Publications by authors named "Jingjing Ling"

23 Publications

  • Page 1 of 1

Fish Waste Based Lipopeptide Production and the Potential Application as a Bio-Dispersant for Oil Spill Control.

Front Bioeng Biotechnol 2020 3;8:734. Epub 2020 Jul 3.

NRPOP Laboratory, Faculty of Engineering and Applied Science, Memorial University of Newfoundland, St. John's, NL, Canada.

There is a growing acceptance worldwide for the application of dispersants as a marine oil spill response strategy. The development of more effective dispersants with less toxicity and higher biodegradability would be a step forward in improving public acceptance and regulatory approvals for their use. By applying advances in environmental biotechnology, a bio-dispersant agent with a lipopeptide biosurfactant produced by N3-1P as the key component was formulated in this study. The economic feasibility of producing biosurfactant (a high-added-value bioproduct) from fish waste-based peptone as a nutrient substrate was evaluated. Protein hydrolyzate was prepared from cod liver and head wastes obtained from fish processing facilities. Hydrolysis conditions (i.e., time, temperature, pH and enzyme to substrate level) for preparing protein hydrolyzates were optimized by response surface methodology using a factorial design. The critical micelle dilution (CMD) value for biosurfactant produced from the fish liver and head waste generated peptones was 54.72 and 47.59 CMD, respectively. Biosurfactant product generated by fish liver peptone had a low critical micelle concentration of 0.18 g L and could reduce the surface tension of distilled water to 27.9 mN/m. Structure characterization proved that the generated biosurfactant product belongs to the lipopeptide class. An alternative to the key surfactant dioctyl sulfosuccinate sodium (DOSS) used in Corexit 9500 has been proposed based on a binary mixture of lipopeptides and DOSS that exhibited synergistic effects. Using the standard baffled flask test, a high dispersion efficiency of 76.8% for Alaska North Slope oil was achieved at a biodispersant composition of 80/20 (v/v) of lipopeptides/DOSS. The results show that fish waste can be utilized to produce a more effective, environmentally acceptable and cost-efficient biodispersant that can be applied to oil spills in the marine environment.
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http://dx.doi.org/10.3389/fbioe.2020.00734DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347989PMC
July 2020

A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity.

Mol Immunol 2019 10 10;114:513-523. Epub 2019 Sep 10.

Program in Cellular and Molecular Medicine, Boston Children's Hospital and Harvard Medical School, Boston, MA, 02115, USA. Electronic address:

A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitroand interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e's activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility.
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http://dx.doi.org/10.1016/j.molimm.2019.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774866PMC
October 2019

Immunometabolism features of metabolic deregulation and cancer.

J Cell Mol Med 2019 02 18;23(2):694-701. Epub 2018 Nov 18.

Department of Pharmacy, COMSATS University Islamabad, Lahore, Pakistan.

Immunometabolism is a branch dealing at the interface of immune functionalities and metabolic regulations. Considered as a bidirectional trafficking, metabolic contents and their precursors bring a considerable change in immune cells signal transductions which as a result affect the metabolic organs and states as an implication. Lipid metabolic ingredients form a major chunk of daily diet and have a proven contribution in immune cells induction, which then undergo metabolic pathway shuffling inside their ownself. Lipid metabolic states activate relevant metabolic pathways inside immune cells that in turn prime appropriate responses to outside environment in various states including lipid metabolic disorders itself and cancers as an extension. Although data on Immunometabolism are still growing, but scientific community need to adjust and readjust according to recent data on given subject. This review attempts to provide current important data on Immunometabolism and consequently its metabolic ramifications. Incumbent data on various lipid metabolic deregulations like obesity, metabolic syndrome, obese asthma and atherosclerosis are analysed. Further, metabolic repercussions on cancers and its immune modalities are also analysed.
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http://dx.doi.org/10.1111/jcmm.13977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6349168PMC
February 2019

Targeted Delivery of Cyclotides via Conjugation to a Nanobody.

ACS Chem Biol 2018 10 5;13(10):2973-2980. Epub 2018 Oct 5.

Whitehead Institute for Biomedical Research , 9 Cambridge Center , Cambridge , Massachusetts 02142 , United States.

Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.
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http://dx.doi.org/10.1021/acschembio.8b00653DOI Listing
October 2018

Nanobody-Antigen Conjugates Elicit HPV-Specific Antitumor Immune Responses.

Cancer Immunol Res 2018 07 23;6(7):870-880. Epub 2018 May 23.

Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, Massachusetts.

High-risk human papillomavirus-associated cancers express viral oncoproteins (e.g., E6 and E7) that induce and maintain the malignant phenotype. The viral origin of these proteins makes them attractive targets for development of a therapeutic vaccine. Camelid-derived single-domain antibody fragments (nanobodies or VHHs) that recognize cell surface proteins on antigen-presenting cells (APC) can serve as targeted delivery vehicles for antigens attached to them. Such VHHs were shown to induce CD4 and CD8 T-cell responses against model antigens conjugated to them via sortase, but antitumor responses had not yet been investigated. Here, we tested the ability of an anti-CD11b VHH (VHH) to target APCs and serve as the basis for a therapeutic vaccine to induce CD8 T-cell responses against HPV tumors. Mice immunized with VHH conjugated to an H-2D-restricted immunodominant E7 epitope (E7) had more E7-specific CD8 T cells compared with those immunized with E7 peptide alone. These CD8 T cells acted prophylactically and conferred protection against a subsequent challenge with HPV E7-expressing tumor cells. In a therapeutic setting, VHH-E7 vaccination resulted in greater numbers of CD8 tumor-infiltrating lymphocytes compared with mice receiving E7 peptide alone in HPV tumor-bearing mice, as measured by noninvasive VHH-based immune-positron emission tomography (immunoPET), which correlated with tumor regression and survival outcome. Together, these results demonstrate that VHHs can serve as a therapeutic cancer vaccine platform for HPV-induced cancers. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030498PMC
July 2018

IL-17 induces cellular stress microenvironment of melanocytes to promote autophagic cell apoptosis in vitiligo.

FASEB J 2018 09 3;32(9):4899-4916. Epub 2018 Apr 3.

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China.

Vitiligo is a depigmentary disorder that develops as a result of the progressive disappearance of epidermal melanocytes. Stress can precipitate or exacerbate a skin disease through psychosomatic mechanisms. Stress exposure induces vitiligo-like symptoms in mice, as cellular damage to melanocytes causes synthetic pigment loss. Stress also increases IL-17, IL-1β, and antimelanocyte IgG in model mouse serum. Up-regulation of the IL-1β transcript in patients suggests its possible role in autoimmune pathogenesis of vitiligo. We demonstrate that IL-17 promoted IL-1β secretion from keratinocytes. Mitochondrial dysfunction, which can induce the excessive production of reactive oxygen species (ROS), is emerging as a mechanism that underlies various inflammatory and autoimmune diseases. In this study, we demonstrate that IL-17 inhibits melanogenesis of zebrafish, normal human epidermal melanocytes, and B16F10 cells. IL-17 increased mitochondrial dysfunction and ROS accumulation, which was related to autophagy induction. Autophagy is needed for autophagic apoptosis of B16F10 cells induced by IL-17. To inhibit ROS generation, B16F10 cells were pretreated with N-acetyl-l-cysteine (NAC), which inhibited autophagy. 3-Methyladenine (3-MA) also had an inhibiting effect on autophagy. NAC or 3-MA pretreatments inhibited IL-17-mediated cell apoptosis. In summary, IL-17 induces the cellular stress microenvironment in melanocytes to promote autophagic cell apoptosis in vitiligo.-Zhou, J., An, X., Dong, J., Wang, Y., Zhong, H., Duan, L., Ling, J., Ping, F., Shang, J. IL-17 induces cellular stress microenvironment of melanocytes to promote autophagic cell apoptosis in vitiligo.
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http://dx.doi.org/10.1096/fj.201701242RRDOI Listing
September 2018

Targeted antigen delivery by an anti-class II MHC VHH elicits focused αMUC1(Tn) immunity.

Chem Sci 2017 Aug 26;8(8):5591-5597. Epub 2017 May 26.

Whitehead Institute for Biomedical Research , 9 Cambridge Center , Cambridge , MA 02142 , USA . Email:

Unusual patterns of glycosylation on the surface of transformed cells contribute to immune modulation and metastasis of malignant tumors. Active immunization against them requires effective antigen presentation, which is complicated by a lack of access to tumor-specific posttranslational modifications through standard genetic approaches and by the low efficiency of passive antigen sampling. We found that antigen targeted to antigen presenting cells class II MHC products can elicit a robust immune response against MUC1(Tn) bearing a defined tumor-associated glycoform, Tn. The two-component vaccine construct was prepared by sortase-mediated protein ligation of a synthetic MUC1(Tn) fragment to a class II MHC-binding single-domain antibody fragment (VHH7) as targeting moiety. We show that VHH7 targets antigen presenting cells , and when conjugated to MUC1(Tn) can elicit a strong αMUC1(Tn) immune response in mice. The resulting sera preferentially recognized the MUC1 epitope with the tumor-associated carbohydrate antigen Tn and were capable of killing cancer cells in a complement-mediated cytotoxicity assay. Immunoglobulin isotype analysis and cytokine release assays suggested a favorable Th1 response. A single boost 12 months after primary immunization triggered a recall response of the same quality, suggesting that long-term αMUC1(Tn) memory had been achieved.
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http://dx.doi.org/10.1039/c7sc00446jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5618788PMC
August 2017

An RNA nanoparticle vaccine against Zika virus elicits antibody and CD8+ T cell responses in a mouse model.

Sci Rep 2017 03 21;7(1):252. Epub 2017 Mar 21.

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA, 02142, USA.

The Zika virus (ZIKV) outbreak in the Americas and South Pacific poses a significant burden on human health because of ZIKV's neurotropic effects in the course of fetal development. Vaccine candidates against ZIKV are coming online, but immunological tools to study anti-ZIKV responses in preclinical models, particularly T cell responses, remain sparse. We deployed RNA nanoparticle technology to create a vaccine candidate that elicited ZIKV E protein-specific IgG responses in C57BL/6 mice as assayed by ELISA. Using this tool, we identified a unique H-2D-restricted epitope to which there was a CD8 T cell response in mice immunized with our modified dendrimer-based RNA nanoparticle vaccine. These results demonstrate that this approach can be used to evaluate new candidate antigens and identify immune correlates without the use of live virus.
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http://dx.doi.org/10.1038/s41598-017-00193-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427874PMC
March 2017

Neurokinin-1 receptor is a novel positive regulator of Wnt/ β-catenin signaling in melanogenesis.

Oncotarget 2016 Dec;7(49):81268-81280

Wuxi People's Hospital affiliated to Nanjing Medical University, Wuxi 214023, P.R. China.

Wnt/β-catenin signaling is essential for melanogenesis in melanocytes. Neurokinin-1 receptor (NK-1R) has recently been demonstrated to be involved in melanin production. However, the cross talk between NK-1R and Wnt/β-catenin is poorly understood. Here, [Sar9, Met(O2)11] substance P (SMSP) was used to activate NK-1R, while L-733060 was used to inhibit it. The effects of NK-1R activation and inhibition on Wnt and its inhibitors were analyzed using western blot and real-time quantitative PCR. The results showed that SMSP positively regulated Wnt/β-catenin signaling by increasing the expression of β-catenin and p-GSK3β protein, which resulted from the weakened expression of the Wnt inhibitor Dickkopf-1 (DKK1). On the contrary, L-733060 lowered the expression of β-catenin and p-GSK3β protein through the up-regulation of DKK1 expression. Furthermore, in L-733060-treated mice, it was found that the pigmentation level as well as the melanogenic proteins and β-catenin protein expression were down-regulated, while the expression of DKK1 was up-regulated. These results showed the interaction between NK-1R and Wnt in human melanocytes in vitro and C57BL/6J mice in vivo, indicating that NK-1R may positively regulate melanogenesis through Wnt/β-catenin signaling pathway.
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http://dx.doi.org/10.18632/oncotarget.13222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5348391PMC
December 2016

Posttranscriptional Regulation of Glycoprotein Quality Control in the Endoplasmic Reticulum Is Controlled by the E2 Ub-Conjugating Enzyme UBC6e.

Mol Cell 2016 09 25;63(5):753-67. Epub 2016 Aug 25.

Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address:

ER-associated degradation (ERAD) is essential for protein quality control in the ER, not only when the ER is stressed, but also at steady state. We report a new layer of homeostatic control, in which ERAD activity itself is regulated posttranscriptionally and independently of the unfolded protein response by adjusting the endogenous levels of EDEM1, OS-9, and SEL1L (ERAD enhancers). Functional UBC6e requires its precise location in the ER to form a supramolecular complex with Derlin2. This complex targets ERAD enhancers for degradation, a function that depends on UBC6e's enzymatic activity. Ablation of UBC6e causes upregulation of active ERAD enhancers and so increases clearance not only of terminally misfolded substrates, but also of wild-type glycoproteins that fold comparatively slowly in vitro and in vivo. The levels of proteins that comprise the ERAD machinery are thus carefully tuned and adjusted to prevailing needs.
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http://dx.doi.org/10.1016/j.molcel.2016.07.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010495PMC
September 2016

Cross-talk between interferon-gamma and interleukin-18 in melanogenesis.

J Photochem Photobiol B 2016 Oct 21;163:133-43. Epub 2016 Aug 21.

Wuxi People's Hospital affiliated to Nanjing Medical University, Wuxi 214023, PR China. Electronic address:

Skin is the largest organ in our body and strategically placed to provide a metabolically active biological barrier against a range of noxious stressors. A lot of inflammatory cytokines, which are increased after ultraviolet (UV) irradiation produced by keratinocytes or other immunocytes, are closely related to pigmentary changes, including interleukin-18 (IL-18) and interferon-γ (IFN-γ). In this study, the effect of cross-talk between IL-18 and IFN-γ on melanogenesis was investigated. Treatment with IL-18 resulted in a dose-dependent increase of melanogenesis, while IFN-γ made an opposite effect. This influence of IL-18 and IFN-γ was mediated by regulations of microphthalmia-associated transcription factor (MITF) and its downstream enzymatic cascade expressions. Furthermore, IFN-γ inhibited basal and IL-18-induced melanogenesis. IFN-γ increased signal transducer and activator of transcription-1 (STAT-1) phosphorylation to play its position in regulating melanin pigmentation, and its inhibitory effect could be prevented by Janus Kinase 1 (JAK 1) inhibitor. IFN-γ could inhibit melanogenesis by decreasing melanocyte dendrite formation. In addition, IFN-γ inhibited the expressions of Rab Pases to suppress the mature and transport of melanosomes. IL-18 could rapidly induce Akt and PTEN phosphorylation and p65 expression in B16F10 cells. When treatment with IL-18 and IFN-γ together, the phosphorylation level of Protein Kinase B (Akt) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) and expression of p65 NF-κB were inhibited, compared with treated with IL-18 only. Our studies indicated that IFN-γ could directly induce B16F10 cells apoptosis in vitro. Furthermore, we demonstrated that IFN-γ markedly up-regulated IL-18 binding protein (BP) production in normal human foreskin-derived epidermal keratinocytes in dose-dependent manner. UVB irradiation induced protease-activated receptor-2 (PAR-2) expression in NHEK, IFN-γ could inhibit this enhancement in a dose-dependent manner. These data suggest that IFN-γ plays a role in regulating inflammation- or UV-induced pigmentary changes, in direct/indirect manner.
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http://dx.doi.org/10.1016/j.jphotobiol.2016.08.024DOI Listing
October 2016

Interferon-γ Attenuates 5-Hydroxytryptamine-Induced Melanogenesis in Primary Melanocyte.

Biol Pharm Bull 2016 ;39(7):1091-9

School of Pharmaceutical Science, Jiangnan University.

Interferon-γ (IFN-γ) is an important cytokine which can be secreted by keratinocytes or macrophages induced by UVB irradiation in skin. Mammalian skin cells have the capability to produce and metabolize 5-hydroxytryptamine (5-HT) whose cutaneous effects are mediated by the interactions with 5-HT receptors. Treatment with 5-HT resulted in a dose-dependent increase of tyrosinase (TYR) activity and melanin contents in normal human foreskin-derived epidermal melanocytes (NHEM), while with IFN-γ a decreased effect resulted. These regulatory results were due to changes of the expression levels of microphthalmia-associated transcription factor (MITF) and its downstream TYR, tyrosinase-related protein 1 (TRP-1) and dopachrome tautomerase (DCT). We proved here that 5-HTR1A/2A participated in the regulation of melanogenesis. IFN-γ could offset the pro-melanogenesis effect of 5-HT in NHEM and the intensity of this neutralization was unanticipated below the baseline level. IFN-γ neutralized the up-regulation effect of 5-HT on MITF and downstream TYR, TRP-1 and DCT. Though functioning as 5-HT1A/2A receptor during the melanogenesis process, IFN-γ played no role in 5-HT1A/2A receptor expressions. Our results also demonstrated that the inhibition of IFN-γ was reversible after its removal. Confusingly, the effect of cross-talk between 5-HT and IFN-γ on NHEM melanogenesis was irreversible. Whether treated with 5-HT for 5 d or 12 d, the pigmentation level neither recovered after displacing the IFN-γ-containing medium. In addition, IFN-γ was able to inhibit the inductive effect of 5-HT on NHEM migration. Taken together, the suppression of IFN-γ on 5-HT-induced melanogenesis further suggests the negative role of IFN-γ in inflammation-associated pigmentary changes.
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http://dx.doi.org/10.1248/bpb.b15-00914DOI Listing
January 2017

Interleukin 10 protects primary melanocyte by activation of Stat-3 and PI3K/Akt/NF-κB signaling pathways.

Cytokine 2016 07 14;83:275-281. Epub 2016 May 14.

Department of Clinical Laboratory Science, Wuxi People's Hospital Affiliated to Nanjing Medical University, Wuxi 214023, PR China. Electronic address:

Vitiligo is a common melanocytopenic disorder of the skin, with acquired focal depigmentation. Normal human skin relies on melanocytes to provide photoprotection and thermoregulation by producing melanin. Interleukin 10 (IL-10) is a pleiotropic immunoregulatory cytokine drawing more and more researchers' attention. The present study was conducted to investigate the effects of IL-10 on melanocytes and elucidate the underlying mechanisms. We proved that IL-10 play no role in regulating melanogenesis of normal human foreskin-derived epidermal melanocytes (NHEM). IL-10 stimulation activated the JAK/Stat-3 and PI3K/Akt signaling pathways. Moreover, IL-10 treatment increased translocation of p65 NF-κB into the nuclear compartment, and up-regulated expression of the pro-survival proteins Bcl-2 and Bcl-xL. IL-10 restored anti-apoptotic proteins expression and suppressed cytochrome c release in H2O2-induced apoptosis. In conclusion, IL-10 may provide pro-survival cues to melanocytes and be applied in the treatment of vitiligo and other depigmenting disorders.
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http://dx.doi.org/10.1016/j.cyto.2016.05.013DOI Listing
July 2016

Crystal structure of a substrate-engaged SecY protein-translocation channel.

Nature 2016 Mar 7;531(7594):395-399. Epub 2016 Mar 7.

Howard Hughes Medical Institute and Harvard Medical School, Department of Cell Biology, 240 Longwood Avenue, Boston, MA 02115, USA.

Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61α, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation. How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (~10 Å) or are of insufficient quality. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.
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http://dx.doi.org/10.1038/nature17163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4855518PMC
March 2016

Structurally Defined αMHC-II Nanobody-Drug Conjugates: A Therapeutic and Imaging System for B-Cell Lymphoma.

Angew Chem Int Ed Engl 2016 Feb 14;55(7):2416-20. Epub 2016 Jan 14.

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA, 02142, USA.

Antibody-drug conjugates (ADCs) of defined structure hold great promise for cancer therapies, but further advances are constrained by the complex structures of full-sized antibodies. Camelid-derived single-domain antibody fragments (VHHs or nanobodies) offer a possible solution to this challenge by providing expedited target screening and validation through switching between imaging and therapeutic activities. We used a nanobody (VHH7) specific for murine MHC-II and rendered "sortase-ready" for the introduction of oligoglycine-modified cytotoxic payloads or NIR fluorophores. The VHH7 conjugates outcompeted commercial monoclonal antibodies (mAbs) for internalization and exhibited high specificity and cytotoxicity against A20 murine B-cell lymphoma. Non-invasive NIR imaging with a VHH7-fluorophore conjugate showed rapid tumor targeting on both localized and metastatic lymphoma models. Subsequent treatment with the nanobody-drug conjugate efficiently controlled tumor growth and metastasis without obvious systemic toxicity.
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http://dx.doi.org/10.1002/anie.201509432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820396PMC
February 2016

Combinatorial bead-based peptide libraries improved for rapid and robust screenings.

Comb Chem High Throughput Screen 2014 ;17(6):520-30

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos 138669, Singapore.

In pursuit of utilizing combinatorial peptide libraries on beads, rapid and robust screening is one of the key steps for the success of high-throughput process. We have introduced improved structural features that greatly facilitate a MALDI-MS/MS-based sequencing, associated with easy and fast synthesis and analysis of such libraries. Whilst commonly used MS-based analysis involves in sophisticated procedures such as ladder synthesis, encoding tags are not required in our MS/MS-based sequencing platform. Fragment peaks in an acquired MS/MS should be outstanding in line with correct identification of parent mass in the preceding MS. To meet these requirements a one-bead-one-compound (OBOC) peptide library was designed by placing a positively charged arginine at C-terminus. As well as enhancing the overall ionization efficiency, arginine appended in all y-ion fragments generates a series of doublet peaks under MS/MS environments, which can speed up the sequencing process in conjunction with high accuracy. It is another strong benefit that the designed library significantly suppresses the adverse formation of sodium ion adducts, which seriously jeopardizes the sequencing, especially of peptides containing negatively charged amino acids. A peptide library constructed with D-amino acids was applied to screening against a clinically significant biomarker, C-reactive protein (CRP). Through the screening of focused libraries narrowed down from a comprehensive library, several hexamer peptide ligands were successfully identified and their binding affinity and specificity towards CRP were validated by surface plasmon resonance (SPR) and dot blot experiments.
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http://dx.doi.org/10.2174/1386207317666140113114403DOI Listing
April 2015

The activation of p38 and JNK by ROS, contribute to OLO-2-mediated intrinsic apoptosis in human hepatocellular carcinoma cells.

Food Chem Toxicol 2014 Jan 5;63:38-47. Epub 2013 Nov 5.

Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China. Electronic address:

In this study, we describe that a novel synthesized compound, olean-28,13β-olide 2 (OLO-2), exhibits selective cytotoxic activity via inducing apoptosis in human hepatocellular carcinoma (HCC) cell lines but not normal human hepatic cells in vitro. Exposure of human HCC HepG2 cells to OLO-2 results in significant loss of mitochondrial transmembrane potential (ΔΨm), the release of cytochrome c, the recruitment of B-cell lymphoma 2 (Bcl-2) assaciated X protein (Bax) and the downregulation of Bcl-2. The apoptosis induced by OLO-2 is associated with the activation of caspase-3/9 and the nuclear translocation of apoptosis inducing factor (AIF). Moreover, the increase of phosphorylated p38 and c-Jun N-terminal kinase (JNK) is observed. OLO-2-induced the externalization of phosphatidyl-serine (PS) and the loss of ΔΨm are blocked by p38 inhibitor SB203580 or JNK inhibitor SP600125. In addition, OLO-2 provokes the generation of reactive oxygen species (ROS) in HepG2 cells, while the antioxidant N-acetyl cysteine (NAC) almost completely blocks OLO-2-induced apoptosis and the activation of p38 and JNK. Taken together, the present study demonstrates that OLO-2 exhibits its cytotoxic activity through intrinsic apoptosis via ROS generation and the activation of p38 and JNK. Its potential to be a candidate of anti-cancer agent is worth being further investigated.
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http://dx.doi.org/10.1016/j.fct.2013.10.043DOI Listing
January 2014

A perfluoroaryl-cysteine S(N)Ar chemistry approach to unprotected peptide stapling.

J Am Chem Soc 2013 Apr 16;135(16):5946-9. Epub 2013 Apr 16.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

We report the discovery of a facile transformation between perfluoroaromatic molecules and a cysteine thiolate, which is arylated at room temperature. This new approach enabled us to selectively modify cysteine residues in unprotected peptides, providing access to variants containing rigid perfluoroaromatic staples. This stapling modification performed on a peptide sequence designed to bind the C-terminal domain of an HIV-1 capsid assembly polyprotein (C-CA) showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog. Importantly, chemical stability of the formed staples allowed us to use this motif in the native chemical ligation-mediated synthesis of a small protein affibody that is capable of binding the human epidermal growth factor 2 receptor.
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http://dx.doi.org/10.1021/ja400119tDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3675880PMC
April 2013

Bacterial adhesion on honeycomb-structured poly(L-lactic acid) surface with ag nanoparticles.

J Biomed Nanotechnol 2012 Oct;8(5):791-9

Jiangsu Key Laboratory for Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.

Polymeric materials with ordered porous structure have received increasing interest due to their potential applications in biomaterials. However, the enhancement of cell proliferation by this porous structure also raises worries about the increase of bacterial adhesion. For their further use as biomaterials in vivo, it is essential to assess the bacterial adhesion on porous polymer films and find strategies to inhibit the bacterial retention. Honeycomb-structured poly(L-lactic acid) (PLLA) films with Ag nanoparticles were used in this purpose. The Ag+ release rate of Ag/PLLA films was analyzed. In vitro bacterial adhesions of S. aureus and E. coli on flat PLLA films, flat Ag/PLLA films, and honeycomb-structured Ag/PLLA films were compared. Ag nanoparticles activity appears to be more effective against bacteria in honeycomb-structured films compared with flat films of the same material. This activity is particularly significant when E. col is used. The results suggest that the honeycomb surface topography, as well as the enhanced release of Ag+, can greatly contribute to the anti-adhesion property of PLLA surface. These honeycomb-structured Ag/PLLA films also have no significant cytotoxicity. The decoration of Ag nanoparticles in honeycomb structure provides an effective and safe strategy to reduce the bacterial adhesion on porous polymer surface.
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http://dx.doi.org/10.1166/jbn.2012.1432DOI Listing
October 2012

Discovery of a potential anti-ischemic stroke agent: 3-pentylbenzo[c]thiophen-1(3H)-one.

J Med Chem 2012 Aug 7;55(16):7173-81. Epub 2012 Aug 7.

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, P. R. China.

The development of novel antithrombotic agents with strong free radical scavenging activity is of great significance for the treatment of ischemic stroke. In the present study, 3-alkyl/arylalkyl-substituted benzo[c]thiophen-1(3H)-ones (5a-h) were designed and synthesized. The most active compound 5d significantly inhibited the adenosine diphosphate (ADP) induced and arachidonic acid (AA) induced in vitro platelet aggregation, superior to clinically used antiplatelet drug aspirin (ASP) and anti-ischemic stroke drugs 3-n-butylphthalide (NBP) and edaravone (Eda). More importantly, in comparison with both NBP and Eda, 5d exhibited stronger antithrombotic and free radical scavenging activities and better or comparable neuroprotective effects against ischemia/reperfusion (I/R) in rats by ameliorating neurobehavioral function, reducing infarct size and brain-water content, attenuating cerebral damage, and normalizing the levels of oxidative enzymes. Overall, our findings may provide an alternative strategy for the design of novel anti-ischemic stroke agents more potent than drugs like NBP and Eda.
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http://dx.doi.org/10.1021/jm300681rDOI Listing
August 2012

NG, a novel PABA/NO-based oleanolic acid derivative, induces human hepatoma cell apoptosis via a ROS/MAPK-dependent mitochondrial pathway.

Eur J Pharmacol 2012 Sep 20;691(1-3):61-8. Epub 2012 Jul 20.

State Key Laboratory of Natural Medicines, Department of Pharmacology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China.

O(2)-(2,4-dinitro-5-{[2-(12-en-28-β-D- galactopyranosyl-oleanolate-3-yl) -oxy-2-oxoethyl]amino}phenyl)1-(N-hydroxyethylmethylamino)diazen-1-ium-1,2- diolate (NG), a novel PABA/NO-based derivative of oleanolic acid (OA), has been found to show potent antitumor activity both in vivo and in vitro. In the present study, NG could significantly reduce tumor volume and weight in the H22 solid tumor mouse model. Meanwhile, NG showed selective effects on the HepG2 cells including NO generation, cytotoxic effect and apoptosis, which were prevented by hemoglobin (NO scavenger). Moreover, NG-induced apoptosis of HepG2 cells was characteristic of intracellular reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential (Δψm) and enhanced Bax-to-Bcl-2 ratio. The release of apoptotic inducing factor (AIF) and cytochrome c (Cyt c) from mitochondria and the activation of caspase-3, 9 were also detected, indicating that NG may induce apoptosis through a mitochondrial-mediated pathway. Simultaneously, NG treatment could lead to the activation of the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK but not ERK1/2. Treatment with SP600125 (an inhibitor of JNK) and SB203580 (an inhibitor of p38) prior to NG was found to reverse NG-induced apoptosis. Moreover, it was found that antioxidant N-acetylcysteine (NAC) blocked the induction of apoptosis and partly reversed the activation of JNK and p38, up-regulation of Bax, down-regulation of Bcl-2 and the activation of caspase-3 in NG-treated cells. Taking together, these findings suggest that NO can be released from NG, which induces apoptosis through a ROS/MAPK-mediated mitochondrial pathway.
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http://dx.doi.org/10.1016/j.ejphar.2012.07.031DOI Listing
September 2012

Protein thioester synthesis enabled by sortase.

J Am Chem Soc 2012 Jul 19;134(26):10749-52. Epub 2012 Jun 19.

Department of Chemistry, Massachusetts Institute of Technology, 16-573a, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

Proteins containing a C-terminal thioester are important intermediates in semisynthesis. Currently there is one main method for the synthesis of protein thioesters that relies upon the use of engineered inteins. Here we report a simple strategy, utilizing sortase A, for routine preparation of recombinant proteins containing a C-terminal (α)thioester. We used our method to prepare two different anthrax toxin cargo proteins: one containing an (α)thioester and another containing a D-polypeptide segment situated between two protein domains. We show that both variants can translocate through protective antigen pore. This new method to synthesize a protein thioester allows for interfacing of sortase-mediated ligation and native chemical ligation.
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http://dx.doi.org/10.1021/ja302354vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3465687PMC
July 2012

Process automation toward ultra-high-throughput screening of combinatorial one-bead-one-compound (OBOC) peptide libraries.

J Lab Autom 2012 Jun 14;17(3):186-200. Epub 2012 Feb 14.

Institute of Bioengineering and Nanotechnology, Singapore.

With an aim to develop peptide-based protein capture agents that can replace antibodies for in vitro diagnosis, an ultra-high-throughput screening strategy has been investigated by automating labor-intensive, time-consuming processes that are the construction of peptide libraries, sorting of positive beads, and peptide sequencing through analysis of tandem mass spectrometry data. Although instruments for automation, such as peptide synthesizers and automatic bead sorters, have been used in some groups, the overall process has not been well optimized to minimize time, cost, and efforts, as well as to maximize product quality and performance. Herein we suggest and explore several solutions to the existing problems with the automation of the key processes. The overall process optimization has been done successfully in orchestration with the technologies such as rapid cleavage of peptides from beads and semiautomatic peptide sequencing that we have developed previously. This optimization allowed one-round screening, from peptide library construction to peptide sequencing, to be completed within 4 to 5 days. We also successfully identified a 6-mer ligand for carcinoembryonic antigen-cell adhesion molecule 5 (CEACAM 5) through three-round screenings, including one-round screening of a focused library.
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http://dx.doi.org/10.1177/2211068211433503DOI Listing
June 2012