Overexpressed Nup88 stabilized through interaction with Nup62 promotes NF-κB dependent pathways in cancer.

Bidirectional nucleo-cytoplasmic transport, regulating several vital cellular processes, is mediated by the Nuclear Pore Complex (NPC) comprising the nucleoporin (Nup) proteins. Nup88, a constituent nucleoporin, is overexpressed in many cancers, and a positive correlation exists between progressive stages of cancer and Nup88 levels. While a significant link of Nup88 overexpression in head and neck cancer exists but mechanistic details of Nup88 roles in tumorigenesis are sparse. Here, we report that Nup88 and Nup62 levels are significantly elevated in head and neck cancer patient samples and cell lines. We demonstrate that the elevated levels of Nup88 or Nup62 impart proliferation and migration advantages to cells. Interestingly, Nup88-Nup62 engage in a strong interaction independent of Nup-glycosylation status and cell-cycle stages. We report that the interaction with Nup62 stabilizes Nup88 by inhibiting the proteasome-mediated degradation of overexpressed Nup88. Overexpressed Nup88 stabilized by interaction with Nup62 can interact with NF-κB (p65) and sequesters p65 partly into nucleus of unstimulated cells. NF-κB targets like Akt, c-myc, IL-6 and BIRC3 promoting proliferation and growth are induced under Nup88 overexpression conditions. In conclusion, our data indicates that simultaneous overexpression of Nup62 and Nup88 in head and neck cancer stabilizes Nup88. Stabilized Nup88 interacts and activates p65 pathway, which perhaps is the underlying mechanism in Nup88 overexpressing tumors.

Human Behavior-Inspired Linchpin-Directed Catalysis for Traceless Precision Labeling of Lysine in Native Proteins.

The complex social ecosystem regulates the spectrum of human behavior. However, it becomes relatively easier to understand if we disintegrate the contributing factors, such as locality and interacting partners. Interestingly, it draws remarkable similarity with the behavior of a residue placed in a social setup of functional groups in a protein. Can it inspire principles for creating a unique environment for the precision engineering of proteins? We demonstrate that localization-regulated interacting partner(s) could render precise and traceless single-site modification of structurally diverse native proteins. The method targets a combination of high-frequency Lys residues through an array of reversible and irreversible reactions. However, excellent simultaneous control over chemoselectivity, site selectivity, and modularity ensures that the user-friendly protocol renders acyl group installation, including post-translational modifications (PTMs), on a single Lys. Besides, it offers a chemically orthogonal handle for the installation of probes. Also, a purification protocol integration delivers analytically pure single-site tagged protein bioconjugates. The precise labeling of a surface Lys residue ensures that the structure and enzymatic activities remain conserved post-bioconjugation. For example, the precise modification of insulin does not affect its uptake and downstream signaling pathway. Further, the method enables the synthesis of homogeneous antibody-fluorophore and antibody-drug conjugates (AFC and ADC; K183 and K249 labeling). The trastuzumab-rhodamine B conjugate displays excellent serum stability along with antigen-specific cellular imaging. Further, the trastuzumab-emtansine conjugate offers highly specific antiproliferative activity toward HER-2 positive SKBR-3 breast cancer cells. This work validates that disintegrate theory can create a comprehensive platform to enrich the chemical toolbox to meet the technological demands at the chemistry, biology, and medicine interface.

Traceless cysteine-linchpin enables precision engineering of lysine in native proteins.

The maintenance of machinery requires its operational understanding and a toolbox for repair. The methods for the precision engineering of native proteins meet a similar requirement in biosystems. Its success hinges on the principles regulating chemical reactions with a protein. Here, we report a technology that delivers high-level control over reactivity, chemoselectivity, site-selectivity, modularity, dual-probe installation, and protein-selectivity. It utilizes cysteine-based chemoselective Linchpin-Directed site-selective Modification of lysine residue in a protein (LDMC-K). The efficiency of the end-user-friendly protocol is evident in quantitative conversions within an hour. A chemically orthogonal C-S bond-formation and bond-dissociation are essential among multiple regulatory attributes. The method offers protein selectivity by targeting a single lysine residue of a single protein in a complex biomolecular mixture. The protocol renders analytically pure single-site probe-engineered protein bioconjugate. Also, it provides access to homogeneous antibody conjugates (AFC and ADC). The LDMC-K-ADC exhibits highly selective anti-proliferative activity towards breast cancer cells.

In vitro and in cellulae methods for determining the target protein SUMOylation.

Post-translational modifications (PTMs) provide a critical means of calibrating the functional proteome and, thus, are extensively utilized by the eukaryotes to exert spatio-temporal regulation on the cellular machinery rapidly. Ubiquitination and phosphorylation are examples of the well-documented PTMs. SUMOylation, the reversible conjugation of the Small Ubiquitin-related MOdifier (SUMO) at a specific lysine residue on a target protein, bears striking similarity with ubiquitination and follows an enzymatic cascade for the attachment of SUMO to the target protein. Unlike Ubiquitination, SUMOylation can modulate the target protein's structure, stability, activity, localization, and interaction. Thus, SUMOylation regulates cellular events such as signal transduction, cell-cycle progression, transcription, nucleocytoplasmic transport, and stress responses. Accordingly, deregulation of SUMOylation is an avenue for diseases, which makes the investigation of SUMO and its substrates within the cell essential. However, the low extent of SUMOylation has posed a significant challenge in detecting SUMO modification within the cell. Bioinformatics tools can help predict SUMOylation, and mass-spectrometric analysis can identify a pool of cellular protein SUMOylome. Nevertheless, the biochemical methods for observing the enhanced level of in vitro SUMOylation help validate protein SUMOylation, critical lysine(s) utilized in the process, and its effect on substrate protein function. This chapter provides a detailed account of biochemical methods commonly utilized to detect SUMOylated proteins that are central for understanding the biological functions and mechanism of regulation of SUMO targets.

Residue-specific N-terminal glycine to aldehyde transformation renders analytically pure single-site labeled proteins.

Here, we present N-Gly-specific glyoxamide generation in native proteins, isolated or in a complex mixture. The resulting aldehyde enables parallel installation of probes and a purification platform to render analytically pure single-site tagged proteins. It renders N-Gly engineered insulin without perturbing its structure, receptor binding, and downstream signaling pathway.

In Pursuit of Distinctiveness: Transmembrane Nucleoporins and Their Disease Associations.

The bi-directional nucleocytoplasmic shuttling of macromolecules like molecular signals, transcription factors, regulatory proteins, and RNAs occurs exclusively through Nuclear Pore Complex (NPC) residing in the nuclear membrane. This magnanimous complex is essentially a congregation of ~32 conserved proteins termed Nucleoporins (Nups) present in multiple copies and mostly arranged as subcomplexes to constitute a functional NPC. Nups participate in ancillary functions such as chromatin organization, transcription regulation, DNA damage repair, genome stabilization, and cell cycle control, apart from their central role as nucleocytoplasmic conduits. Thus, Nups exert a role in the maintenance of cellular homeostasis. In mammals, precisely three nucleoporins traverse the nuclear membrane, are called transmembrane Nups (TM-Nups), and are involved in multiple cellular functions. Owing to their vital roles in cellular processes and homeostasis, dysregulation of nucleoporin function is implicated in various diseases. The deregulated functioning of TM-Nups can thus act as an opportune window for the development of diseases. Indeed, mounting evidence exhibits a strong association of TM-Nups in cancer and numerous other physiological disorders. These findings have provided much-needed insights into the novel mechanisms of disease progression. While nucleoporin's functions have often been summarized in the disease context, a focus on TM-Nups has always lacked. This review emphasizes the elucidation of distinct canonical and non-canonical functions of mammalian TM-Nups and the underlying mechanisms of their disease association.

SUMO and SUMOylation Pathway at the Forefront of Host Immune Response.

Pathogens pose a continuous challenge for the survival of the host species. In response to the pathogens, the host immune system mounts orchestrated defense responses initiating various mechanisms both at the cellular and molecular levels, including multiple post-translational modifications (PTMs) leading to the initiation of signaling pathways. The network of such pathways results in the recruitment of various innate immune components and cells at the site of infection and activation of the adaptive immune cells, which work in synergy to combat the pathogens. Ubiquitination is one of the most commonly used PTMs. Host cells utilize ubiquitination for both temporal and spatial regulation of immune response pathways. Over the last decade, ubiquitin family proteins, particularly small ubiquitin-related modifiers (SUMO), have been widely implicated in host immune response. SUMOs are ubiquitin-like (Ubl) proteins transiently conjugated to a wide variety of proteins through SUMOylation. SUMOs primarily exert their effect on target proteins by covalently modifying them. However, SUMO also engages in a non-covalent interaction with the SUMO-interacting motif (SIM) in target proteins. Unlike ubiquitination, SUMOylation alters localization, interactions, functions, or stability of target proteins. This review provides an overview of the interplay of SUMOylation and immune signaling and development pathways in general. Additionally, we discuss in detail the regulation exerted by covalent SUMO modifications of target proteins, and SIM mediated non-covalent interactions with several effector proteins. In addition, we provide a comprehensive review of the literature on the importance of the SUMO pathway in the development and maintenance of a robust immune system network of the host. We also summarize how pathogens modulate the host SUMO cycle to sustain infectability. Studies dealing mainly with SUMO pathway proteins in the immune system are still in infancy. We anticipate that the field will see a thorough and more directed analysis of the SUMO pathway in regulating different cells and pathways of the immune system. Our current understanding of the importance of the SUMO pathway in the immune system necessitates an urgent need to synthesize specific inhibitors, bioactive regulatory molecules, as novel therapeutic targets.

Antihypernociceptive effects of Petersianthus macrocarpus stem bark on neuropathic pain induced by chronic constriction injury in rats.

Petersianthus macrocarpus (Lecythidaceae) stem bark is traditionally used in West and Central Africa for the treatment of boils and pain. The present study examined the chemical composition of the aqueous and methanolic stem bark extracts of P. macrocarpus by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) . Their antinociceptive effect was evaluated using chronic constriction injury (CCI)-induced neuropathic pain in a rat model. On the ninth day post-surgery, the pain perception (allodynia and hyperalgesia) of the animals was assessed after the administration of aqueous and methanolic extracts at the doses of 100 and 200 mg/kg. In addition, the effect of the extracts was evaluated on nitric oxide activity and on the expression of pro-inflammatory cytokines (TNF-α, IL-1ß, and NF-κB). The LC-ESI-MS analysis revealed the presence of ellagic acid as the major constituent in the methanol extract. Both extracts at the employed doses (100 and 200 mg/kg), significantly (p < 0.01 and p < 0.001) reduced the spontaneous pain, tactile and cold allodynia, and mechanical hyperalgesia. The methanolic extract used at the dose of 200 mg/kg significantly reduced the nitric oxide level (p < 0.001) and the gene expression levels of NF-κB (p < 0.05) and TNF-α (p < 0.01) in the brain. These data may indicate that stem bark extracts of P. macrocarpus possess a potent anti-hypernociceptive effect on CCI neuropathic pain. The inhibition of the nitric oxide pathway as well as the reduction in NF-κB and TNF-α gene expression in the brain may at least partially contribute to this effect. The results further support the use of this plant by traditional healers in pain conditions.

Linchpins empower promiscuous electrophiles to enable site-selective modification of histidine and aspartic acid in proteins.

The conservation of chemoselectivity becomes invalid for multiple electrophilic warheads during protein bioconjugation. Consequently, it leads to unpredictable heterogeneous labeling of proteins. Here, we report that a linchpin can create a unique chemical space to enable site-selectivity for histidine and aspartic acid modifications overcoming the pre-requisite of chemoselectivity.

Molecular and Phenotypic Characterization Following RNAi Mediated Knockdown in Drosophila.

Loss of function studies shed significant light on the involvement of a gene or gene product in different cellular processes. Short hairpin RNA (shRNA) mediated RNA interference (RNAi) is a classical yet straightforward technique frequently used to knock down a gene for assessing its function. Similar perturbations in gene expression can be achieved by siRNA, microRNA, or CRISPR-Cas9 methods also. In Drosophila genetics, the UAS-GAL4 system is utilized to express RNAi and make ubiquitous and tissue-specific knockdowns possible. The UAS-GAL4 system borrows genetic components of S. cerevisiae, hence rule out the possibility of accidental expression of the system. In particular, this technique uses a target-specific shRNA, and the expression of the same is governed by the upstream activating sequence (UAS). Controlled expression of GAL4, regulated by specific promoters, can drive the interfering RNA expression ubiquitously or in a tissue-specific manner. The knockdown efficiency is measured by RNA isolation and semiquantitative RT-PCR reaction followed by agarose gel electrophoresis. We have employed immunostaining procedure also to assess knockdown efficiency. RNAi provides researchers with an option to decrease the gene product levels (equivalent to hypomorph condition) and study the outcomes. UAS-GAL4 based RNAi method provides spatio-temporal regulation of gene expression and helps deduce the function of a gene required during early developmental stages also.

Implantable HDAC-inhibiting chemotherapeutics derived from hydrophobic amino acids for localized anticancer therapy.

Epigenetic targeting of different cancers by inhibiting particular histone deacetylase (HDAC) isozymes is a promising treatment approach against cancer. Development of locally-implantable molecular inhibitors of HDAC (henceforth called HDACi) promises high tumour site concentration and reduced systemic degradation of the HDACi. Herein, we report the design of such implantable HDACi based on amphiphilic derivatives of hydrophobic amino acids endowed with a hydroxamic acid (hxa)-based zinc-binding residue. The amino acids present in HDACi influenced the HDAC isozyme that could be inhibited most effectively; the l-phenylalanine derivative 4e inhibited the HDAC6 isozyme most potently (IC50 ∼ 88 nM), while the l-isoleucine derivative 4h was most effective against the isozyme HDAC2 (IC50 ∼ 94 nM). We also noticed that the l-Phe derivative 4e was up to 5× more potent towards inhibiting HDAC6 than its optical antipode 4f derived from d-Phe. This was rationalized in terms of the varying extent of penetration of the enantiomeric inhibitors inside the catalytic tunnel of the enzyme. Since the isozymes HDAC6 and HDAC2 are overexpressed in different cancer cells, 4e and 4h elicited selective anticancer activity in different cancer cell lines. Additive therapeutic action of the combination therapy of 4e and 4h was observed on lung cancer cells that overexpress both these isozymes. Further, 4e formed implantable self-assembled hydrogels that achieved sustained and selective killing of cancer cells in the vicinity of implantation.

Drosophila ELYS regulates Dorsal dynamics during development.

Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes into functional nuclear pores toward the end of mitosis. Using cellular, molecular, and genetic tools, including fluorescence and Electron microscopy, quantitative PCR, and RNAi-mediated depletion, we report here that the ELYS ortholog (dElys) plays critical roles during Drosophila development. dElys localized to the nuclear rim in interphase cells, but during mitosis it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated dElys depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that dElys depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes reaper and hid The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon dElys depletion. Moreover, we noted that dElys has conserved structural features, but contains a noncanonical AT-hook-like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by dElys during development.

A single amino acid Gly-tag enables metal-free protein purification.

Analytically pure proteins are indispensable for diverse applications, including therapeutics. Here, we report a methodology where a single amino acid, glycine, enables metal-free protein purification. This robust platform is enabled by a Gly-tag resin for site-specific capture, enrichment, and release through chemically triggered C-C bond dissociation by resonance-assisted electron density polarization.

Single-site glycine-specific labeling of proteins.

Labeling of native proteins invites interest from diverse segments of science. However, there remains the significant unmet challenge in precise labeling at a single site of a protein. Here, we report the site-specific labeling of natural or easy-to-engineer N-terminus Gly in proteins with remarkable efficiency and selectivity. The method generates a latent nucleophile from N-terminus imine that reacts with an aldehyde to deliver an aminoalcohol under physiological conditions. It differentiates N-Gly as a unique target amongst other proteinogenic amino acids. The method allows single-site labeling of proteins in isolated form and extends to lysed cells. It administers an orthogonal aldehyde group primed for late-stage tagging with an affinity tag, 19F NMR probe, and a fluorophore. A user-friendly protocol delivers analytically pure tagged proteins. The mild reaction conditions do not alter the structure and function of the protein. The cellular uptake of fluorophore-tagged insulin and its ability to activate the insulin-receptor mediated signaling remains unperturbed.

SUMOylation of periplakin is critical for efficient reorganization of keratin filament network.

The architecture of the cytoskeleton and its remodeling are tightly regulated by dynamic reorganization of keratin-rich intermediate filaments. Plakin family proteins associate with the network of intermediate filaments (IFs) and affect its reorganization during migration, differentiation, and response to stress. The smallest plakin, periplakin (PPL), interacts specifically with intermediate filament proteins K8, K18, and vimentin via its C-terminal linker domain. Here, we show that periplakin is SUMOylated at a conserved lysine in its linker domain (K1646) preferentially by small ubiquitin-like modifier 1 (SUMO1). Our data indicate that PPL SUMOylation is essential for the proper reorganization of the keratin IF network. Stresses perturbing intermediate-filament and cytoskeletal architecture induce hyper--SUMOylation of periplakin. Okadaic acid induced hyperphosphorylation-dependent collapse of the keratin IF network results in a similar hyper-SUMOylation of PPL. Strikingly, exogenous overexpression of a non-SUMOylatable periplakin mutant (K1646R) induced aberrant bundling and loose network interconnections of the keratin filaments. Time-lapse imaging of cells expressing the K1646R mutant showed the enhanced sensitivity of keratin filament collapse upon okadaic acid treatment. Our data identify an important regulatory role for periplakin SUMOylation in dynamic reorganization and stability of keratin IFs.

Single-Site Labeling of Native Proteins Enabled by a Chemoselective and Site-Selective Chemical Technology.

Chemical biology research often requires precise covalent attachment of labels to the native proteins. Such methods are sought after to probe, design, and regulate the properties of proteins. At present, this demand is largely unmet due to the lack of empowering chemical technology. Here, we report a chemical platform that enables site-selective labeling of native proteins. Initially, a reversible intermolecular reaction places the "chemical linchpins" globally on all the accessible Lys residues. These linchpins have the capability to drive site-selective covalent labeling of proteins. The linchpin detaches within physiological conditions and capacitates the late-stage installation of various tags. The chemical platform is modular, and the reagent design regulates the site of modification. The linchpin is a multitasking group and facilitates purification of the labeled protein eliminating the requirement of additional chromatography tag. The methodology allows the labeling of a single protein in a mixture of proteins. The precise modification of an accessible residue in protein ensures that their structure remains unaltered. The enzymatic activity of myoglobin, cytochrome C, aldolase, and lysozyme C remains conserved after labeling. Also, the cellular uptake of modified insulin and its downstream signaling process remain unperturbed. The linchpin directed modification (LDM) provides a convenient route for the conjugation of a fluorophore and drug to a Fab and monoclonal antibody. It delivers trastuzumab-doxorubicin and trastuzumab-emtansine conjugates with selective antiproliferative activity toward Her-2 positive SKBR-3 breast cancer cells.

An Endogenous Reactive Oxygen Species (ROS)-Activated Histone Deacetylase Inhibitor Prodrug for Cancer Chemotherapy.

Suberoylanilide hydroxamic acid (SAHA, vorinostat) is a potent small-molecule pan-inhibitor of histone deacetylases (HDACs) approved for treatment of cutaneous T-cell lymphoma (CTCL). However, SAHA exhibits poor selectivity for cancer cells over noncancer cells. With an aim to improving its selectivity for cancer cells, we generated a novel SAHA prodrug (SAHA-OBP) that is activated in the presence of hydrogen peroxide, a reactive oxygen species (ROS) known to be overexpressed in cancer cells. The high endogenous ROS content in cancer cells triggers rapid removal of the 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonyl (OBP) cap to release active SAHA. The SAHA-OBP prodrug demonstrates selective activity against multiple cancer cell lines such as HeLa, MCF-7, MDA-MB-231, and B16-F10, while remaining benign toward noncancer cells. The downstream effects of SAHA released from SAHA-OBP in cancer cells is the induction of apoptosis. SAHA-OBP was also found to be effective on multicellular tumor spheroids (MCTS). The SAHA prodrug designed in this study undergoes rapid ROS-dependent activation and imparts much-needed selectivity to SAHA for cancer cells.

Diffuse panbronchiolitis research in MEDLINE (1969-2017): Report of a bibliometric analysis.

BACKGROUND: Given the well documented and undesired impact of diffuse panbronchiolitis (DPB), there is a need to create a statistical inventory of research output on DPB. The aim of this study was to identify and analyze different trends in publication over time, with technological additions. METHODS: Articles indexed in MEDLINE regarding DPB were retrieved using PubMed. Citation results were categorized by article type, year of publication, language, and country. RESULTS: A total of 657 publications, published between 1969 and 2017 were retrieved from PubMed; the year with the highest number of articles published was 1992 (n = 32). Japan accounted for 68.34% (n = 449) publications, followed by the United States of America (USA; 6.70%, n = 44) and China (5.48%, n = 36). English (n = 401) was the most commonly used language in the publications, followed by Japanese (n = 223) and Chinese (n = 16). Articles were published in 218 different journals; The Japanese Journal of Thoracic Diseases published the maximum number of articles, accounting for 13.7% (n = 90) citations. Sugiyama Y was the most productive author and contributed to 12 publications. CONCLUSIONS: This was the first bibliometric analysis of DPB. Between 1991 and 2000, the number of publications on DPB literature reached a peak, and although the frequency of publication has decreased recently, the quantity of DPB research during the past 48 years is adequate and satisfactory. Overall, the publications on DPB have undergone exponential growth over the last 30 years.