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991.
《Expert review of proteomics》2013,10(5):545-559
Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein–protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms. 相似文献
992.
《Nucleosides, nucleotides & nucleic acids》2013,32(6-7):921-925
Photoanlogues of the initiation substrates of the RNA polymerase II, N3Ar‐ NH(CH2)nNHpppA where N3Ar is 5‐azido‐2‐nitrobenzoyl group (n = 2 or 4) were synthesized, allowing the preparation of photoreactive oligonucleotides in situ by RNA polymerase II for application as photolabels. Photolysis of p‐nitro‐substituted aromatic azide in aqueous medium was investigated. Using the azoxy‐coupling reaction it was possible to determine whether a nitrene or p‐nitrophenyl hydroxylamine azoxy compound is the trappable intermediate that is generated at ambient temperature in aqueous solution. 相似文献
993.
《Autophagy》2013,9(1):170-172
A long-standing quest in the autophagy field is to define the membrane origin of the autophagosome. We have established a cell-free assay based on LC3 lipidation that recapitulates multiple regulatory hallmarks of early autophagosome biogenesis. Using a systematic membrane fractionation approach, we have identified the ER-Golgi intermediate compartment (ERGIC) as the most efficient membrane substrate for LC3 lipidation. Further studies indicate that the ERGIC plays an essential role to trigger LC3 lipidation and autophagosome biogenesis by recruiting the key early autophagic factor ATG14. 相似文献
994.
Chenxi Shen Stephanie Y. Jo Chenzhong Liao Jay L. Hess Zaneta Nikolovska-Coleska 《The Journal of biological chemistry》2013,288(42):30585-30596
The MLL fusion proteins, AF9 and ENL, activate target genes in part via recruitment of the histone methyltransferase DOT1L (disruptor of telomeric silencing 1-like). Here we report biochemical, biophysical, and functional characterization of the interaction between DOT1L and MLL fusion proteins, AF9/ENL. The AF9/ENL-binding site in human DOT1L was mapped, and the interaction site was identified to a 10-amino acid region (DOT1L865–874). This region is highly conserved in DOT1L from a variety of species. Alanine scanning mutagenesis analysis shows that four conserved hydrophobic residues from the identified binding motif are essential for the interactions with AF9/ENL. Binding studies demonstrate that the entire intact C-terminal domain of AF9/ENL is required for optimal interaction with DOT1L. Functional studies show that the mapped AF9/ENL interacting site is essential for immortalization by MLL-AF9, indicating that DOT1L interaction with MLL-AF9 and its recruitment are required for transformation by MLL-AF9. These results strongly suggest that disruption of interaction between DOT1L and AF9/ENL is a promising therapeutic strategy with potentially fewer adverse effects than enzymatic inhibition of DOT1L for MLL fusion protein-associated leukemia. 相似文献
995.
Desmosomes are dynamic junctions between cells that maintain the structural integrity of skin and heart tissues by withstanding shear forces. Mutations in component genes cause life-threatening conditions including arrhythmogenic right ventricular cardiomyopathy, and desmosomal proteins are targeted by pathogenic autoantibodies in skin blistering diseases such as pemphigus. Here, we review a set of newly discovered pathogenic alterations and discuss the structural repercussions of debilitating mutations on desmosomal proteins. The architectures of native desmosomal assemblies have been visualized by cryo-electron microscopy and cryo-electron tomography, and the network of protein domain interactions is becoming apparent. Plakophilin and desmoplakin mutations have been discovered to alter binding interfaces, structures, and stabilities of folded domains that have been resolved by X-ray crystallography and NMR spectroscopy. The flexibility within desmoplakin has been revealed by small-angle X-ray scattering and fluorescence assays, explaining how mechanical stresses are accommodated. These studies have shown that the structural and functional consequences of desmosomal mutations can now begin to be understood at multiple levels of spatial and temporal resolution. This review discusses the recent structural insights and raises the possibility of using modeling for mechanism-based diagnosis of how deleterious mutations alter the integrity of solid tissues. 相似文献
996.
Everett Clinton Smith Stacy E. Smith James R. Carter Stacy R. Webb Kathleen M. Gibson Lance M. Hellman Michael G. Fried Rebecca Ellis Dutch 《The Journal of biological chemistry》2013,288(50):35726-35735
Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. 相似文献
997.
Omri Faingold Avraham Ashkenazi Nathali Kaushansky Avraham Ben-Nun Yechiel Shai 《The Journal of biological chemistry》2013,288(46):32852-32860
An immunosuppressive motif was recently found within the HIV-1 gp41 fusion protein (termed immunosuppressive loop-associated determinant core motif (ISLAD CM)). Peptides containing the motif interact with the T-cell receptor (TCR) complex; however, the mechanism by which the motif exerts its immunosuppressive activity is yet to be determined. Recent studies showed that interactions between protein domains in the membrane milieu are not always sterically controlled. Therefore, we utilized the unique membrane leniency toward association between d- and l-stereoisomers to investigate the detailed mechanism by which ISLAD CM inhibits T-cell activation. We show that a d-enantiomer of ISLAD CM (termed ISLAD d-CM) inhibited the proliferation of murine myelin oligodendrocyte glycoprotein (MOG)-(35–55)-specific line T-cells to the same extent as the l-motif form. Moreover, the d- and l-forms preferentially bound spleen-derived T-cells over B-cells by 13-fold. Furthermore, both forms of ISLAD CM co-localized with the TCR on activated T-cells and interacted with the transmembrane domain of the TCR. FRET experiments revealed the importance of basic residues for the interaction between ISLAD CM forms and the TCR transmembrane domain. Ex vivo studies demonstrated that ISLAD d-CM administration inhibited the proliferation (72%) and proinflammatory cytokine secretion of pathogenic MOG(35–55)-specific T-cells. This study provides insights into the immunosuppressive mechanism of gp41 and demonstrates that chirality-independent interactions in the membrane can take place in diverse biological systems. Apart from HIV pathogenesis, the d-peptide reported herein may serve as a potential tool for treating T-cell-mediated pathologies. 相似文献
998.
A critical step in the influenza virus replication cycle is the cleavage activation of the HA precursor. Cleavage activation of influenza HA enables fusion with the host endosome, allowing for release of the viral genome into the host cell. To date, studies have determined that HA activation is driven by trypsin-like host cell proteases, as well as yet to be identified bacterial proteases. Although the number of host proteases that can activate HA is growing, there is still uncertainty regarding which secreted proteases are able to support multicycle replication of influenza. In this study, we have determined that the kallikrein-related peptidases 5 and 12 are secreted from the human respiratory tract and have the ability to cleave and activate HA from the H1, H2, and H3 subtypes. Each peptidase appears to have a preference for particular influenza subtypes, with kallikrein 5 cleaving the H1 and H3 subtypes most efficiently and kallikrein 12 cleaving the H1 and H2 subtypes most efficiently. Cleavage analysis using HA cleavage site peptide mimics revealed that the amino acids neighboring the arginine cleavage site affect cleavage efficiency. Additionally, the thrombolytic zymogens plasminogen, urokinase, and plasma kallikrein have all been shown to cleave and activate influenza but are found circulating mainly as inactive precursors. Kallikrein 5 and kallikrein 12 were examined for their ability to activate the thrombolytic zymogens, and both resulted in activation of each zymogen, with kallikrein 12 being a more potent activator. Activation of the thrombolytic zymogens may therefore allow for both direct and indirect activation of the HA of human-adapted influenza viruses by kallikrein 5 and kallikrein 12. 相似文献
999.
Kim C. Mansky Eric D. Jensen Julia Davidova Masato Yamamoto Rajaram Gopalakrishnan 《The Journal of biological chemistry》2013,288(14):9826-9834
Although PKD is broadly expressed and involved in numerous cellular processes, its function in osteoclasts has not been previously reported. In this study, we found that PKD2 is the main PKD isoform expressed in osteoclastic cells. PKD phosphorylation, indicative of the activated state, increased after 2–3 days of treatment of bone marrow macrophages with M-CSF and RANKL, corresponding to the onset of preosteoclast fusion. RNAi against PKD2 and treatment with the PKD inhibitor CID755673 showed that PKD activity is dispensable for induction of bone marrow macrophages into tartrate-resistant acid phosphatase-positive preosteoclasts in culture but is required for the transition from mononucleated preosteoclasts to multinucleated osteoclasts. Loss of PKD activity reduced expression of DC-STAMP in RANKL-stimulated cultures. Overexpression of DC-STAMP was sufficient to rescue treatment with CID755673 and restore fusion into multinucleated osteoclasts. From these data, we conclude that PKD activity promotes differentiation of osteoclast progenitors through increased expression of DC-STAMP. 相似文献
1000.