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  1. Cooperativity transitions driven by higher-order oligomer formations in ligand-induced receptor dimerization
    Watabe M, Arjunan SNV, Chew WX, Kaizu K, Takahashi K
    Phys Rev E, 2019 Dec;100(6-1):062407.
    PMID: 31962468 DOI: 10.1103/PhysRevE.100.062407
    While cooperativity in ligand-induced receptor dimerization has been linked with receptor-receptor couplings via minimal representations of physical observables, effects arising from higher-order oligomer, e.g., trimer and tetramer, formations of unobserved receptors have received less attention. Here we propose a dimerization model of ligand-induced receptors in multivalent form representing physical observables under basis vectors of various aggregated receptor states. Our simulations of multivalent models not only reject Wofsy-Goldstein parameter conditions for cooperativity, but show that higher-order oligomer formations can shift cooperativity from positive to negative.
    Matched MeSH terms: Protein Multimerization*
  2. Fulltext Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation
    Surya W, Chooduang S, Choong YK, Torres J, Boonserm P
    PLoS One, 2016;11(6):e0158356.
    PMID: 27341696 DOI: 10.1371/journal.pone.0158356
    The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes.
    Matched MeSH terms: Protein Multimerization*
  3. Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form
    Lee YV, Choi SB, Wahab HA, Choong YS
    J Chem Inf Model, 2017 09 25;57(9):2351-2357.
    PMID: 28820943 DOI: 10.1021/acs.jcim.7b00265
    Tuberculosis (TB) still remains a global threat due to the emergence of a drug-resistant strain. Instead of focusing on the drug target of active stage TB, we are highlighting the isocitrate lyase (ICL) at the dormant stage TB. ICL is one of the persistent factors for Mycobacterium tuberculosis (MTB) to survive during the dormant phase. In addition, the absence of ICL in human has made ICL a potential drug target for TB therapy. However, the dynamic details of ICL which could give insights to the ICL-ligand interaction have yet to be solved. Therefore, a series of ICL dimer dynamics studies through molecular dynamics simulation were performed in this work. The ICL active site entrance gate closure is contributed to by hydrogen bonding and electrostatic interactions with the C-terminal. Analysis suggested that the open-closed behavior of the ICL active site entrance depends on the type of ligand present in the active site. We also observed four residues (Ser91, Asp108, Asp153, and Cys191) which could possibly be the nucleophiles for nucleophilic attack on the cleavage of isocitrate at the C2-C3bond. We hope that the elucidation of ICL dynamics can benefit future works such as lead identification or antibody design against ICL for TB therapeutics.
    Matched MeSH terms: Protein Multimerization*
  4. TMDIM: an improved algorithm for the structure prediction of transmembrane domains of bitopic dimers
    Cao H, Ng MCK, Jusoh SA, Tai HK, Siu SWI
    J Comput Aided Mol Des, 2017 Sep;31(9):855-865.
    PMID: 28864946 DOI: 10.1007/s10822-017-0047-0
    [Formula: see text]-Helical transmembrane proteins are the most important drug targets in rational drug development. However, solving the experimental structures of these proteins remains difficult, therefore computational methods to accurately and efficiently predict the structures are in great demand. We present an improved structure prediction method TMDIM based on Park et al. (Proteins 57:577-585, 2004) for predicting bitopic transmembrane protein dimers. Three major algorithmic improvements are introduction of the packing type classification, the multiple-condition decoy filtering, and the cluster-based candidate selection. In a test of predicting nine known bitopic dimers, approximately 78% of our predictions achieved a successful fit (RMSD <2.0 Å) and 78% of the cases are better predicted than the two other methods compared. Our method provides an alternative for modeling TM bitopic dimers of unknown structures for further computational studies. TMDIM is freely available on the web at https://cbbio.cis.umac.mo/TMDIM . Website is implemented in PHP, MySQL and Apache, with all major browsers supported.
    Matched MeSH terms: Protein Multimerization
  5. Fulltext γ-Secretase Inhibitors and Modulators Induce Distinct Conformational Changes in the Active Sites of γ-Secretase and Signal Peptide Peptidase
    Gertsik N, Chau DM, Li YM
    ACS Chem. Biol., 2015 Aug 21;10(8):1925-31.
    PMID: 26030233 DOI: 10.1021/acschembio.5b00321
    γ-Secretase inhibitors (GSIs) and modulators (GSMs) are at the frontline of cancer and Alzheimer's disease research, respectively. While both are therapeutically promising, not much is known about their interactions with proteins other than γ-secretase. Signal peptide peptidase (SPP), like γ-secretase, is a multispan transmembrane aspartyl protease that catalyzes regulated intramembrane proteolysis. We used active site-directed photophore walking probes to study the effects of different GSIs and GSMs on the active sites of γ-secretase and SPP and found that nontransition state GSIs inhibit labeling of γ-secretase by activity-based probes but enhance labeling of SPP. The opposite is true of GSMs, which have little effect on the labeling of γ-secretase but diminish labeling of SPP. These results demonstrate that GSIs and GSMs are altering the structure of not only γ-secretase but also SPP, leading to potential changes in enzyme activity and specificity that may impact the clinical outcomes of these molecules.
    Matched MeSH terms: Protein Multimerization/drug effects
  6. Fulltext Activating CARD14 Mutations Are Associated with Generalized Pustular Psoriasis but Rarely Account for Familial Recurrence in Psoriasis Vulgaris
    Berki DM, Liu L, Choon SE, David Burden A, Griffiths CEM, Navarini AA, et al.
    J Invest Dermatol, 2015 Dec;135(12):2964-2970.
    PMID: 26203641 DOI: 10.1038/jid.2015.288
    Caspase recruitment family member 14 (CARD14, also known as CARMA2), is a scaffold protein that mediates NF-κB signal transduction in skin keratinocytes. Gain-of-function CARD14 mutations have been documented in familial forms of psoriasis vulgaris (PV) and pityriasis rubra pilaris (PRP). More recent investigations have also implicated CARD14 in the pathogenesis of pustular psoriasis. Follow-up studies, however, have been limited, so that it is not clear to what extent CARD14 alleles account for the above conditions. Here, we sought to address this question by carrying out a systematic CARD14 analysis in an extended patient cohort (n=416). We observed no disease alleles in subjects with familial PV (n=159), erythrodermic psoriasis (n=23), acral pustular psoriasis (n=100), or sporadic PRP (n=29). Conversely, our analysis of 105 individuals with generalized pustular psoriasis (GPP) identified a low-frequency variant (p.Asp176His) that causes constitutive CARD14 oligomerization and shows a significant association with GPP in Asian populations (P=8.4×10(-5); odds ratio=6.4). These data indicate that the analysis of CARD14 mutations could help stratify pustular psoriasis cohorts but would be mostly uninformative in the context of psoriasis and sporadic PRP.
    Matched MeSH terms: Protein Multimerization
  7. Fulltext The Sec61 translocon limits IRE1α signaling during the unfolded protein response
    Sundaram A, Plumb R, Appathurai S, Mariappan M
    Elife, 2017 05 15;6.
    PMID: 28504640 DOI: 10.7554/eLife.27187
    IRE1α is an endoplasmic reticulum (ER) localized endonuclease activated by misfolded proteins in the ER. Previously, we demonstrated that IRE1α forms a complex with the Sec61 translocon, to which its substrate XBP1u mRNA is recruited for cleavage during ER stress (Plumb et al., 2015). Here, we probe IRE1α complexes in cells with blue native PAGE immunoblotting. We find that IRE1α forms a hetero-oligomeric complex with the Sec61 translocon that is activated upon ER stress with little change in the complex. In addition, IRE1α oligomerization, activation, and inactivation during ER stress are regulated by Sec61. Loss of the IRE1α-Sec61 translocon interaction as well as severe ER stress conditions causes IRE1α to form higher-order oligomers that exhibit continuous activation and extended cleavage of XBP1u mRNA. Thus, we propose that the Sec61-IRE1α complex defines the extent of IRE1α activity and may determine cell fate decisions during ER stress conditions.
    Matched MeSH terms: Protein Multimerization
  8. Expression and characterization of functional domains of FK506-binding protein 35 from Plasmodium knowlesi
    Goh CKW, Silvester J, Wan Mahadi WNS, Chin LP, Ying LT, Leow TC, et al.
    Protein Eng. Des. Sel., 2018 12 01;31(12):489-498.
    PMID: 31120120 DOI: 10.1093/protein/gzz008
    The FK506-binding protein of Plasmodium knowlesi (Pk-FKBP35) is considerably a viable antimalarial drug target, which belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) protein family member. Structurally, this protein consists of an N-terminal FK506-binding domain (FKBD) and a C-terminal tetratricopeptide repeat domain (TPRD). This study aims to decipher functional properties of these domains as a platform for development of novel antimalarial drugs. Accordingly, full-length Pk-FKBP35 as well as its isolated domains, Pk-FKBD and Pk-TPRD were overexpressed, purified, and characterized. The results showed that catalytic PPIase activity was confined to the full-length Pk-FKBP35 and Pk-FKBD, suggesting that the catalytic activity is structurally regulated by the FKBD. Meanwhile, oligomerization analysis revealed that Pk-TPRD is essential for dimerization. Asp55, Arg60, Trp77 and Phe117 in the Pk-FKBD were considerably important for catalysis as underlined by significant reduction of PPIase activity upon mutations at these residues. Further, inhibition activity of Pk-FKBP35 towards calcineurin phosphatase activity revealed that the presence of FKBD is essential for the inhibitory property, while TPRD may be important for efficient binding to calcineurin. We then discussed possible roles of FKBP35 in Plasmodium cells and proposed mechanisms by which the immunosuppressive drug, FK506, interacts with the protein.
    Matched MeSH terms: Protein Multimerization
  9. Identification of regions affecting enzyme activity, substrate binding, dimer stabilization and polyhydroxyalkanoate (PHA) granule morphology in the PHA synthase of Aquitalea sp. USM4
    Lim H, Chuah JA, Chek MF, Tan HT, Hakoshima T, Sudesh K
    Int J Biol Macromol, 2021 Sep 01;186:414-423.
    PMID: 34246679 DOI: 10.1016/j.ijbiomac.2021.07.041
    Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized by microorganisms as intracellular energy reservoirs under stressful environmental conditions. PHA synthase (PhaC) is the key enzyme responsible for PHA biosynthesis, but the importance of its N- and C-terminal ends still remains elusive. Six plasmid constructs expressing truncation variants of Aquitalea sp. USM4 PhaC (PhaC1As) were generated and heterologously expressed in Cupriavidus necator PHB-4. Removal of the first six residues at the N-terminus enabled the modulation of PHA composition without altering the PHA content in cells. Meanwhile, deletion of 13 amino acids from the C-terminus greatly affected the catalytic activity of PhaC1As, retaining only 1.1-7.4% of the total activity. Truncation(s) at the N- and/or C-terminus of PhaC1As gradually diminished the incorporation of comonomer units, and revealed that the N-terminal region is essential for PhaC1As dimerization whereas the C-terminal region is required for stabilization. Notably, transmission electron microscopy analysis showed that PhaC modification affected the morphology of intracellular PHA granules, which until now is only known to be regulated by phasins. This study provided substantial evidence and highlighted the significance of both the N- and C-termini of PhaC1As in regulating intracellular granule morphology, activity, substrate specificity, dimerization and stability of the synthase.
    Matched MeSH terms: Protein Multimerization
  10. Fulltext JMJD8 is a novel endoplasmic reticulum protein with a JmjC domain
    Yeo KS, Tan MC, Lim YY, Ea CK
    Sci Rep, 2017 11 13;7(1):15407.
    PMID: 29133832 DOI: 10.1038/s41598-017-15676-z
    Jumonji C (JmjC) domain-containing proteins have been shown to regulate cellular processes by hydroxylating or demethylating histone and non-histone targets. JMJD8 belongs to the JmjC domain-only family that was recently shown to be involved in angiogenesis and TNF-induced NF-κB signaling. Here, we employed bioinformatic analysis and immunofluorescence microscopy to examine the physiological properties of JMJD8. We demonstrated that JMJD8 localizes to the lumen of endoplasmic reticulum and that JMJD8 forms dimers or oligomers in vivo. Furthermore, we identified potential JMJD8-interacting proteins that are known to regulate protein complex assembly and protein folding. Taken together, this work demonstrates that JMJD8 is the first JmjC domain-containing protein found in the lumen of the endoplasmic reticulum that may function in protein complex assembly and protein folding.
    Matched MeSH terms: Protein Multimerization
  11. MicroRNA regulation of CTP synthase and cytoophidium in Drosophila melanogaster
    Dzaki N, Woo WK, Thangadurai S, Azzam G
    Exp Cell Res, 2019 12 15;385(2):111688.
    PMID: 31678212 DOI: 10.1016/j.yexcr.2019.111688
    CTPsyn is a crucial metabolic enzyme which synthesizes CTP nucleotides. It has the extraordinary ability to compartmentalize into filaments termed cytoophidia. Though the structure is evolutionarily conserved across kingdoms, the mechanisms behind their formation remain unknown. MicroRNAs (miRNAs) are short single-stranded RNA capable of directing mRNA silencing and degradation. D. melanogaster has a high total gene count to miRNA gene number ratio, alluding to the possibility that CTPsyn too may come under their regulation. A thorough miRNA overexpression involving 123 miRNAs was conducted, followed by CTPsyn-specific staining upon cytoophidia-rich egg chambers. This revealed a small group of candidates which confer either a lengthening or truncating effect on cytoophidia, suggesting they may play a role in regulating CTPsyn. MiR-975 and miR-1014 are both cytoophidia-elongating, whereas miR-190 and miR-932 are cytoophidia-shortening. Though target prediction shows that miR-975 and miR-932 do indeed have binding sites on CTPsyn mRNA, in vitro assays instead revealed a low probability of this being true, instead indicating that the effects asserted by overexpressed miRNAs indirectly reach CTPsyn and its cytoophidia through the actions of middling elements. In silico target prediction and qPCR quantification indicated that, at least for miR-932 and miR-1014, these undetermined elements may be players in fat metabolism. This is the first study to thoroughly investigate miRNAs in connection to CTPsyn expression and activity in any species. The findings presented could serve as a basis for further queries into not only the fundamental aspects of the enzyme's regulation, but may uncover new facets of closely related pathways as well.
    Matched MeSH terms: Protein Multimerization
  12. Fulltext Role of α-helical structure in organic solvent-activated homodimer of elastase strain K
    Rahman RN, Salleh AB, Basri M, Wong CF
    Int J Mol Sci, 2011;12(9):5797-814.
    PMID: 22016627 DOI: 10.3390/ijms12095797
    Recombinant elastase strain K overexpressed from E. coli KRX/pCon2(3) was purified to homogeneity by a combination of hydrophobic interaction chromatography and ion exchange chromatography, with a final yield of 48% and a 25-fold increase in specific activity. The purified protein had exhibited a first ever reported homodimer size of 65 kDa by SDS-PAGE and MALDI-TOF, a size which is totally distinct from that of typically reported 33 kDa monomer from P. aeruginosa. The organic solvent stability experiment had demonstrated a stability pattern which completely opposed the rules laid out in previous reports in which activity stability and enhancement were observed in hydrophilic organic solvents such as DMSO, methanol, ethanol and 1-propanol. The high stability and enhancement of the enzyme in hydrophilic solvents were explained from the view of alteration in secondary structures. Elastinolytic activation and stability were observed in 25 and 50% of methanol, respectively, despite slight reduction in α-helical structure caused upon the addition of the solvent. Further characterization experiments had postulated great stability and enhancement of elastase strain K in broad range of temperatures, pHs, metal ions, surfactants, denaturing agents and substrate specificity, indicating its potential application in detergent formulation.
    Matched MeSH terms: Protein Multimerization*
  13. Fulltext Expression of p53 N-terminal isoforms in B-cell precursor acute lymphoblastic leukemia and its correlation with clinicopathological profiles
    Oh L, Hainaut P, Blanchet S, Ariffin H
    BMC Cancer, 2020 Feb 10;20(1):110.
    PMID: 32041553 DOI: 10.1186/s12885-020-6599-8
    BACKGROUND: TP53 mutations occur in only about 3% of primary and 10-20% of relapse B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). However, alternative mechanisms may contribute to functionally impairing the p53 pathway in the absence of a mutation. Candidate mechanisms include overexpression of p53 mRNA variants encoding either dominant-negative p53 protein isoforms such as Delta40p53 and Delta133p53, or modulatory isoforms such as p53beta, which counteract the effects of Delta133p53 on replicative senescence in T-lymphocytes.

    METHODS: We used semi-quantitative reverse-transcriptase PCR (RT-PCR) and Western blot to investigate the expression of full length p53 (TAp53), Delta40p53, Delta133p53 or p53beta in diagnostic marrow from a clinical cohort of 50 BCP-ALL patients without TP53 mutation (29 males and 21 females, age range 2-14 years) and in the bone marrow cells of 4 healthy donors (used as controls).

    RESULTS: Irrespective of isoforms, levels of p53 mRNA were low in controls but were increased by 2 to 20-fold in primary or relapse BCP-ALL. TAp53 was increased in primary BCP-ALL, Delta40p53 was elevated in relapse BCP-ALL, whereas Delta133p53 and p53beta were increased in both. Next, mRNA levels were used as a basis to infer the ratio between protein isoform levels. This inference suggested that, in primary BCP-ALL, p53 was predominantly in active oligomeric conformations dominated by TAp53. In contrast, p53 mostly existed in inactive quaternary conformations containing ≥2 Delta40 or Delta133p53 in relapse BCP-ALL. Western blot analysis of blasts from BCP-ALL showed a complex pattern of N-terminally truncated p53 isoforms, whereas TAp53beta was detected as a major isoform. The hypothesis that p53 is in an active form in primary B-ALL was consistent with elevated level of p53 target genes CDKN1A and MDM2 in primary cases, whereas in relapse BCP-ALL, only CDKN1A was increased as compared to controls.

    CONCLUSION: Expression of p53 isoforms is deregulated in BCP-ALL in the absence of TP53 mutation, with increased expression of alternative isoforms in relapse BCP-ALL. Variations in isoform expression may contribute to functional deregulation of the p53 pathway in BCP-ALL, specifically contributing to its down-regulation in relapse forms.

    Matched MeSH terms: Protein Multimerization/genetics
  14. Fulltext Alzheimer's Disease Determination by a Dual Probe on Gold Nanourchins and Nanohorn Hybrids
    Qiu Z, Shen Q, Jiang C, Yao L, Sun X, Li J, et al.
    Int J Nanomedicine, 2021;16:2311-2322.
    PMID: 33776435 DOI: 10.2147/IJN.S302396
    Background: Alzheimer's disease (AD) is a neurodegenerative chronic disorder that causes dementia and problems in thinking, cognitive impairment and behavioral changes. Amyloid-beta (Aβ) is a peptide involved in AD progression, and a high level of Aβ is highly correlated with severe AD. Identifying and quantifying Aβ levels helps in the early treatment of AD and reduces the factors associated with AD.

    Materials and Methods: This research introduced a dual probe detection system involving aptamers and antibodies to identify Aβ. Aptamers and antibodies were attached to the gold (Au) urchin and hybrid on the carbon nanohorn-modified surface. The nanohorn was immobilized on the sensor surface by using an amine linker, and then a Au urchin dual probe was immobilized.

    Results: This dual probe-modified surface enhanced the current flow during Aβ detection compared with the surface with antibody as the probe. This dual probe interacted with higher numbers of Aβ peptides and reached the detection limit at 10 fM with R2=0.992. Furthermore, control experiments with nonimmune antibodies, complementary aptamer sequences and control proteins did not display the current responses, indicating the specific detection of Aβ.

    Conclusion: Aβ-spiked artificial cerebrospinal fluid showed a similar response to current changes, confirming the selective identification of Aβ.

    Matched MeSH terms: Protein Multimerization
  15. Fulltext Crystal structure of an antigenic outer-membrane protein from Salmonella Typhi suggests a potential antigenic loop and an efflux mechanism
    Guan HH, Yoshimura M, Chuankhayan P, Lin CC, Chen NC, Yang MC, et al.
    Sci Rep, 2015 Nov 13;5:16441.
    PMID: 26563565 DOI: 10.1038/srep16441
    ST50, an outer-membrane component of the multi-drug efflux system from Salmonella enterica serovar Typhi, is an obligatory diagnostic antigen for typhoid fever. ST50 is an excellent and unique diagnostic antigen with 95% specificity and 90% sensitivity and is used in the commercial diagnosis test kit (TYPHIDOT(TM)). The crystal structure of ST50 at a resolution of 2.98 Å reveals a trimer that forms an α-helical tunnel and a β-barrel transmembrane channel traversing the periplasmic space and outer membrane. Structural investigations suggest significant conformational variations in the extracellular loop regions, especially extracellular loop 2. This is the location of the most plausible antibody-binding domain that could be used to target the design of new antigenic epitopes for the development of better diagnostics or drugs for the treatment of typhoid fever. A molecule of the detergent n-octyl-β-D-glucoside is observed in the D-cage, which comprises three sets of Asp361 and Asp371 residues at the periplasmic entrance. These structural insights suggest a possible substrate transport mechanism in which the substrate first binds at the periplasmic entrance of ST50 and subsequently, via iris-like structural movements to open the periplasmic end, penetrates the periplasmic domain for efflux pumping of molecules, including poisonous metabolites or xenobiotics, for excretion outside the pathogen.
    Matched MeSH terms: Protein Multimerization
  16. An in silico approach towards the identification of novel inhibitors of the TLR-4 signaling pathway
    Mahita J, Harini K, Rao Pichika M, Sowdhamini R
    J Biomol Struct Dyn, 2016 Jun;34(6):1345-62.
    PMID: 26264972 DOI: 10.1080/07391102.2015.1079243
    Precise functioning and fine-tuning of Toll-like receptor 4 (TLR4) signaling is a critical requirement for the smooth functioning of the innate immune system, since aberrant TLR4 activation causes excessive production of pro-inflammatory cytokines and interferons. This can result in life threatening conditions such as septic shock and other inflammatory disorders. The TRIF-related adaptor molecule (TRAM) adaptor protein is unique to the TLR4 signaling pathway and abrogation of TRAM-mediated TLR4 signaling is a promising strategy for developing therapeutics aimed at disrupting TRAM interactions with other components of the TLR4 signaling complex. The VIPER motif from the vaccinia virus-producing protein, A46 has been reported to disrupt TRAM-TLR4 interactions. We have exploited this information, in combination with homology modeling and docking approaches, to identify a potential binding site on TRAM lined by the BB loop and αC helix. Virtual screening of commercially available small molecules targeting the binding site enabled to short-list 12 small molecules to abrogate TRAM-mediated TLR4 signaling. Molecular dynamics and molecular mechanics calculations have been performed for the analysis of these receptor-ligand interactions.
    Matched MeSH terms: Protein Multimerization
  17. Disruption of repressive p130-DREAM complexes by human papillomavirus 16 E6/E7 oncoproteins is required for cell-cycle progression in cervical cancer cells
    Nor Rashid N, Yusof R, Watson RJ
    J Gen Virol, 2011 Nov;92(Pt 11):2620-2627.
    PMID: 21813705 DOI: 10.1099/vir.0.035352-0
    Human papillomaviruses (HPVs) with tropism for mucosal epithelia are the major aetiological factors in cervical cancer. Most cancers are associated with so-called high-risk HPV types, in particular HPV16, and constitutive expression of the HPV16 E6 and E7 oncoproteins is critical for malignant transformation in infected keratinocytes. E6 and E7 bind to and inactivate the cellular tumour suppressors p53 and Rb, respectively, thus delaying differentiation and inducing proliferation in suprabasal keratinocytes to enable HPV replication. One member of the Rb family, p130, appears to be a particularly important target for E7 in promoting S-phase entry. Recent evidence indicates that p130 regulates cell-cycle progression as part of a large protein complex termed DREAM. The composition of DREAM is cell cycle-regulated, associating with E2F4 and p130 in G0/G1 and with the B-myb transcription factor in S/G2. In this study, we addressed whether p130-DREAM is disrupted in HPV16-transformed cervical cancer cells and whether this is a critical function for E6/E7. We found that p130-DREAM was greatly diminished in HPV16-transformed cervical carcinoma cells (CaSki and SiHa) compared with control cell lines; however, when E6/E7 expression was targeted by specific small hairpin RNAs, p130-DREAM was reformed and the cell cycle was arrested. We further demonstrated that the profound G1 arrest in E7-depleted CaSki cells was dependent on p130-DREAM reformation by also targeting the expression of the DREAM component Lin-54 and p130. The results show that continued HPV16 E6/E7 expression is necessary in cervical cancer cells to prevent cell-cycle arrest by a repressive p130-DREAM complex.
    Matched MeSH terms: Protein Multimerization
  18. Purification and functional analysis of recombinant Acholeplasma laidlawii histone-like HU protein
    Levitskiy SA, Sycheva AM, Kharlampieva DD, Oberto J, Kamashev DE, Serebryakova MV, et al.
    Biochimie, 2011 Jul;93(7):1102-9.
    PMID: 21443922 DOI: 10.1016/j.biochi.2011.03.005
    HU is a most abundant DNA-binding protein in bacteria. This protein is conserved either in its heterodimeric form or in one of its homodimeric forms in all bacteria, in plant chloroplasts, and in some viruses. HU protein non-specifically binds and bends DNA as a hetero- or homodimer and can participate in DNA supercoiling and DNA condensation. It also takes part in some DNA functions such as replication, recombination, and repair. HU does not recognize any specific sequences but shows some specificity to cruciform DNA and to repair intermediates, e.g., nick, gap, bulge, 3'-overhang, etc. To understand the features of HU binding to DNA and repair intermediates, a fast and easy HU proteins purification procedure is required. Here we report overproduction and purification of the HU homodimers. The method of HU purification allows obtaining a pure recombinant non-tagged protein cloned in Escherichia coli. We applied this method for purification of Acholeplasma laidlawii HU and demonstrated that this protein possesses a DNA-binding activity and is free of contaminating nuclease activity. Besides that we have shown that expression of A. laidlawii ihf_hu gene in a slow-growing hupAB E. coli strain restores the wild-type growth indicating that aclHU can perform the basic functions of E. coli HU in vivo.
    Matched MeSH terms: Protein Multimerization
  19. Crystal structure of fuculose aldolase from the Antarctic psychrophilic yeast Glaciozyma antarctica PI12
    Jaafar NR, Littler D, Beddoe T, Rossjohn J, Illias RM, Mahadi NM, et al.
    Acta Crystallogr F Struct Biol Commun, 2016 11 01;72(Pt 11):831-839.
    PMID: 27827354
    Fuculose-1-phosphate aldolase (FucA) catalyses the reversible cleavage of L-fuculose 1-phosphate to dihydroxyacetone phosphate (DHAP) and L-lactaldehyde. This enzyme from mesophiles and thermophiles has been extensively studied; however, there is no report on this enzyme from a psychrophile. In this study, the gene encoding FucA from Glaciozyma antarctica PI12 (GaFucA) was cloned and the enzyme was overexpressed in Escherichia coli, purified and crystallized. The tetrameric structure of GaFucA was determined to 1.34 Å resolution. The overall architecture of GaFucA and its catalytically essential histidine triad are highly conserved among other fuculose aldolases. Comparisons of structural features between GaFucA and its mesophilic and thermophilic homologues revealed that the enzyme has typical psychrophilic attributes, indicated by the presence of a high number of nonpolar residues at the surface and a lower number of arginine residues.
    Matched MeSH terms: Protein Multimerization
  20. Fulltext Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop
    Cale EM, Gorman J, Radakovich NA, Crooks ET, Osawa K, Tong T, et al.
    Immunity, 2017 05 16;46(5):777-791.e10.
    PMID: 28514685 DOI: 10.1016/j.immuni.2017.04.011
    Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.
    Matched MeSH terms: Protein Multimerization
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