Component of Starch.

Starch granules contain two Glc polymers, amylopectin and amylose. Amylose makes up approximately 10% to 30% (w/w) of all natural starches thus far examined, but mutants of crop and model plants that produce amylose-free starch are generally indistinguishable from their wild-type counterparts with respect to growth, starch content, and granule morphology. Since the function and adaptive significance of amylose are unknown, we asked whether there is natural genetic variation in amylose synthesis within a wild, uncultivated species. We examined polymorphisms among the 1,135 sequenced accessions of Arabidopsis (Arabidopsis thaliana) in GRANULE-BOUND STARCH SYNTHASE (GBSS), encoding the enzyme responsible for amylose synthesis. We identified 18 accessions that are predicted to have polymorphisms in GBSS that affect protein function, and five of these accessions produced starch with no or extremely low amylose (< 0.5% [w/w]). Eight further accessions had amylose contents that were significantly lower or higher than that of Col-0 (9% [w/w]), ranging from 5% to 12% (w/w). We examined the effect of the polymorphisms on GBSS function and uncovered three mechanisms by which GBSS sequence variation led to different amylose contents: (1) altered GBSS abundance, (2) altered GBSS activity, and (3) altered affinity of GBSS for binding PROTEIN TARGETING TO STARCH1-a protein that targets GBSS to starch granules. These findings demonstrate that amylose in leaves is not essential for the viability of some naturally occurring Arabidopsis genotypes, at least over short timescales and under some environmental conditions and open an opportunity to explore the adaptive significance of amylose.

Neutrophils from patients with the cardiac clinical form of Chagas disease release less NETs than neutrophils from healthy individuals.

INTRODUCTION: Chagas disease (CD) is a global health concern with approximately 12 000 deaths per year worldwide. In the chronic phase, about 30% of patients develop the cardiac clinical form, which presents symptoms associated with the presence of inflammatory cells in the cardiac tissue. Neutrophils are inflammatory cells able to modulate the chronic immune response against pathogens. These cells are capable of interacting with Trypanosoma cruzi, the aetiological agent of CD, and perform several effector functions, such as NET release. However, few studies have been carried out to investigate the role of these cells in the disease. AIMS: To investigate the release of NETs by neutrophils from CD patients by measuring the amount of DNA and elastase released. METHODS AND RESULTS: Measurement of DNA release by neutrophils from chronic CD patients presenting the indeterminate (IND group; n = 18) and cardiac (CARD group; n = 15) clinical forms and nonchagasic subjects (n = 18) stimulated with soluble antigen of T. cruzi was quantified using the Quant-iT™ PicoGreen® dsDNA assay kit. Patients from CARD group release less DNA (117.3 ± 21.85 ng/mL; *P = .0131) than neutrophils from control (177.7 ± 58.41 ng/mL). Elastase enzyme degranulation was measured using the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (SAAVNA). Absorbance values of elastase degranulation activity showed that only cells from healthy individuals presented a high release profile of elastase. Also, we found a negative correlation between DNA released concentration and risk of death (r = -.6574; *P = .0173); the lower the neutrophil DNA release from chagasic patients with cardiac event, the higher the risk of death. CONCLUSION: These preliminary data show that patients with the cardiac form of CD release less NETs than nonchagasic individuals, raising the possibility that lower release of NETs enhances risk of death in CD patients with cardiac events.

Localization of BCR-ABL to Stress Granules Contributes to Its Oncogenic Function.

The oncogenic tyrosine kinase BCR-ABL activates a variety of signaling pathways and plays a causative role in the pathogenesis of chronic myelogenous leukemia (CML); however, the subcellular distribution of this chimeric protein remains controversial. Here, we report that BCR-ABL is localized to stress granules and that its granular localization contributes to BCR-ABL-dependent leukemogenesis. BCR-ABL-positive granules were not colocalized with any markers for membrane-bound organelles but were colocalized with HSP90a, a component of RNA granules. The number of such granules increased with thapsigargin treatment, confirming that the granules were stress granules. Given that treatment with the ABL kinase inhibitor imatinib and elimination of the N-terminal region of BCR-ABL abolished granule formation, kinase activity and the coiled-coil domain are required for granule formation. Whereas wild-type BCR-ABL rescued the growth defect in IL-3-depleted Ba/F3 cells, mutant BCR-ABL lacking the N-terminal region failed to do so. Moreover, forced tetramerization of the N-terminus-deleted mutant could not restore the growth defect, indicating that granule formation, but not tetramerization, through its N-terminus is critical for BCR-ABL-dependent oncogenicity. Our findings together provide new insights into the pathogenesis of CML by BCR-ABL and open a window for developing novel therapeutic strategies for this disease.Key words: BCR-ABL, subcellular localization, stress granule.

Defining the Role of Stress Granules in Innate Immune Suppression by the Herpes Simplex Virus 1 Endoribonuclease VHS.

In response to virus-induced shutoff host protein synthesis, dynamic aggregates containing mRNA, RNA-binding proteins and translation factors termed stress granules (SGs) often accumulate within the cytoplasm. SGs typically form following phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), a substrate of the double-stranded RNA (dsRNA)-activated kinase protein kinase R (PKR). The detection of innate immune sensors and effectors like PKR at SGs suggests a role in pathogen nucleic acid sensing. However, the functional importance of SGs in host innate responses is unclear and has primarily been examined in response to infection with select RNA viruses. During infection with the DNA virus herpes simplex virus 1 (HSV-1), the virus-encoded virion host shutoff (VHS) endoribonuclease is required to restrict interferon production, PKR activation, and SG formation, although the relationship between these activities remains incompletely understood. Here, we show that in cells infected with a VHS-deficient HSV-1 (ΔVHS) dsRNA accumulated and localized to SGs. Surprisingly, formation of dsRNA and its concentration at SGs was not required for beta interferon mRNA induction, indicating that suppression of type I interferon induction by VHS does not stem from its control of dsRNA accumulation. Instead, STING signaling downstream of cGMP-AMP synthase (cGAS)-dependent DNA sensing is required for beta interferon induction. In contrast, significantly less PKR activation is observed when SG assembly is disrupted by ISRIB, an inhibitor of phosphorylated eIF2α-mediated translation repression, or depleting SG scaffolding proteins G3BP1 or TIA1. This demonstrates that PKR activation is intimately linked to SG formation and that SGs form important hubs to potentiate PKR activation during infection.IMPORTANCE Formation of cytoplasmic stress granules that are enriched for innate immune sensors and effectors is suppressed during many viral infections. It is unclear, however, to what extent this is a side effect of viral efforts to maintain protein synthesis or intentional disruption of a hub for innate immune sensing. In this study, we utilize a herpes simplex virus 1 mutant lacking the RNA nuclease VHS which upon infection induces SGs, PKR activation, and beta interferon to address this question. We show that dsRNA is localized to SGs and that SGs can function to promote PKR activation in the context of a DNA virus infection, but we find no evidence to support their importance for interferon induction during HSV-1 infection.

Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules.

Rice (Oryza sativa) endosperm is mainly occupied by homogeneous polygonal starch from inside to outside. However, morphologically different (heterogeneous) starches have been identified in some rice mutants. How these heterogeneous starches form remains unknown. A high-amylose rice line (TRS) generated through the antisense inhibition of starch branching synthase I (SBEI) and SBEIIb contains four heterogeneous starches: polygonal, aggregate, elongated, and hollow starch; these starches are regionally distributed in the endosperm from inside to outside. Here, we investigated the relationship between SBE dosage and the morphological architecture of heterogeneous starches in TRS endosperm from the view of the molecular structure of starch. The results indicated that their molecular structures underwent regular changes, including gradually increasing true amylose content but decreasing amylopectin content and gradually increasing the ratio of amylopectin long chain but decreasing the ratio of amylopectin short chain. Granule-bound starch synthase I (GBSSI) amounts in the four heterogeneous starches were not significantly different from each other, but SBEI, SBEIIa, and SBEIIb showed a gradually decreasing trend. Further immunostaining analysis revealed that the gradually decreasing SBEs acting on the formation of the four heterogeneous granules were mainly due to the spatial distribution of the three SBEs in the endosperm. It was suggested that the decreased amylopectin in starch might remove steric hindrance and provide extra space for abundant amylose accumulation when the GBSSI amount was not elevated. Furthermore, extra amylose coupled with altered amylopectin structure possibly led to morphological changes in heterogeneous granules.

Lkb1 regulates granule cell migration and cortical folding of the cerebellar cortex.

Cerebellar growth and foliation require the Hedgehog-driven proliferation of granule cell precursors (GCPs) in the external granule layer (EGL). However, that increased or extended GCP proliferation generally does not elicit ectopic folds suggests that additional determinants control cortical expansion and foliation during cerebellar development. Here, we find that genetic loss of the serine-threonine kinase Liver Kinase B1 (Lkb1) in GCPs increased cerebellar cortical size and foliation independent of changes in proliferation or Hedgehog signaling. This finding is unexpected given that Lkb1 has previously shown to be critical for Hedgehog pathway activation in cultured cells. Consistent with unchanged proliferation rate of GCPs, the cortical expansion of Lkb1 mutants is accompanied by thinning of the EGL. The plane of cell division, which has been implicated in diverse processes from epithelial surface expansions to gyrification of the human cortex, remains unchanged in the mutants when compared to wild-type controls. However, we find that Lkb1 mutants display delayed radial migration of post-mitotic GCPs that coincides with increased cortical size, suggesting that aberrant cell migration may contribute to the cortical expansion and increase foliation. Taken together, our results reveal an important role for Lkb1 in regulating cerebellar cortical size and foliation in a Hedgehog-independent manner.

Cytosine methylation by DNMT2 facilitates stability and survival of HIV-1 RNA in the host cell during infection.

The enigmatic methyltransferase, DNMT2 (DNA methyltransferase 2), structurally resembles a DNA methyltransferase, but has been shown to be a tRNA methyltransferase targeting cytosine within a specific CpG in different tRNA molecules. We had previously shown that, during environmental stress conditions, DNMT2 is re-localized from the nucleus to the cytoplasmic stress granules (SGs) and is associated with RNA-processing proteins. In the present study, we show that DNMT2 binds and methylates various mRNA species in a sequence-independent manner and gets re-localized to SGs in a phosphorylation-dependent manner. Importantly, our results indicate that HIV-1 enhances its survivability in the host cell by utilizing this RNA methylation capability of DNMT2 to increase the stability of its own genome. Upon infection, DNMT2 re-localizes from the nucleus to the SGs and methylates HIV-1 RNA. This DNMT2-dependent methylation provided post-transcriptional stability to the HIV-1 RNA. Furthermore, DNMT2 overexpression increased the HIV-1 viral titre. This would suggest that HIV hijacks the RNA-processing machinery within the SGs to ensure its own survival in the host cell. Thus, our findings provide for a novel mechanism by which virus tries to modulate the host cell machinery to its own advantage.

Regulation of Human Endonuclease V Activity and Relocalization to Cytoplasmic Stress Granules.

Endonuclease V (EndoV) is an enzyme with specificity for inosines in nucleic acids. Whereas the bacterial homologs are active on both DNA and RNA, the mammalian variants only cleave RNA, at least when assayed with recombinant proteins. Here we show that ectopically expressed, as well as endogenously expressed human (h)EndoV, share the same enzymatic properties as the recombinant protein and cleaves RNA with inosine but not DNA. In search for proteins interacting with hEndoV, polyadenylate-binding protein C1 (PABPC1) was identified. The association between PABPC1 and hEndoV is RNA dependent and furthermore, PABPC1 stimulates hEndoV activity and affinity for inosine-containing RNA. Upon cellular stress, PABPC1 relocates to cytoplasmic stress granules that are multimolecular aggregates of stalled translation initiation complexes formed to aid cell recovery. Arsenite, as well as other agents, triggered relocalization also of hEndoV to cytoplasmic stress granules. As inosines in RNA are highly abundant, hEndoV activity is likely regulated in cells to avoid aberrant cleavage of inosine-containing transcripts. Indeed, we find that hEndoV cleavage is inhibited by normal intracellular ATP concentrations. The ATP stores inside a cell do not overlay stress granules and we suggest that hEndoV is redistributed to stress granules as a strategy to create a local environment low in ATP to permit hEndoV activity.

Role of granule proteases in the life and death of neutrophils.

Neutrophils constitute a crucial component of the innate immune defenses against microbes. Produced in the bone marrow and patrolling in blood vessels, neutrophils are recruited to injured tissues and are immediately active to contain pathogen invasion. Neutrophils undergo programmed cell death by multiple, context-specific pathways, which have consequences on immunopathology and disease outcome. Studies in the last decade indicate additional functions for neutrophils - or a subset of neutrophils - in modulating adaptive responses and tumor progression. Neutrophil granules contain abundant amounts of various proteases, which are directly implicated in protective and pathogenic functions of neutrophils. It now emerges that neutral serine proteases such as cathepsin G and proteinase-3 also contribute to the neutrophil life cycle, but do so via different pathways than that of the aspartate protease cathepsin D and that of mutants of the serine protease elastase. The aim of this review is to appraise the present knowledge of the function of neutrophil granule proteases and their inhibitors in neutrophil cell death, and to integrate these findings in the current understandings of neutrophil life cycle and programmed cell death pathways.

Lytic granule loading of CD8+ T cells is required for HIV-infected cell elimination associated with immune control.

Virus-specific CD8+ T cells probably mediate control over HIV replication in rare individuals, termed long-term nonprogressors (LTNPs) or elite controllers. Despite extensive investigation, the mechanisms responsible for this control remain incompletely understood. We observed that HIV-specific CD8+ T cells of LTNPs persisted at higher frequencies than those of treated progressors with equally low amounts of HIV. Measured on a per-cell basis, HIV-specific CD8+ T cells of LTNPs efficiently eliminated primary autologous HIV-infected CD4+ T cells. This function required lytic granule loading of effectors and delivery of granzyme B to target cells. Defective cytotoxicity of progressor effectors could be restored after treatment with phorbol ester and calcium ionophore. These results establish an effector function and mechanism that clearly segregate with immunologic control of HIV. They also demonstrate that lytic granule contents of memory cells are a critical determinant of cytotoxicity that must be induced for maximal per-cell killing capacity.

Flow cytometric assessment of myeloperoxidase in bovine blood neutrophils and monocytes.

Myeloperoxidase (MPO) is a lysosomal peroxidase enzyme mainly stored in the azurophilic granules of neutrophils playing an important role in innate immunity for first-line protection against microorganisms in many species including cattle. As such, determination of MPO has become of great interest for the diagnosis of infectious and inflammatory diseases in multiple species such as humans. In cattle, MPO determination is rarely done because methods to assess MPO in this species are limited: functional assays have been described earlier, but so far, the quantification of MPO at the single cell level has not been done yet. In the present paper, an innovative flow cytometric method to assess MPO in blood leukocytes of dairy cattle is described. A commercial anti-bovine MPO was used following density gradient separation to isolate polymorphonuclear (PMN) and mononuclear (MN) leukocytes from blood. Identification of PMN and MN, subdivided in monocytes and lymphocytes, was based on the expression of the surface markers CH138A and CD172A. The optimized protocol was subsequently evaluated on blood samples of 17 Holstein Friesian heifers. Myeloperoxidase expression was measured flow cytometrically and visualized by fluorescence microscopic imaging of sorted PMN and MN populations. We suggest this innovative method to be useful in the field for early detection of cows at higher risk for inflammatory diseases such as mastitis and metritis during the transition period.

Syk Is Recruited to Stress Granules and Promotes Their Clearance through Autophagy.

Syk is a cytoplasmic kinase that serves multiple functions within the immune system to couple receptors for antigens and antigen-antibody complexes to adaptive and innate immune responses. Recent studies have identified additional roles for the kinase in cancer cells, where its expression can either promote or suppress tumor cell growth, depending on the context. Proteomic analyses of Syk-binding proteins identified several interacting partners also found to be recruited to stress granules. We show here that the treatment of cells with inducers of stress granule formation leads to the recruitment of Syk to these protein-RNA complexes. This recruitment requires the phosphorylation of Syk on tyrosine and results in the phosphorylation of proteins at or near the stress granule. Grb7 is identified as a Syk-binding protein involved in the recruitment of Syk to the stress granule. This recruitment promotes the formation of autophagosomes and the clearance of stress granules from the cell once the stress is relieved, enhancing the ability of cells to survive the stress stimulus.

Does ß-hexosaminidase function only as a degranulation indicator in mast cells? The primary role of ß-hexosaminidase in mast cell granules.

ß-Hexosaminidase, which is generally present in the lysosome, is essential for glycoprotein metabolism in the maintenance of cell homeostasis. In mast cells (MCs), large amounts of ß-hexosaminidase are present in the granules as opposed to the lysosome, and the biological role of MC ß-hexosaminidase has yet to be fully elucidated. Therefore, we investigated the biological role of ß-hexosaminidase in MC granules. Bone marrow-derived MCs from C57BL/6 (BL/6-BMMC) or ß-hexosaminidase gene-deficient (hexb(-/-)-BMMC) mice were transplanted into MC-deficient (WBB6F1/J-Kit(W)/Kit(W-v) [W/W(v)]) mice to generate MC-reconstituted models. In asthma model experiments, no differences were observed in the symptoms of BL/6, W/W(v), BL/6-BMMC-reconstituted W/W(v), or hexb(-/-)-BMMC-reconstituted W/W(v) mice. In Staphylococcus epidermidis experimental infection model experiments, the severity of symptoms and frequency of death were markedly higher in W/W(v) and hexb(-/-)-BMMC-reconstituted W/W(v) mice than in BL/6 and BL/6-BMMC-reconstituted W/W(v) mice. The growth of S. epidermidis in an in vitro study was clearly inhibited by addition of BL/6-BMMC lysate, but not by addition of hexb(-/-)-BMMC lysate. Moreover, suppression of bacterial proliferation was completely recovered when bacteria were incubated with hexb(-/-)-BMMC lysate plus ß-hexosaminidase. Transmission electron microscopy indicated that the cell wall of S. epidermidis was heavily degraded following coincubation of bacteria with BL/6-BMMC lysate, but not following coincubation with hexb(-/-)-BMMC lysate. These findings strongly suggest that MC granule ß-hexosaminidase is crucial for defense against bacterial invasion, but is not involved in the allergic response. Our results also suggest that the bactericidal mechanism of ß-hexosaminidase involves degradation of bacterial cell wall peptidoglycan.

Tryptase, a novel angiogenic factor stored in mast cell granules.

Human mast cells (MCs) are a rich reservoir of neutral proteases, packed in large amounts in their granules and comprising a high fraction of all cellular proteins. Among these proteases, tryptase is involved in angiogenesis after its release from activated MC granules, as it has been demonstrated in different in vitro and in vivo assays. Moreover, tryptase-positive MCs increase in number and vascularization increases in a linear fashion in different solid and hematological tumors. This complex interplay between MCs and tumor angiogenesis have led to consider the therapeutic use of angiogenesis inhibitors, which specifically target the angiogenic activity of tryptase, such as gabexate mesilate and nafamostat mesilate, two inhibitors of trypsin-like serine proteases.

Human LINE-1 restriction by APOBEC3C is deaminase independent and mediated by an ORF1p interaction that affects LINE reverse transcriptase activity.

LINE-1 (L1) retrotransposons are mobile genetic elements whose extensive proliferation resulted in the generation of ≈ 34% of the human genome. They have been shown to be a cause of single-gene diseases. Moreover, L1-encoded endonuclease can elicit double-strand breaks that may lead to genomic instability. Mammalian cells adopted strategies restricting mobility and deleterious consequences of uncontrolled retrotransposition. The human APOBEC3 protein family of polynucleotide cytidine deaminases contributes to intracellular defense against retroelements. APOBEC3 members inhibit L1 retrotransposition by 35-99%. However, genomic L1 retrotransposition events that occurred in the presence of L1-restricting APOBEC3 proteins are devoid of detectable G-to-A hypermutations, suggesting one or multiple deaminase-independent L1 restricting mechanisms. We set out to uncover the mechanism of APOBEC3C (A3C)-mediated L1 inhibition and found that it is deaminase independent, requires an intact dimerization site and the RNA-binding pocket mutation R122A abolishes L1 restriction by A3C. Density gradient centrifugation of L1 ribonucleoprotein particles, subcellular co-localization of L1-ORF1p and A3C and co-immunoprecipitation experiments indicate that an RNA-dependent physical interaction between L1 ORF1p and A3C dimers is essential for L1 restriction. Furthermore, we demonstrate that the amount of L1 complementary DNA synthesized by L1 reverse transcriptase is reduced by ≈ 50% if overexpressed A3C is present.

Myeloperoxidase in human neutrophil host defence.

Human neutrophils represent the predominant leucocyte in circulation and the first responder to infection. Concurrent with ingestion of microorganisms, neutrophils activate and assemble the NADPH oxidase at the phagosome, thereby generating superoxide anion and hydrogen peroxide. Concomitantly, granules release their contents into the phagosome, where the antimicrobial proteins and enzymes synergize with oxidants to create an environment toxic to the captured microbe. The most rapid and complete antimicrobial action by human neutrophils against many organisms relies on the combined efforts of the azurophilic granule protein myeloperoxidase and hydrogen peroxide from the NADPH oxidase to oxidize chloride, thereby generating hypochlorous acid and a host of downstream reaction products. Although individual components of the neutrophil antimicrobial response exhibit specific activities in isolation, the situation in the environment of the phagosome is far more complicated, a consequence of multiple and complex interactions among oxidants, proteins and their by-products. In most cases, the cooperative interactions among the phagosomal contents, both from the host and the microbe, culminate in loss of viability of the ingested organism.