Integrative proteomics reveals an increase in non-degradative ubiquitylation in activated CD4+ T cells.

Despite gathering evidence that ubiquitylation can direct non-degradative outcomes, most investigations of ubiquitylation in T cells have focused on degradation. Here, we integrated proteomic and transcriptomic datasets from primary mouse CD4+ T cells to establish a framework for predicting degradative or non-degradative outcomes of ubiquitylation. Di-glycine remnant profiling was used to reveal ubiquitylated proteins, which in combination with whole-cell proteomic and transcriptomic data allowed prediction of protein degradation. Analysis of ubiquitylated proteins identified by di-glycine remnant profiling indicated that activation of CD4+ T cells led to an increase in non-degradative ubiquitylation. This correlated with an increase in non-proteasome-targeted K29, K33 and K63 polyubiquitin chains. This study revealed over 1,200 proteins that were ubiquitylated in primary mouse CD4+ T cells and highlighted the relevance of non-proteasomally targeted ubiquitin chains in T cell signaling.

The diabetes susceptibility gene Clec16a regulates mitophagy.

Clec16a has been identified as a disease susceptibility gene for type 1 diabetes, multiple sclerosis, and adrenal dysfunction, but its function is unknown. Here we report that Clec16a is a membrane-associated endosomal protein that interacts with E3 ubiquitin ligase Nrdp1. Loss of Clec16a leads to an increase in the Nrdp1 target Parkin, a master regulator of mitophagy. Islets from mice with pancreas-specific deletion of Clec16a have abnormal mitochondria with reduced oxygen consumption and ATP concentration, both of which are required for normal ß cell function. Indeed, pancreatic Clec16a is required for normal glucose-stimulated insulin release. Moreover, patients harboring a diabetogenic SNP in the Clec16a gene have reduced islet Clec16a expression and reduced insulin secretion. Thus, Clec16a controls ß cell function and prevents diabetes by controlling mitophagy. This pathway could be targeted for prevention and control of diabetes and may extend to the pathogenesis of other Clec16a- and Parkin-associated diseases.

The E3 ubiquitin ligase Cul4b promotes CD4+ T cell expansion by aiding the repair of damaged DNA.

The capacity for T cells to become activated and clonally expand during pathogen invasion is pivotal for protective immunity. Our understanding of how T cell receptor (TCR) signaling prepares cells for this rapid expansion remains limited. Here we provide evidence that the E3 ubiquitin ligase Cullin-4b (Cul4b) regulates this process. The abundance of total and neddylated Cul4b increased following TCR stimulation. Disruption of Cul4b resulted in impaired proliferation and survival of activated T cells. Additionally, Cul4b-deficient CD4+ T cells accumulated DNA damage. In T cells, Cul4b preferentially associated with the substrate receptor DCAF1, and Cul4b and DCAF1 were found to interact with proteins that promote the sensing or repair of damaged DNA. While Cul4b-deficient CD4+ T cells showed evidence of DNA damage sensing, downstream phosphorylation of SMC1A did not occur. These findings reveal an essential role for Cul4b in promoting the repair of damaged DNA to allow survival and expansion of activated T cells.

Neutrophils promote clearance of nuclear debris following acid-induced lung injury.

Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared with wild type. Size analysis of DNA debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation, they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intratracheal administration of DNase I. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.

Differentiating children with sepsis with and without acute respiratory distress syndrome using proteomics.

Both sepsis and acute respiratory distress syndrome (ARDS) rely on imprecise clinical definitions leading to heterogeneity, which has contributed to negative trials. Because circulating protein/DNA complexes have been implicated in sepsis and ARDS, we aimed to develop a proteomic signature of DNA-bound proteins to discriminate between children with sepsis with and without ARDS. We performed a prospective case-control study in 12 children with sepsis with ARDS matched to 12 children with sepsis without ARDS on age, severity of illness score, and source of infection. We performed co-immunoprecipitation and downstream proteomics in plasma collected ≤ 24 h of intensive care unit admission. Expression profiles were generated, and a random forest classifier was used on differentially expressed proteins to develop a signature which discriminated ARDS. The classifier was tested in six independent blinded samples. Neutrophil and nucleosome proteins were over-represented in ARDS, including two S100A proteins, superoxide dismutase (SOD), and three histones. Random forest produced a 10-protein signature that accurately discriminated between children with sepsis with and without ARDS. This classifier perfectly assigned six independent blinded samples as having ARDS or not. We validated higher expression of the most informative discriminating protein, galectin-3-binding protein, in children with ARDS. Our methodology has applicability to isolation of DNA-bound proteins from plasma. Our results support the premise of a molecular definition of ARDS, and give preliminary insight into why some children with sepsis, but not others, develop ARDS.

Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis.

Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis.

A core viral protein binds host nucleosomes to sequester immune danger signals.

Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling.

Itch attenuates CD4 T-cell proliferation in mice by limiting WBP2 protein stability.

To mount an antipathogen response, CD4 T cells must undergo rapid cell proliferation; however, poorly controlled expansion can result in diseases such as autoimmunity. One important regulator of T-cell activity is the E3 ubiquitin ligase Itch. Itch deficient patients suffer from extensive autoinflammation. Similarly, Itch deficient mice exhibit inflammation characterized by high numbers of activated CD4 T cells. While the role of Itch in limiting CD4 T-cell cytokine production has been extensively studied, it is less clear whether and how Itch regulates proliferation of these cells. We determined that Itch deficient CD4 T cells are hyperproliferative in vitro and in vivo, due to increased S phase entry. Whole cell proteomics analysis of Itch deficient primary mouse CD4 T cells revealed increased abundance of the ß-catenin coactivator WW domain-binding protein 2 (WBP2). Furthermore, Itch deficient cells demonstrate increased WBP2 protein stability, and Itch and WBP2 interact in CD4 T cells. Knockdown of WBP2 in CD4 T cells caused reduced proliferation. Together, our data support that Itch attenuates CD4 T cell proliferation by promoting WBP2 degradation. This study identifies novel roles for Itch and WBP2 in regulating CD4 T cell proliferation, providing insight into how Itch may prevent inflammation.

Urine Proteomics: Evaluation of Different Sample Preparation Workflows for Quantitative, Reproducible, and Improved Depth of Analysis.

The growing field of urinary proteomics shows promise to expand the number of biomarkers for the diagnosis and prognosis of a number of human diseases. With the rapid developments in mass spectrometry methods for proteome quantification, there exists an opportunity for improved sample processing and separation workflows to make important contributions to urine proteomic analyses. Here we evaluate the performance of four sample preparation methods: MStern, PreOmics in-StageTip (iST), suspension-trapping (S-Trap), and conventional urea In-Solution trypsin hydrolysis for nondepleted urine samples. Data-dependent acquisition (DDA) mode on a QExactive HF mass spectrometer was used for single-shot label-free data acquisition. Our results demonstrate a high degree of reproducibility within each workflow. PreOmics iST yields the best digestion efficiency, whereas the S-Trap workflow gives the greatest number of peptide and protein identifications. Using the S-Trap method and starting with ∼0.5 mL, we identify ∼1500 protein groups and ∼17 700 peptides from DDA analysis with a single injection on the mass spectrometer.

Identifying Host Factors Associated with DNA Replicated During Virus Infection.

Viral DNA genomes replicating in cells encounter a myriad of host factors that facilitate or hinder viral replication. Viral proteins expressed early during infection modulate host factors interacting with viral genomes, recruiting proteins to promote viral replication, and limiting access to antiviral repressors. Although some host factors manipulated by viruses have been identified, we have limited knowledge of pathways exploited during infection and how these differ between viruses. To identify cellular processes manipulated during viral replication, we defined proteomes associated with viral genomes during infection with adenovirus, herpes simplex virus and vaccinia virus. We compared enrichment of host factors between virus proteomes and confirmed association with viral genomes and replication compartments. Using adenovirus as an illustrative example, we uncovered host factors deactivated by early viral proteins, and identified a subgroup of nucleolar proteins that aid virus replication. Our data sets provide valuable resources of virus-host interactions that affect proteins on viral genomes.

Unbiased Proteomics of Early Lewy Body Formation Model Implicates Active Microtubule Affinity-Regulating Kinases (MARKs) in Synucleinopathies.

Parkinson's disease (PD) patients progressively accumulate intracytoplasmic inclusions formed by misfolded α-synuclein known as Lewy bodies (LBs). LBs also contain other proteins that may or may not be relevant in the disease process. To identify proteins involved early in LB formation, we performed proteomic analysis of insoluble proteins in a primary neuron culture model of α-synuclein pathology. We identified proteins previously found in authentic LBs in PD as well as several novel proteins, including the microtubule affinity-regulating kinase 1 (MARK1), one of the most enriched proteins in this model of LB formation. Activated MARK proteins (MARKs) accumulated in LB-like inclusions in this cell-based model as well as in a mouse model of LB disease and in LBs of postmortem synucleinopathy brains. Inhibition of MARKs dramatically exacerbated α-synuclein pathology. These findings implicate MARKs early in synucleinopathy pathogenesis and as potential therapeutic drug targets.SIGNIFICANCE STATEMENT Neurodegenerative diseases are diagnosed definitively only in postmortem brains by the presence of key misfolded and aggregated disease proteins, but cellular processes leading to accumulation of these proteins have not been well elucidated. Parkinson's disease (PD) patients accumulate misfolded α-synuclein in LBs, the diagnostic signatures of PD. Here, unbiased mass spectrometry was used to identify the microtubule affinity-regulating kinase family (MARKs) as activated and insoluble in a neuronal culture PD model. Aberrant activation of MARKs was also found in a PD mouse model and in postmortem PD brains. Further, inhibition of MARKs led to increased pathological α-synuclein burden. We conclude that MARKs play a role in PD pathogenesis.

Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP.

Infection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood. Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 µM and 45 µM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (KD = 0.72 µM), soluble VWF (KD = 0.58 µM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n = 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n = 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values <.001). There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman ρ = 0.7932, P < .0001). Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders.

Population pharmacokinetics of exendin-(9-39) and clinical dose selection in patients with congenital hyperinsulinism.

AIMS: Congenital hyperinsulinism (HI) is the most common cause of persistent hypoglycaemia in infants and children. Exendin-(9-39), an inverse glucagon-like peptide 1 (GLP-1) agonist, is a novel therapeutic agent for HI that has demonstrated glucose-raising effect. We report the first population pharmacokinetic (PopPK) model of the exendin-(9-39) in patients with HI and propose the optimal dosing regimen for future clinical trials in neonates with HI. METHODS: A total of 182 pharmacokinetic (PK) observations from 26 subjects in three clinical studies were included for constructing the PopPK model using first order conditional estimation (FOCE) with interaction method in nonlinear mixed-effects modelling (NONMEM). Exposure metrics (area under the curve [AUC] and maximum plasma concentration [Cmax ]) at no observed adverse effect levels (NOAELs) in rats and dogs were determined in toxicology studies. RESULTS: Observed concentration-time profiles of exendin-(9-39) were described by a linear two-compartmental PK model. Following allometric scaling of PK parameters, age and creatinine clearance did not significantly affect clearance. The calculated clearance and elimination half-life for adult subjects with median weight of 69 kg were 11.8 l h-1 and 1.81 h, respectively. The maximum recommended starting dose determined from modelling and simulation based on the AUC0-last at the NOAEL and predicted AUC0-inf using the PopPK model was 27 mg kg-1  day-1 intravenously. CONCLUSIONS: This is the first study to investigate the PopPK of exendin-(9-39) in humans. The final PopPK model was successfully used with preclinical toxicology findings to propose the optimal dosing regimen of exendin-(9-39) for clinical studies in neonates with HI, allowing for a more targeted dosing approach to achieve desired glycaemic response.

Medication contaminants as a potential cause of anaphylaxis to vincristine.

Vincristine (VCR) is a vinca alkaloid and common chemotherapeutic that is used to treat multiple pediatric and adult malignancies. Despite its common use, cases of anaphylaxis to VCR are rare and typically isolated to a single individual. We report a series of eight patients with adverse reactions to VCR over the course of 11 months at a single institution, four of which progressed to anaphylaxis and one of which resulted in cardiac arrest. Mass spectrometry analysis of medication lots was performed to test for possible contaminant(s). Our findings highlight the risk of anaphylaxis during therapy with VCR.

Improved surfaceome coverage with a label-free nonaffinity-purified workflow.

The proteins of the cellular plasma membrane (PM) perform important functions relating to homeostasis and intercellular communication. Due to its overall low cellular abundance, amphipathic character, and low membrane-to-cytoplasm ratio, the PM proteome has been challenging to isolate and characterize, and is poorly represented in standard LC-MS/MS analyses. In this study, we employ sucrose gradient ultracentrifugation for the enrichment of the PM proteome, without chemical labeling and affinity purification, together with GeLCMS and use subsequent bioinformatics tools to select proteins associated with the PM/cell surface, herein referred to as the surfaceome. Using this methodology, we identify over 1900 cell surface associated proteins in a human acute myeloid leukemia cell line. These surface proteins comprise almost 50% of all detected cellular proteins, a number that substantially exceeds the depth of coverage in previously published studies describing the leukemia surfaceome.

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation.

The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high Mr disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringle-like domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils.

The Neuroprotective Marine Compound Psammaplysene A Binds the RNA-Binding Protein HNRNPK.

In previous work, we characterized the strong neuroprotective properties of the marine compound Psammaplysene A (PA) in in vitro and in vivo models of neurodegeneration. Based on its strong neuroprotective activity, the current work attempts to identify the physical target of PA to gain mechanistic insight into its molecular action. Two distinct methods, used in parallel, to purify protein-binding partners of PA led to the identification of HNRNPK as a direct target of PA. Based on surface plasmon resonance, we find that the binding of PA to HNRNPK is RNA-dependent. These findings suggest a role for HNRNPK-dependent processes in neurodegeneration/neuroprotection, and warrant further study of HNRNPK in this context.

Neutrophils promote alveolar epithelial regeneration by enhancing type II pneumocyte proliferation in a model of acid-induced acute lung injury.

Alveolar epithelial regeneration is essential for resolution of the acute respiratory distress syndrome (ARDS). Although neutrophils have traditionally been considered mediators of epithelial damage, recent studies suggest they promote type II pneumocyte (AT2) proliferation, which is essential for regenerating alveolar epithelium. These studies did not, however, evaluate this relationship in an in vivo model of alveolar epithelial repair following injury. To determine whether neutrophils influence alveolar epithelial repair in vivo, we developed a unilateral acid injury model that creates a severe yet survivable injury with features similar to ARDS. Mice that received injections of the neutrophil-depleting Ly6G antibody had impaired AT2 proliferation 24 and 72 h after acid instillation, which was associated with decreased reepithelialization and increased alveolar protein concentration 72 h after injury. As neutrophil depletion itself may alter the cytokine response, we questioned the contribution of neutrophils to alveolar epithelial repair in neutropenic granulocyte-colony stimulating factor (G-CSF)-/- mice. We found that the loss of G-CSF recapitulated the neutrophil response of Ly6G-treated mice and was associated with defective alveolar epithelial repair, similar to neutrophil-depleted mice, and was reversed by administration of exogenous G-CSF. To approach the mechanisms, we employed an unbiased protein analysis of bronchoalveolar lavage fluid from neutrophil-depleted and neutrophil-replete mice 12 h after inducing lung injury. Pathway analysis identified significant differences in multiple signaling pathways that may explain the differences in epithelial repair. These data emphasize an important link between the innate immune response and tissue repair in which neutrophils promote alveolar epithelial regeneration.

Integrated analysis of proteome and transcriptome changes in the mucopolysaccharidosis type VII mouse hippocampus.

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by the deficiency of ß-glucuronidase. In this study, we compared the changes relative to normal littermates in the proteome and transcriptome of the hippocampus in the C57Bl/6 mouse model of MPS VII, which has well-documented histopathological and neurodegenerative changes. A completely different set of significant changes between normal and MPS VII littermates were found in each assay. Nevertheless, the functional annotation terms generated by the two methods showed agreement in many of the processes, which also corresponded to known pathology associated with the disease. Additionally, assay-specific changes were found, which in the proteomic analysis included mitochondria, energy generation, and cytoskeletal differences in the mutant, while the transcriptome differences included immune, vesicular, and extracellular matrix changes. In addition, the transcriptomic changes in the mutant hippocampus were concordant with those in a MPS VII mouse caused by the same mutation but on a different background inbred strain.

Quaternary arrangement of an active, native group II intron ribonucleoprotein complex revealed by small-angle X-ray scattering.

The stable ribonucleoprotein (RNP) complex formed between the Lactococcus lactis group II intron and its self-encoded LtrA protein is essential for the intron's genetic mobility. In this study, we report the biochemical, compositional, hydrodynamic and structural properties of active group II intron RNP particles (+A) isolated from its native host using a novel purification scheme. We employed small-angle X-ray scattering to determine the structural properties of these particles as they exist in solution. Using sucrose as a contrasting agent, we derived a two-phase quaternary model of the protein-RNA complex. This approach revealed that the spatial properties of the complex are largely defined by the RNA component, with the protein dimer located near the center of mass. A transfer RNA fusion engineered into domain II of the intron provided a distinct landmark consistent with this interpretation. Comparison of the derived +A RNP shape with that of the previously reported precursor intron (ΔA) particle extends previous findings that the loosely packed precursor RNP undergoes a dramatic conformational change as it compacts into its active form. Our results provide insights into the quaternary arrangement of these RNP complexes in solution, an important step to understanding the transition of the group II intron from the precursor to a species fully active for DNA invasion.