Publications by authors named "Xiaodi Deng"

19 Publications

  • Page 1 of 1

One size does not fit all: navigating the multi-dimensional space to optimize T-cell engaging protein therapeutics.

MAbs 2021 Jan-Dec;13(1):1871171

Pfizer Worldwide R&D , BioMedicine Design, CA, USA.

T-cell engaging biologics is a class of novel and promising immune-oncology compounds that leverage the immune system to eradicate cancer. Here, we compared and contrasted a bispecific diabody-Fc format, which displays a relatively short antigen-binding arm distance, with our bispecific IgG platform. By generating diverse panels of antigen-expressing cells where B cell maturation antigen is either tethered to the cell membrane or located to the juxtamembrane region and masked by elongated structural spacer units, we presented a systematic approach to investigate the role of antigen epitope location and molecular formats in immunological synapse formation and cytotoxicity. We demonstrated that diabody-Fc is more potent for antigen epitopes located in the membrane distal region, while bispecific IgG is more efficient for membrane-proximal epitopes. Additionally, we explored other parameters, including receptor density, antigen-binding affinity, and kinetics. Our results show that molecular format and antigen epitope location, which jointly determine the intermembrane distance between target cells and T cells, allow decoupling of cytotoxicity and cytokine release, while antigen-binding affinities appear to be positively correlated with both readouts. Our work offers new insight that could potentially lead to a wider therapeutic window for T-cell engaging biologics in general.
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http://dx.doi.org/10.1080/19420862.2020.1871171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889206PMC
February 2021

Direct control of CAR T cells through small molecule-regulated antibodies.

Nat Commun 2021 01 29;12(1):710. Epub 2021 Jan 29.

Pfizer, La Jolla, CA, USA.

Antibody-based therapeutics have experienced a rapid growth in recent years and are now utilized in various modalities spanning from conventional antibodies, antibody-drug conjugates, bispecific antibodies to chimeric antigen receptor (CAR) T cells. Many next generation antibody therapeutics achieve enhanced potency but often increase the risk of adverse events. Antibody scaffolds capable of exhibiting inducible affinities could reduce the risk of adverse events by enabling a transient suspension of antibody activity. To demonstrate this, we develop conditionally activated, single-module CARs, in which tumor antigen recognition is directly modulated by an FDA-approved small molecule drug. The resulting CAR T cells demonstrate specific cytotoxicity of tumor cells comparable to that of traditional CARs, but the cytotoxicity is reversibly attenuated by the addition of the small molecule. The exogenous control of conditional CAR T cell activity allows continual modulation of therapeutic activity to improve the safety profile of CAR T cells across all disease indications.
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http://dx.doi.org/10.1038/s41467-020-20671-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846603PMC
January 2021

High-Throughput Platform to Identify Antibody Conjugation Sites from Antibody-Drug Conjugate Libraries.

Bioconjug Chem 2020 04 27;31(4):1199-1208. Epub 2020 Mar 27.

Discovery Biotherapeutics, Bristol-Myers Squibb, 700 Bay Road, Redwood City, California 94063, United States.

Antibody-drug conjugates (ADCs) are a therapeutic modality that traditionally enable the targeted delivery of highly potent cytotoxic agents to specific cells such as tumor cells. More recently, antibodies have been used to deliver molecules such as antibiotics, antigens, and adjuvants to bacteria or specific immune cell subsets. Site-directed mutagenesis of proteins permits more precise control over the site and stoichiometry of their conjugation, giving rise to homogeneous chemically defined ADCs. Identification of favorable sites for conjugation in antibodies is essential as reaction efficiency and product stability are influenced by the tertiary structure of immunoglobulin G (IgG). Current methods to evaluate potential conjugation sites are time-consuming and labor intensive, involving multistep processes for individually produced reactions. Here, we describe a highly efficient method for identification of conjugatable genetic variants by analyzing pooled ADC libraries using mass spectrometry. This approach provides a versatile platform to rapidly uncover new conjugation sites for site-specific ADCs.
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http://dx.doi.org/10.1021/acs.bioconjchem.0c00146DOI Listing
April 2020

VISTA is an acidic pH-selective ligand for PSGL-1.

Nature 2019 10 23;574(7779):565-570. Epub 2019 Oct 23.

Immuno-Oncology Discovery, Bristol-Myers Squibb, Redwood City, CA, USA.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement.
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http://dx.doi.org/10.1038/s41586-019-1674-5DOI Listing
October 2019

Considering B7-CD28 as a family through sequence and structure.

Exp Biol Med (Maywood) 2019 12 17;244(17):1577-1583. Epub 2019 Jun 17.

Bristol-Myers Squibb (BMS), Redwood City, CA 94063, USA.

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http://dx.doi.org/10.1177/1535370219855970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920668PMC
December 2019

Transcriptomic but not genomic variability confers phenotype of breast cancer stem cells.

Cancer Commun (Lond) 2018 09 19;38(1):56. Epub 2018 Sep 19.

Center of Genome and Personalized Medicine, Institute of Cancer Stem Cell, Dalian Medical University, Dalian, 116044, Liaoning, P. R. China.

Background: Breast cancer stem cells (BCSCs) are considered responsible for cancer relapse and drug resistance. Understanding the identity of BCSCs may open new avenues in breast cancer therapy. Although several discoveries have been made on BCSC characterization, the factors critical to the origination of BCSCs are largely unclear. This study aimed to determine whether genomic mutations contribute to the acquisition of cancer stem-like phenotype and to investigate the genetic and transcriptional features of BCSCs.

Methods: We detected potential BCSC phenotype-associated mutation hotspot regions by using whole-genome sequencing on parental cancer cells and derived serial-generation spheres in increasing order of BCSC frequency, and then performed target deep DNA sequencing at bulk-cell and single-cell levels. To identify the transcriptional program associated with BCSCs, bulk-cell and single-cell RNA sequencing was performed.

Results: By using whole-genome sequencing of bulk cells, potential BCSC phenotype-associated mutation hotspot regions were detected. Validation by target deep DNA sequencing, at both bulk-cell and single-cell levels, revealed no genetic changes specifically associated with BCSC phenotype. Moreover, single-cell RNA sequencing showed profound transcriptomic variability in cancer cells at the single-cell level that predicted BCSC features. Notably, this transcriptomic variability was enriched during the transcription of 74 genes, revealed as BCSC markers. Breast cancer patients with a high risk of relapse exhibited higher expression levels of these BCSC markers than those with a low risk of relapse, thereby highlighting the clinical significance of predicting breast cancer prognosis with these BCSC markers.

Conclusions: Transcriptomic variability, not genetic mutations, distinguishes BCSCs from non-BCSCs. The identified 74 BCSC markers have the potential of becoming novel targets for breast cancer therapy.
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http://dx.doi.org/10.1186/s40880-018-0326-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146522PMC
September 2018

Spatial Vulnerabilities of the Genome to Spontaneous Mutations Revealed with Improved Duplex Sequencing.

Genetics 2018 10 3;210(2):547-558. Epub 2018 Aug 3.

Center of Genome and Personalized Medicine, Institute of Cancer Stem Cell, Dalian Medical University, Liaoning 116044, China

Investigation of spontaneous mutations by next-generation sequencing technology has attracted extensive attention lately due to the fundamental roles of spontaneous mutations in evolution and pathological processes. However, these studies only focused on the mutations accumulated through many generations during long-term (possibly be years of) culturing, but not the freshly generated mutations that occur at very low frequencies. In this study, we established a molecularly barcoded deep sequencing strategy to detect low abundant spontaneous mutations in genomes of bacteria cell cultures. Genome-wide spontaneous mutations in 15 cell culture samples were defined with a high confidence ( < 0.01). We also developed a hotspot-calling approach based on the run-length encoding algorithm to find the genomic regions that are vulnerable to the spontaneous mutations. The hotspots for the mutations appeared to be highly conserved across the bacteria samples. Further biological annotation of these regions indicated that most of the spontaneous mutations were located at the repeat domains or nonfunctional domains of the genomes, suggesting the existence of mechanisms that could somehow prevent the occurrence of mutations in crucial genic areas. This study provides a more faithful picture of mutation occurrence and spectra in a single expansion process without long-term culturing.
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http://dx.doi.org/10.1534/genetics.118.301345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6216599PMC
October 2018

Conflicts of CpG density and DNA methylation are proximally and distally involved in gene regulation in human and mouse tissues.

Epigenetics 2018 25;13(7):721-741. Epub 2018 Aug 25.

a Center of Genome and Personalized Medicine , Institute of Cancer Stem Cell, Dalian Medical University , Dalian , China.

The relationship between CpG content and DNA methylation has attracted considerable interest in recent years. Direct or indirect methods have been developed to investigate their regulatory functions based on various hypotheses, large cohort studies, and meta-analyses. However, all of these analyses were performed at units of CpG blocks and, thus, the influence of finer genome structure has been neglected. Herein, we present a novel algorithm of base-pair resolution to systematically investigate the relationship between CpG contents and DNA methylation. By introducing the concept of 'complementary index' we examined the methylomes of 34 adult and 7 embryonic tissues and successfully fitted the relationship of DNA methylation and CpG density into a nonlinear mathematical model. A further algorithm was developed to locate the regions where CpG density does not match expectations from the model, termed 'conflict of gap' (COG) regions. Interestingly, COGs are highly concordant in human and mouse and their distributions display a tissue-specific pattern. Based on COG methylation patterns we correctly classified tissues according to their function or origin. We demonstrate that COGs based on our method can reveal more and deeper information than traditional differential methylation region (DMR) approaches. We also found that when COGs are located near to transcription start site (TSS), these regions can determine which promoters will be utilized for initiating gene transcription. Furthermore, COGs located far from the TSS perform as enhancers in terms of histone modification, sequence conservation, transcription factor binding, and DNase I-hypersensitivity.
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http://dx.doi.org/10.1080/15592294.2018.1500057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224217PMC
March 2019

Germline-encoded neutralization of a Staphylococcus aureus virulence factor by the human antibody repertoire.

Nat Commun 2016 11 18;7:13376. Epub 2016 Nov 18.

Rinat R&D, Pfizer Inc., 230 East Grand Avenue, South San Francisco, California 94080, USA.

Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.
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http://dx.doi.org/10.1038/ncomms13376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120205PMC
November 2016

Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms.

MAbs 2016 14;8(2):264-77. Epub 2015 Dec 14.

b Crystal Bioscience , CA , USA.

The ability of monoclonal antibodies (mAbs) to target specific antigens with high precision has led to an increasing demand to generate them for therapeutic use in many disease areas. Historically, the discovery of therapeutic mAbs has relied upon the immunization of mammals and various in vitro display technologies. While the routine immunization of rodents yields clones that are stable in serum and have been selected against vast arrays of endogenous, non-target self-antigens, it is often difficult to obtain species cross-reactive mAbs owing to the generally high sequence similarity shared across human antigens and their mammalian orthologs. In vitro display technologies bypass this limitation, but lack an in vivo screening mechanism, and thus may potentially generate mAbs with undesirable binding specificity and stability issues. Chicken immunization is emerging as an attractive mAb discovery method because it combines the benefits of both in vivo and in vitro display methods. Since chickens are phylogenetically separated from mammals, their proteins share less sequence homology with those of humans, so human proteins are often immunogenic and can readily elicit rodent cross-reactive clones, which are necessary for in vivo proof of mechanism studies. Here, we compare the binding characteristics of mAbs isolated from chicken immunization, mouse immunization, and phage display of human antibody libraries. Our results show that chicken-derived mAbs not only recapitulate the kinetic diversity of mAbs sourced from other methods, but appear to offer an expanded repertoire of epitopes. Further, chicken-derived mAbs can bind their native serum antigen with very high affinity, highlighting their therapeutic potential.
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http://dx.doi.org/10.1080/19420862.2015.1118596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966639PMC
December 2016

Role of Conserved Proline Residues in Human Apolipoprotein A-IV Structure and Function.

J Biol Chem 2015 Apr 2;290(17):10689-702. Epub 2015 Mar 2.

From the Departments of Molecular Genetics, Biochemistry and Microbiology and

Apolipoprotein (apo)A-IV is a lipid emulsifying protein linked to a range of protective roles in obesity, diabetes, and cardiovascular disease. It exists in several states in plasma including lipid-bound in HDL and chylomicrons and as monomeric and dimeric lipid-free/poor forms. Our recent x-ray crystal structure of the central domain of apoA-IV shows that it adopts an elongated helical structure that dimerizes via two long reciprocating helices. A striking feature is the alignment of conserved proline residues across the dimer interface. We speculated that this plays important roles in the structure of the lipid-free protein and its ability to bind lipid. Here we show that the systematic conversion of these prolines to alanine increased the thermodynamic stability of apoA-IV and its propensity to oligomerize. Despite the structural stabilization, we noted an increase in the ability to bind and reorganize lipids and to promote cholesterol efflux from cells. The novel properties of these mutants allowed us to isolate the first trimeric form of an exchangeable apolipoprotein and characterize it by small-angle x-ray scattering and chemical cross-linking. The results suggest that the reciprocating helix interaction is a common feature of all apoA-IV oligomers. We propose a model of how self-association of apoA-IV can result in spherical lipoprotein particles, a model that may have broader applications to other exchangeable apolipoprotein family members.
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http://dx.doi.org/10.1074/jbc.M115.637058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409236PMC
April 2015

The structure of human apolipoprotein A-IV as revealed by stable isotope-assisted cross-linking, molecular dynamics, and small angle x-ray scattering.

J Biol Chem 2014 Feb 14;289(9):5596-608. Epub 2014 Jan 14.

From the Departments of Molecular Genetics, Biochemistry and Microbiology and.

Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a "clasp" mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states.
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http://dx.doi.org/10.1074/jbc.M113.541037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937636PMC
February 2014

Structure of protein related to Dan and Cerberus: insights into the mechanism of bone morphogenetic protein antagonism.

Structure 2013 Aug 11;21(8):1417-29. Epub 2013 Jul 11.

Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Medical Sciences Building, Cincinnati, OH 45267, USA.

The bone morphogenetic proteins (BMPs) are secreted ligands largely known for their functional roles in embryogenesis and tissue development. A number of structurally diverse extracellular antagonists inhibit BMP ligands to regulate signaling. The differential screening-selected gene aberrative in neuroblastoma (DAN) family of antagonists represents the largest group of BMP inhibitors; however, little is known of how they mechanistically inhibit BMP ligands. Here, we present the structure of the DAN family member, protein related to Dan and Cerberus (PRDC), solved by X-ray crystallography. The structure reveals a growth factor-like appearance with an unexpected dimerization mechanism that is formed through extensive β strand contacts. Using site-directed mutagenesis coupled with in vitro and in vivo activity assays, we identified a BMP-binding epitope on PRDC. We also determined that PRDC binds heparin with high affinity and that heparin binding to PRDC interferes with BMP antagonism. These results offer insight for how DAN family antagonists functionally inhibit BMP ligands.
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http://dx.doi.org/10.1016/j.str.2013.06.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749838PMC
August 2013

Small-angle X-ray scattering of apolipoprotein A-IV reveals the importance of its termini for structural stability.

J Biol Chem 2013 Feb 3;288(7):4854-66. Epub 2013 Jan 3.

Department of Molecular Genetics, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267, USA.

ApoA-IV is an amphipathic protein that can emulsify lipids and has been linked to protective roles against cardiovascular disease and obesity. We previously reported an x-ray crystal structure of apoA-IV that was truncated at its N and C termini. Here, we have extended this work by demonstrating that self-associated states of apoA-IV are stable and can be structurally studied using small-angle x-ray scattering. Both the full-length monomeric and dimeric forms of apoA-IV were examined, with the dimer showing an elongated rod core with two nodes at opposing ends. The monomer is roughly half the length of the dimer with a single node. Small-angle x-ray scattering visualization of several deletion mutants revealed that removal of both termini can have substantial conformational effects throughout the molecule. Additionally, the F334A point mutation, which we previously showed increases apoA-IV lipid binding, also exhibited large conformational effects on the entire dimer. Merging this study's low-resolution structural information with the crystal structure provides insight on the conformation of apoA-IV as a monomer and as a dimer and further defines that a clasp mechanism may control lipid binding and, ultimately, protein function.
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http://dx.doi.org/10.1074/jbc.M112.436709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3576090PMC
February 2013

The structure of dimeric apolipoprotein A-IV and its mechanism of self-association.

Structure 2012 May;20(5):767-79

Department of Molecular Genetics, Biochemistry, and Microbiology, College of Medicine, University of Cincinnati, OH 45267, USA.

Apolipoproteins are key structural elements of lipoproteins and critical mediators of lipid metabolism. Their detergent-like properties allow them to emulsify lipid or exist in a soluble lipid-free form in various states of self-association. Unfortunately, these traits have hampered high-resolution structural studies needed to understand the biogenesis of cardioprotective high-density lipoproteins (HDLs). We derived a crystal structure of the core domain of human apolipoprotein (apo)A-IV, an HDL component and important mediator of lipid absorption. The structure at 2.4 Å depicts two linearly connected 4-helix bundles participating in a helix swapping arrangement that offers a clear explanation for how the protein self-associates as well as clues to the structure of its monomeric form. This also provides a logical basis for antiparallel arrangements recently described for lipid-containing particles. Furthermore, we propose a "swinging door" model for apoA-IV lipid association.
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http://dx.doi.org/10.1016/j.str.2012.02.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3354570PMC
May 2012

Improving the diffraction of apoA-IV crystals through extreme dehydration.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Jan 24;68(Pt 1):105-10. Epub 2011 Dec 24.

Department of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio, USA.

Apolipoproteins are the protein component of high-density lipoproteins (HDL), which are necessary for mobilizing lipid-like molecules throughout the body. Apolipoproteins undergo self-association, especially at higher concentrations, making them difficult to crystallize. Here, the crystallization and diffraction of the core fragment of apolipoprotein A-IV (apoA-IV), consisting of residues 64-335, is presented. ApoA-IV(64-335) crystallized readily in a variety of hexagonal (P6) morphologies with similar unit-cell parameters, all containing a long axis of nearly 550 Å in length. Preliminary diffraction experiments with the different crystal morphologies all resulted in limited streaky diffraction to 3.5 Å resolution. Crystal dehydration was applied to the different morphologies with variable success and was also used as a quality indicator of crystal-growth conditions. The results show that the morphologies that withstood the most extreme dehydration conditions showed the greatest improvement in diffraction. One morphology in particular was able to withstand dehydration in 60% PEG 3350 for over 12 h, which resulted in well defined intensities to 2.7 Å resolution. These results suggest that the approach of integrating dehydration with variation in crystal-growth conditions might be a general technique to optimize diffraction.
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http://dx.doi.org/10.1107/S1744309111048706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253848PMC
January 2012

Ectopic expression of LIM-nebulette (LASP2) reveals roles in cell migration and spreading.

Cell Motil Cytoskeleton 2008 Oct;65(10):827-40

Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.

LIM-nebulette (LASP2) is a small focal adhesion protein and a member of the nebulin family of actin binding proteins. This recently identified splice variant of the nebulette locus is widely expressed and highly enriched in neuronal tissue. Other than that LIM-nebulette is a focal adhesion protein and interacts with zyxin, nothing is known about its function. Given that LIM-nebulette has an identical modular organization and overlapping tissue distributions to that of LASP1, we have analyzed the role of LIM-nebulette in comparison with that of LASP1. We find that LIM-nebulette is a dynamic focal adhesion protein that increases the rate of attachment and spreading of fibroblasts on fibronectin coated surfaces. Additionally, LIM-nebulette is recruited from the cortical cytoskeleton in non-motile cells to focal adhesions at the leading edge of stimulated cells. In confluent cultures of HeLa and NIH3T3 cells, LIM-nebulette co-localizes with alpha-catenin in putative adherens junctions, whereas LASP1 is devoid of these areas. Interestingly, overexpression of LIM-nebulette in PC6 cells inhibits neurite outgrowth in response to growth factors. Collectively, our data indicate that LIM-nebulette and LASP1 have distinct roles in the actin cytoskeleton.
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http://dx.doi.org/10.1002/cm.20304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2603443PMC
October 2008

Targeting of nebulin fragments to the cardiac sarcomere.

Exp Cell Res 2007 Mar 30;313(5):896-909. Epub 2006 Dec 30.

Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 S. Limestone, Lexington, KY 40536, USA.

Nebulin, a vertebrate skeletal muscle actin binding protein, plays an important role in thin filament architecture. Recently, a number of reports have indicated evidence for nebulin expression in vertebrate hearts. To investigate the ability of nebulin to interact with cardiac myofilaments, we have expressed nebulin cDNA fragments tagged with green fluorescent protein (GFP) in chicken cardiomyocytes and PtK2 cells. Nebulin fragments from both the superrepeats and single repeats were expressed minus and plus the nebulin linker. Nebulin fragment incorporation was monitored by fluorescent microscopy and compared with the distribution of actin, alpha-actinin and titin. Expression of nebulin N-terminal superrepeats displayed a punctate cytoplasmic distribution in PtK2 cells and cardiomyocytes. Addition of the nebulin linker to the superrepeats resulted in association of the punctate staining with the myofibrils. Nebulin C-terminal superrepeats plus and minus the linker localized with stress fibers of PtK2 cells and associated with the cardiac myofilaments at the level of the Z-line. Expression of the single repeats plus and minus the nebulin linker region resulted in both a Z-line distribution and an A-band distribution. These data suggest that N-terminal superrepeat nebulin modules are incapable of supporting interactions with the cardiac myofilaments; whereas the C-terminal nebulin modules can. The expression of the N-terminal or C-terminal superrepeats did not alter the distribution of actin, alpha-actinin or titin in either atrial or ventricular cultures.
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http://dx.doi.org/10.1016/j.yexcr.2006.12.016DOI Listing
March 2007

Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death.

Mol Cell Biol 2004 Aug;24(15):6592-607

Dept. of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Park Rd. 1C, Smithville, TX 78957, USA.

It was recently demonstrated that during apoptosis, active caspase 9 and caspase 3 rapidly accumulate in the mitochondrion-enriched membrane fraction (D. Chandra and D. G. Tang, J. Biol. Chem.278:17408-17420, 2003). We now show that active caspase 8 also becomes associated with the membranes in apoptosis caused by multiple stimuli. In MDA-MB231 breast cancer cells treated with etoposide (VP16), active caspase 8 is detected only in the membrane fraction, which contains both mitochondria and endoplasmic reticulum (ER), as revealed by fractionation studies. Immunofluorescence microscopy, however, shows that procaspase 8 and active caspase 8 predominantly colocalize with the mitochondria. Biochemical analysis demonstrates that both procaspase 8 and active caspase 8 are localized mainly on the outer mitochondrial membrane (OMM) as integral proteins. Functional analyses with dominant-negative mutants, small interfering RNAs, peptide inhibitors, and Fas-associated death domain (FADD)- and caspase 8-deficient Jurkat T cells establish that the mitochondrion-localized active caspase 8 results mainly from the FADD-dependent and tumor necrosis factor receptor-associated death domain-dependent mechanisms and that caspase 8 activation plays a causal role in VP16-induced caspase 3 activation and cell death. Finally, we present evidence that the OMM-localized active caspase 8 can activate cytosolic caspase 3 and ER-localized BAP31. Cleavage of BAP31 leads to the generation of ER- localized, proapoptotic BAP20, which may mediate mitochondrion-ER cross talk through a Ca(2+)-dependent mechanism.
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http://dx.doi.org/10.1128/MCB.24.15.6592-6607.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC444870PMC
August 2004