Full-length Counterparts Research Articles

Cryptic proteins translated from deletion-containing viral genomes dramatically expand the influenza virus proteome.

Productive infections by RNA viruses require faithful replication of the entire genome. Yet many RNA viruses also produce deletion-containing viral genomes (DelVGs), aberrant replication products with large internal deletions. DelVGs interfere with the replication of wild-type virus and their presence in patients is associated with better clinical outcomes. The DelVG RNA itself is hypothesized to confer this interfering activity. DelVGs antagonize replication by out-competing the full-length genome and triggering innate immune responses. Here, we identify an additionally inhibitory mechanism mediated by a new class of viral proteins encoded by DelVGs. We identified hundreds of cryptic viral proteins translated from DelVGs. These DelVG-encoded proteins (DPRs) include canonical viral proteins with large internal deletions, as well as proteins with novel C-termini translated from alternative reading frames. Many DPRs retain functional domains shared with their full-length counterparts, suggesting they may have activity during infection. Mechanistic studies of DPRs derived from the influenza virus protein PB2 showed that they poison replication of wild-type virus by acting as dominant-negative inhibitors of the viral polymerase. These findings reveal that DelVGs have a dual inhibitory mechanism, acting at both the RNA and protein level. They further show that DPRs have the potential to dramatically expand the functional proteomes of diverse RNA viruses.

Inhibitory effect of truncated isoforms on GPCR dimerization predicted by combinatorial computational strategy

G protein-coupled receptors (GPCRs) play a pivotal role in fundamental biological processes and disease development. GPCR isoforms, derived from alternative splicing, can exhibit distinct signaling patterns. Some highly-truncated isoforms can impact functional performance of full-length receptors, suggesting their intriguing regulatory roles. However, how these truncated isoforms interact with full-length counterparts remains largely unexplored. Here, we computationally investigated the interaction patterns of three human GPCRs from three different classes, ADORA1 (Class A), mGlu2 (Class C) and SMO (Class F) with their respective truncated isoforms because their homodimer structures have been experimentally determined, and they have truncated isoforms deposited and identified at protein level in Uniprot database. Combining the neural network-based AlphaFold2 and two physics-based protein-protein docking tools, we generated multiple complex structures and assessed the binding affinity in the context of atomistic molecular dynamics simulations. Our computational results suggested all the four studied truncated isoforms showed potent binding to their counterparts and overlapping interfaces with homodimers, indicating their strong potential to block homodimerization of their counterparts. Our study offers insights into functional significance of GPCR truncated isoforms and supports the ubiquity of their regulatory roles.

Enhanced Brain Retention of Aβ4‐x Proteoforms and its Contribution to Amyloid Deposits in Alzheimer’s Disease

The deposition of amyloid‐β (Aβ) in brain parenchyma and cerebral vasculature together with intraneuronal neurofibrillary tangles and the gradual loss of synapsis are central neuropathological hallmarks of Alzheimer’s disease (AD). Although it remains unclear what primarily triggers and drives the progression of AD, different lines of investigation point out to a central role of oligomeric Aβ conformations for the disease pathogenesis. Recent studies revealed that the molecular heterogeneity of Aβ deposits is significantly more complex than originally anticipated, extending well beyond the classic Aβ1‐40/Aβ1‐42 dichotomy and being substantially expanded by the presence of multiple post‐translational modifications that increase the proteome diversity. Although N‐terminal truncations at glutamates 3 and 11 and their subsequent cyclation to pyroglutamate are perhaps the most extensively studied modifications, many others have been reported, with species beginning at phenylalanine 4 and bearing an intact C‐terminus being especially relevant. Indeed, Aβ4‐42 peptides were reported as major components of amyloid plaque cores and the limited studies available seem to indicate their relative abundance in patients with AD, Down’s syndrome, and vascular dementia. In this work we assessed the topographic distribution of these species with in‐house‐generated anti‐Aβ4‐x monoclonal 18H6 – specific for Aβ species starting at position 4 and blind for the full‐length Aβ40 and Aβ42 – and used in conjunction with thioflavin S and antibodies specific for the C‐terminus 40 and 42 in confirmed AD cases. All types of Aβ deposits – parenchymal plaques, pre‐amyloid lesions and CAA – contained the Aβ epitopes tested, albeit expressed in different proportions. These co‐localizations were further stressed by combining double immunohistochemical analysis with Thioflavin S staining, showing the preferential location of Aβ4‐x species in CAA and cored plaques with fibrillar amyloid conformation, strongly suggesting poor clearance characteristics. To investigate the brain clearance of Aβ4‐x species in comparison with full‐length Aβ40 and Aβ42 homologues, peptides were synthesized, biochemically and biophysically characterized, radiolabeled with [125I]Na and utilized for in vivo brain clearance experiments in 5–6‐week‐old C57Bl/6 mice. Brain efflux of all monomeric Aβ species tested was fast, with >80% cleared within the 60 min duration of the experiment whereas retention of oligomeric preparations was consistently higher than that of monomeric forms. Notably, clearance of Aβ4‐40 and Aβ4‐42 was significantly lower (<45%) than the full‐length counterparts (61% ‐ 68%), further supporting a pathogenic role for Aβ oligomerization in AD and specifically highlighting the importance of oligomeric Aβ4‐x species exhibiting higher brain retention compared to their full‐length counterparts. Since the process of multimerization is concentration dependent, failing to remove these oligomeric forms of Aβ from the brain will further contribute to exacerbate the amyloidogenic loop and the persistence of amyloid deposits.

RNA Editing-Associated Post-Transcriptional Gene Regulation in Rheumatoid Arthritis.

Background:Rheumatoid arthritis (RA) is an autoimmune disease characterised by systemic inflammation of joints. The observed complexity of RA pathogenesis and studies that have been carried out so far indicate that RA pathogenesis is regulated at multiple levels. Given the role of RNA editing in autoimmune disease, we hypothesised that RNA editing could contribute to RA pathogenesis by regulating gene expression through post-transcriptional mechanisms.Methods:We identified RNA editing events in synovial tissues from early and established RA compared with normal subjects from an available transcriptome data set using REDItools. To investigate the potential effect of these RNA editing events on gene expression, we carried out an analysis of differential exon usage in the vicinity of the differentially edited sites using DEXSeq. We then used STRING to identify putative interactions between differentially edited genes identified from REDItools analysis. We also investigated the possible effects of these RNA editing events on miRNA-target mRNA interactions as predicted by miRanda.Results:Our analysis revealed that there is extensive RNA editing in RA, with 304 and 273 differentially edited events in early RA and established RA, respectively. Of these, 25 sites were within 11 genes in early RA, and 34 sites were within 7 genes in established RA. DEXSeq analysis revealed that RNA editing correlated with differential exon usage in 4 differentially edited genes that have previously also been associated with RA in some measure: ATM, ZEB1, ANXA4, and TIMP3. DEXSeq analysis also revealed enrichment of some non-functional isoforms of these genes, perhaps at the expense of their full-length counterparts. Network analysis using STRING showed that several edited genes were part of the p53 protein-protein interaction network. We also identified several putative miRNA binding sites in the differentially edited genes that were lost upon editing.Conclusions:Our results suggested that the expression of genes involved in DNA repair and cell cycle, including ATM and ZEB1 which are well-known functional regulators of the DNA damage response pathway, could be regulated by RNA editing in RA synovia. This may contribute to an impaired DNA damage response in synovial tissues.

Proteome-wide mapping of short-lived proteins in human cells

Rapid protein degradation enables cells to quickly modulate protein abundance. Dysregulation of short-lived proteins plays essential roles in disease pathogenesis. A focused map of short-lived proteins remains understudied. Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we identified 1,017 short-lived proteins (half-lives ≤ 8 h). These short-lived proteins are less abundant, evolutionarily younger, and less thermally stable than other proteins. We quantified 103 proteins with different stabilities among cell lines. We showed that U2OS and HCT116 cells express truncated forms of ATRX and GMDS, respectively, which have lower stability than their full-length counterparts. This study provides a large-scale resource of human short-lived proteins under translational arrest, leading to untapped avenues of protein regulation for therapeutic interventions.

Email Newsletters: An Analysis of Content From Nine Top News Organizations

Email newsletters are increasingly popular delivery systems for legacy and digital-native news outlets. The newsletter embodies another level of gatekeeping, but little research has explored which stories make it through the email newsletter “gate” and into the subscriber's inbox. This content analysis of newsletter items ( N = 1,231) from select broadcast, cable, print, and digital-native news outlets provides a snapshot of the focus, subject matter, and style of daily newsletters. Newsletter subscribers generally are encouraged to link to the outlet's full-length online content on its website; however, not all outlets attempt to make personal connections with their newsletter readers. In general, the newsletters are largely filled with nationally focused stories on politics and government and mimic the style of their full-length counterparts.

Abstract 2410: The mutant p53 C-terminal domain assists in DNA interactions and cell cycle promotion

Abstract Introduction: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer and has the worst prognosis of any breast cancer sub-type. More than 80% of TNBC patients harbor mutated TP53. Some mutant p53 proteins (mtp53) exhibit gain-of function (GOF) oncogenic characteristics, contributing to the decreased prognosis for TNBC patients, but also providing a potential therapeutic target for this aggressive disease. Our group previously found mtp53 R273H interacts with replicating DNA, and increases chromatin-bound replication proteins MCM2-7, PCNA, and PARP1. Mutant p53 retains an intact C-terminal domain (CTD), the domain which facilitates non-specific DNA binding. We hypothesize that the CTD of mtp53 facilitates increased DNA replication processivity. Methods: To test this hypothesis, we have transiently transfected human cells to compare wild-type p53, and R273H mtp53 full-length and CTD-truncated variants. We used CRISPR/Cas9 to generate endogenous CTD truncations of mtp53 R273H in the breast cancer cell line MDA-MB-468. Specifically, clones were used which have mtp53 CTD truncations (R273HΔ381-388 and R273HΔ347-393) and one clone with a frameshift resulting in no detectable mtp53 protein (R273Hfs387). Various western blotting techniques were used to assess protein level and sub-cellular localization, including the iPOND (isolation of proteins on nascent DNA) assay. Several microscopy techniques were used to visualize protein-protein interactions in cells including immunofluorescence, proximity ligation, and DNA fiber assays. Results: Our data show mtp53 R273H and wild-type p53 proteins with CTD truncations tether less to chromatin than their full-length counterparts. Also, our data shows that the truncated R273HΔ381-388 mtp53 is in close proximity to PARylated proteins (and potentially even PARylated itself), but the R273HΔ347-393 truncation is not in close proximity to PARylated proteins. Additionally, R273HΔ381-388 and R273HΔ347-393 demonstrate delayed cell cycle progression and reduced proliferation compared to MDA-MB-468 cells. We observe DNA replication processivity is decreased in 468 R273HΔ381-388 and R273Hfs387 cells. Processivity, but not proliferation rate, is rescued in 468 R273HΔ347-393 cells. Conclusions: The combination of reduced cell proliferation in all CTD-truncated cells, and reduced DNA replication processivity in R273HΔ381-388 and R273Hfs387 cells, but not in R273HΔ347-393 indicates a nuanced role for the CTD in DNA replication and S-phase progression. Citation Format: Devon Lundine, Zafar Syed, Viola Ellison, George Annor, Gu Xiao, Jill Bargonetti. The mutant p53 C-terminal domain assists in DNA interactions and cell cycle promotion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2410.

Maize NAC-domain retained splice variants act as dominant negatives to interfere with the full-length NAC counterparts

The plant-specific NAC transcription factors play diverse roles in various stress signaling. Alternative splicing is particularly prevalent in plants under stress. However, the investigation of cadmium (Cd) on the differential expression of the splice variants of NACs is in its infancy. Here, we identified three Cd-induced intron retention splice NAC variants which only contained the canonical NAC domain, designated as nacDomains, derived from three Cd-upregulated maize NACs. Subcellular localization analysis indicated that both nacDomain and its full-length NAC counterpart co-localized in the nucleus as manifested in the BiFC assay, thus implied that nacDomains and their corresponding NACs form heterodimers through the identical NAC domain. Further chimeric reporter/effector transient expression assay and Cd-tolerance assay in tobacco leaves collectively indicated that nacDomain-NAC heterodimers were involved in the regulation of NAC function. The results obtained here were in accordance with the model of dominant negative, which suggested that nacDomain act as the dominant negative to antagonize the regulation of NAC on its target gene expression and the Cd-tolerance function performance of NAC transcription factor. These findings proposed a novel insight into understanding the molecular mechanisms of Cd response in plants.

Single-Molecular Heteroamyloidosis of Human Islet Amyloid Polypeptide.

Human amyloids and plaques uncovered post mortem are highly heterogeneous in structure and composition, yet literature concerning the heteroaggregation of amyloid proteins is extremely scarce. This knowledge deficiency is further exacerbated by the fact that peptide delivery is a major therapeutic strategy for targeting their full-length counterparts associated with the pathologies of a range of human diseases, including dementia and type 2 diabetes (T2D). Accordingly, here we examined the coaggregation of full-length human islet amyloid polypeptide (IAPP), a peptide associated with type 2 diabetes, with its primary and secondary amyloidogenic fragments 19-29 S20G and 8-20. Single-molecular aggregation dynamics was obtained by high-speed atomic force microscopy, augmented by transmission electron microscopy, X-ray diffraction, and super-resolution stimulated emission depletion microscopy. The coaggregation significantly prolonged the pause phase of fibril elongation, increasing its dwell time by 3-fold. Surprisingly, unidirectional elongation of mature fibrils, instead of protofilaments, was observed for the coaggregation, indicating a new form of tertiary protein aggregation unknown to existing theoretical models. Further in vivo zebrafish embryonic assay indicated improved survival and hatching, as well as decreased frequency and severity of developmental abnormalities for embryos treated with the heteroaggregates of IAPP with 19-29 S20G, but not with 8-20, compared to the control, indicating the therapeutic potential of 19-29 S20G against T2D.

Peptide-Based Functional Biomaterials for Soft-Tissue Repair.

Synthetically derived peptide-based biomaterials are in many instances capable of mimicking the structure and function of their full-length endogenous counterparts. Combine this with the fact that short mimetic peptides are easier to produce when compared to full length proteins, show enhanced processability and ease of modification, and have the ability to be prepared under well-defined and controlled conditions; it becomes obvious why there has been a recent push to develop regenerative biomaterials from these molecules. There is increasing evidence that the incorporation of peptides within regenerative scaffolds can result in the generation of structural recognition motifs that can enhance cell attachment or induce cell signaling pathways, improving cell infiltration or promote a variety of other modulatory biochemical responses. By highlighting the current approaches in the design and application of short mimetic peptides, we hope to demonstrate their potential in soft-tissue healing while at the same time drawing attention to the advances made to date and the problems which need to be overcome to advance these materials to the clinic for applications in heart, skin, and cornea repair.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Functional expression of TLR5 of different vertebrate species and diversification in intestinal pathogen recognition

Toll-like receptor 5 (TLR5) is activated by bacterial flagellins and plays a crucial role in the first-line defence against pathogenic bacteria and in immune homeostasis, and is highly conserved in vertebrate species. However, little comparative information is available on TLR5 functionality. In this study, we compared TLR5 activation using full-length and chimeric TLR5 of various vertebrate species (human, chicken, mouse, pig, cattle). Chimeric TLR5 receptors, consisting of human transmembrane and intracellular domains, linked to extracellular domains of animal origin, were generated and expressed. The comparison of chimeric TLR5s and their full-length counterparts revealed significant functional disparities. While porcine and chicken full-length TLR5s showed a strongly reduced functionality in human cells, all chimeric receptors were functional when challenged with TLR5 ligand Salmonella FliC. Using chimeric receptors as a tool allowed for the identification of ectodomain-dependent activation potential and partially host species-specific differences in response to various enteric bacterial strains and their purified flagellins. We conclude that both the extra- and intracellular determinants of TLR5 receptors are crucial for compatibility with the species expression background and hence for proper receptor functionality. TLR5 receptors with a common intracellular domain provide a useful system to investigate bacteria- and host-specific differences in receptor activation.

Short peptide analogs as alternatives to collagen in pro-regenerative corneal implants

Short collagen-like peptides (CLPs) are being proposed as alternatives to full-length collagen for use in tissue engineering, on their own as soft hydrogels, or conjugated to synthetic polymer for mechanical strength. However, despite intended clinical use, little is known about their safety and efficacy, mechanism of action or degree of similarity to the full-length counterparts they mimic. Here, we show the functional equivalence of a CLP conjugated to polyethylene glycol (CLP-PEG) to full-length recombinant human collagen in vitro and in promoting stable regeneration of corneal tissue and nerves in a pre-clinical mini-pig model. We also show that these peptide analogs exerted their pro-regeneration effects through stimulating extracellular vesicle production by host cells. Our results support future use of CLP-PEG implants for corneal regeneration, suggesting the feasibility of these or similar peptide analogs in clinical application in the eye and other tissues. Statement of significanceAlthough biomaterials comprising full-length recombinant human collagen and extracted animal collagen have been evaluated and used clinically, these macromolecules provide only a limited number of functional groups amenable to chemical modification or crosslinking and are demanding to process. Synthetic, customizable analogs that are functionally equivalent, and can be readily scaled-up are therefore very desirable for pre-clinical to clinical translation. Here, we demonstrate, using cornea regeneration as our test bed, that collagen-like-peptides conjugated to multifunctional polyethylene glycol (CLP-PEG) when grafted into mini-pigs as corneal implants were functionally equivalent to recombinant human collagen-based implants that were successfully tested in patients. We also show for the first time that these materials affected regeneration through stimulation of extracellular vesicle production by endogenous host cells that have migrated into the CLP-PEG scaffolds.

Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F.

Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine.IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo, resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge.

Role of the N-terminus for the stability of an amyloid-β fibril with three-fold symmetry.

A key player in Alzheimer’s disease is the peptide amyloid-beta (Aβ), whose aggregation into small soluble oligomers, protofilaments, and fibrils finally leads to plaque deposits in human brains. The aggregation behavior of Aβ is strongly modulated by the nature and composition of the peptide’s environment and by its primary sequence properties. The N-terminal residues of Aβ play an important role, because they are known to change the peptide’s aggregation propensity. Since these residues are for the first time completely resolved at the molecular level in a three-fold symmetric fibril structure derived from a patient, we chose that system as template for a systematic investigation of the influence of the N-terminus upon structural stability. Using atomistic molecular dynamics simulations, we examined several fibrillar systems comprising three, six, twelve and an infinite number of layers, both with and without the first eight residues. First, we found that three layers are not sufficient to stabilize the respective Aβ topology. Second, we observed a clear stabilizing effect of the N-terminal residues upon the overall fibril fold: truncated Aβ systems were less stable than their full-length counterparts. The N-terminal residues Arg5, Asp7, and Ser8 were found to form important interfilament contacts stabilizing the overall fibril structure of three-fold symmetry. Finally, similar structural rearrangements of the truncated Aβ species in different simulations prompted us to suggest a potential mechanism involved in the formation of amyloid fibrils with three-fold symmetry.

DPP4 truncated GM-CSF and IL-3 manifest distinct receptor-binding and regulatory functions compared with their full-length forms.

Dipeptidylpeptidase 4 (DPP4/CD26) enzymatically cleaves select penultimate amino acids of proteins, including colony stimulating factors (CSFs), and has been implicated in cellular regulation. To better understand the role of DPP4 regulation of hematopoiesis, we analyzed the activity of DPP4 on the surface of immature blood cells and then comparatively assessed the interactions and functional effects of full-length (FL) and DPP4 truncated factors [(T)-GM-CSF and- IL-3] on both in vitro and in vivo models of normal and leukemic cells. T-GM-CSF and T-IL-3 had enhanced receptor binding, but decreased CSF activity, compared to their FL forms. Importantly, T-GM-CSF and T-IL-3 significantly, and reciprocally, blunted receptor binding and myeloid progenitor cell proliferation activity of both FL-GM-CSF and FL-IL-3 in vitro and in vivo. Similar effects were apparent in vitro using cluster forming cells from patients with Acute Myeloid Leukemia (AML) regardless of cytogenetic or molecular alterations and in vivo utilizing animal models of leukemia. This suggests that DPP4 T-molecules have modified binding and functions compared to their FL counterparts and may serve regulatory roles in normal and malignant hematopoiesis.

Identification of Residues That Affect Oligomerization and/or Enzymatic Activity of Influenza Virus H5N1 Neuraminidase Proteins.

Influenza A virus (IAV) attachment to and release from sialoside receptors is determined by the balance between hemagglutinin (HA) and neuraminidase (NA). The molecular determinants that mediate the specificity and activity of NA are still poorly understood. In this study, we aimed to design the optimal recombinant soluble NA protein to identify residues that affect NA enzymatic activity. To this end, recombinant soluble versions of four different NA proteins from H5N1 viruses were compared with their full-length counterparts. The soluble NA ectodomains were fused to three commonly used tetramerization domains. Our results indicate that the particular oligomerization domain used does not affect the Km value but may affect the specific enzymatic activity. This particularly holds true when the stalk domain is included and for NA ectodomains that display a low intrinsic ability to oligomerize. NA ectodomains extended with a Tetrabrachion domain, which forms a nearly parallel four-helix bundle, better mimicked the enzymatic properties of full-length proteins than when other coiled-coil tetramerization domains were used, which probably distort the stalk domain. Comparison of different NA proteins and mutagenic analysis of recombinant soluble versions thereof resulted in the identification of several residues that affected oligomerization of the NA head domain (position 95) and therefore the specific activity or sialic acid binding affinity (Km value; positions 252 and 347). This study demonstrates the potential of using recombinant soluble NA proteins to reveal determinants of NA assembly and enzymatic activity. The IAV HA and NA glycoproteins are important determinants of host tropism and pathogenicity. However, NA is relatively understudied compared to HA. Analysis of soluble versions of these glycoproteins is an attractive way to study their activities, as they are easily purified from cell culture media and applied in downstream assays. In the present study, we analyzed the enzymatic activity of different NA ectodomains with three commonly used tetramerization domains and compared them with full-length NA proteins. By performing a mutagenic analysis, we identified several residues that affected NA assembly, activity, and/or substrate binding. In addition, our results indicate that the design of the recombinant soluble NA protein, including the particular tetramerization domain, is an important determinant for maintaining the enzymatic properties within the head domain. NA ectodomains extended with a Tetrabrachion domain better mimicked the full-length proteins than when the other tetramerization domains were used.

Functional Analysis of the Chloroplast Division Complex Using Schizosaccharomyces pombe as a Heterologous Expression System.

Chloroplast division is driven by a macromolecular complex that assembles at the midplastid. The FtsZ ring (Z ring) is the central structure in this complex, and is composed of the functionally distinct cytoskeletal proteins FtsZ1 and FtsZ2. Recent studies in the heterologous Schizosaccharomyces pombe system showed that Arabidopsis FtsZ1 and FtsZ2 filaments have distinct assembly and turnover characteristics. To further analyze these FtsZs, we employed this system to compare the assembly and dynamic properties of FtsZ1 and FtsZ2 lacking their N- and/or C-termini with those of their full-length counterparts. Our data provide evidence that the N-terminus of FtsZ2 is critical for its structural dominance over FtsZ1, and that the N- and C-termini promote polymer bundling and turnover of both FtsZs and contribute to their distinct behaviors. We also assessed how ARC6 affects FtsZ2 filament dynamics, and found that it interacts with and stabilizes FtsZ2 filaments in S. pombe independent of its presumed Z-ring tethering function in planta. Finally, we generated FtsZ1-FtsZ2 coexpression constructs to facilitate reconstitution of more complex interaction networks. Our experiments yield new insight into factors influencing FtsZ behavior and highlight the utility of S. pombe for analyzing chloroplast FtsZs and their assembly regulators.

Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM.

Measles virus (MeV) and canine distemper virus (CDV) possess tetrameric attachment proteins (H) and trimeric fusion proteins, which cooperate with either SLAM or nectin 4 receptors to trigger membrane fusion for cell entry. While the MeV H-SLAM cocrystal structure revealed the binding interface, two distinct oligomeric H assemblies were also determined. In one of the conformations, two SLAM units were sandwiched between two discrete H head domains, thus spotlighting two binding interfaces ("front" and "back"). Here, we investigated the functional relevance of both interfaces in activating the CDV membrane fusion machinery. While alanine-scanning mutagenesis identified five critical regulatory residues in the front H-binding site of SLAM, the replacement of a conserved glutamate residue (E at position 123, replaced with A [E123A]) led to the most pronounced impact on fusion promotion. Intriguingly, while determination of the interaction of H with the receptor using soluble constructs revealed reduced binding for the identified SLAM mutants, no effect was recorded when physical interaction was investigated with the full-length counterparts of both molecules. Conversely, although mutagenesis of three strategically selected residues within the back H-binding site of SLAM did not substantially affect fusion triggering, nevertheless, the mutants weakened the H-SLAM interaction recorded with the membrane-anchored protein constructs. Collectively, our findings support a mode of binding between the attachment protein and the V domain of SLAM that is common to all morbilliviruses and suggest a major role of the SLAM residue E123, located at the front H-binding site, in triggering the fusion machinery. However, our data additionally support the hypothesis that other microdomain(s) of both glycoproteins (including the back H-binding site) might be required to achieve fully productive H-SLAM interactions. A complete understanding of the measles virus and canine distemper virus (CDV) cell entry molecular framework is still lacking, thus impeding the rational design of antivirals. Both viruses share many biological features that partially rely on the use of analogous Ig-like host cell receptors, namely, SLAM and nectin 4, for entering immune and epithelial cells, respectively. Here, we provide evidence that the mode of binding between the membrane-distal V domain of SLAM and the attachment protein (H) of morbilliviruses is very likely conserved. Moreover, although structural information revealed two discrete conformational states of H, one of the structures displayed two H-SLAM binding interfaces ("front" and "back"). Our data not only spotlight the front H-binding site of SLAM as the main determinant of membrane fusion promotion but suggest that the triggering efficiency of the viral entry machinery may rely on a local conformational change within the front H-SLAM interactive site rather than the binding affinity.

Unique and Unexpected Functional Activities of Hematopoietically Active Cytokines Truncated By Dipeptidylpeptidase-4 (DPP4)

Dipeptidylpeptidase 4 (DPP4) is a serine peptidase with enzymatic activity leading to the N terminal cleavage of select penultimate amino acids of proteins. We previously published that the number of cytokines, chemokines and growth factors that have putative DPP4 truncation sites have been dramatically underestimated. Functional and mechanistic roles of full length (FL) versus DPP4 truncated (T) factors, as well as the ability of DPP4 T proteins to induce signaling that FL factors can not, have not been previously investigated and may have yet unappreciated clinical application. Here we present novel data demonstrating the here to fore unknown ability of DPP4 cleavage of proteins to alter not only cellular function, but intracellular signaling of growth factors leading to miRNA expression, phosphorylation, and global induction of proteins that the FL form of the protein does not induce. Additionally, and unexpectedly, a 1:1 mixture of the DPP4 T protein and their FL counterpart leads to both overlapping signaling between the FL and T, as well as unique signaling, that is not induced by the FL or T protein alone. This suggests multifaceted, important, and currently unappreciated roles that the DPP4 truncation of proteins may play in the regulation of normal and malignant hematopoiesis as well as other physiologic and pathophysiologic states.From functional data, utilizing the human factor-dependent TF-1 cell line, as well as primary samples from human cord blood (CB) and patients with Acute Myeloid Leukemia (AML), we observed that DPP4 truncation of GM-CSF (T-GM-CSF) and IL-3 (T-IL-3) results in decreased colony stimulating factor (CSF) activity in normal and malignant hematopoietic progenitor cells (HPC). Receptor binding studies confirmed that both T-GM-CSF and T-IL-3 have enhanced receptor affinity compared to their FL form and each can compete to blunt the receptor binding of either FL-GM-CSF or FL-IL-3. In vivo studies demonstrated that either exogenously added T-GM-CSF or T-IL-3 suppressed the effects of exogenously added FL-GM-CSF or FL-IL-3 on progenitor cell numbers per femur and diminished HPC cycling. Investigation of FL vs T mediated signaling alterations was done with TF-1 cells using proteomic and bioinformatic analysis of miRNA expression, and phosphorylation/global protein induction. Both T-GM-CSF and T-IL-3 induced unique signaling that FL-GM-CSF and FL-IL-3 did not, as well as signaling that overlapped with their FL counterparts (Figure 1, Venn Diagram example of global induction of proteins). Additionally, treatment with a 1:1 ratio of the FL/T proteins resulted in both distinctive, and common, signaling compared to that detected with treatment of only FL or T molecules, thus revealing the complexity of the signaling interactions and heretofore unknown activities of DPP4 truncated proteins (Figure 1). Fisher’s exact test or B-H multiple testing correction in IPA software were used to statistically generate comparisons of molecules associated with specific signaling induction, and differential as well as overlapping signaling for all GM-CSF vs IL-3 groups. As examples, for both the GM-CSF and IL-3 groups, molecules associated with PI3K/Akt signaling were increased in T and 1:1 groups vs treatment with FL molecules. In contrast, molecules affiliated with CDC42 signaling were induced by treatment with a 1:1 ratio of FL:T molecules but not in the FL or T groups alone for both GM-CSF and IL-3. Interestingly, phosphorylated proteins associated with Flt3 and IL-9 signaling in hematopoietic progenitors was increased in the T and 1:1 groups for GM-CSF and IL-3 compared to the FL groups. Conversely, phosphorylated molecules involved in nucleic acid metabolism were induced by the FL, T and 1:1 of the GM-CSF groups but only by the FL version of IL-3. Further, phosphorylated molecules associated with antigen presentation were only detected in the FL IL-3 and 1:1 IL-3 groups, and molecules functionally associated with carbohydrate metabolism and cellular therapeutics were only detected in the T and 1:1 groups for both GM-CSF and IL-3, and not in the FL stimulated groups. Collectively, these data substantiate that further investigation into the roles and regulation of DPP4, in normal and malignant hematopoiesis, will allow for a better understanding of the significance, and potential clinical utility, of DPP4 activity altering compounds as well as DPP4 truncated molecules. [Display omitted] DisclosuresBroxmeyer:CordUse: Membership on an entity’s Board of Directors or advisory committees.