Enzymatic Midchain Branching of Polylactosamine Backbones Is Restricted in a Site-Specific Manner in α1,3-Fucosylated Chains†

Biochemistry ◽  
1997 ◽  
Vol 36 (44) ◽  
pp. 13729-13735 ◽  
Author(s):  
Anne Leppänen ◽  
Ritva Niemelä ◽  
Ossi Renkonen
Keyword(s):  
Site Specific ◽  
Specific Manner ◽  
A Site

Optimization of the posttranslational click modification of proteins

2011 ◽  
Vol 76 (9) ◽  
pp. 1089-1101
Author(s):  
Milan Vrabel ◽  
Emine Kaya ◽  
Stefan Prill ◽  
Veronika Ehmke ◽  
Thomas Carell
Keyword(s):  
Mass Spectrometry ◽  
Amino Acids ◽  
Fluorescent Protein ◽  
Click Reaction ◽  
Yellow Fluorescent Protein ◽  
Trna Synthetase ◽  
Site Specific ◽  
Specific Manner ◽  
Modified Proteins ◽  
A Site

In order to develop efficient methods that would enable the synthesis of posttranslationaly modified proteins in a site-specific manner we have adopted the orthogonal pyrrolysyl-tRNA synthetase/tRNA pair to genetically encode various pyrrolysine analogs, which we were able to insert into the yellow fluorescent protein (YFP). These experiments showed that the alkene and alkyne containing amino acids 5 and 6 are superior substrates for the pyrrolysyl-tRNA synthetase and that they can be successfully incorporated into proteins. Using the Cu(I)-catalyzed Huisgen–Meldal–Sharpless click reaction, the alkyne containing YFP was finally glycosylated with various sugars. We confirmed the presence of the modified amino acids as well as the corresponding sugar modifications by HPLC-MS/MS mass spectrometry.

scholarly journals Osterix Regulates Tooth Root Formation in a Site-specific Manner

2015 ◽  
Vol 94 (3) ◽  
pp. 430-438 ◽  
Author(s):  
T.H. Kim ◽  
C.H. Bae ◽  
J.C. Lee ◽  
J.E. Kim ◽  
X. Yang ◽  
...  
Keyword(s):  
Root Formation ◽  
Tooth Root ◽  
Site Specific ◽  
Specific Manner ◽  
A Site

scholarly journals Hypoxia inducible factor signaling in breast tumors controls spontaneous tumor dissemination in a site-specific manner

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Vera M. Todd ◽  
Lawrence A. Vecchi ◽  
Miranda E. Clements ◽  
Katherine P. Snow ◽  
Cayla D. Ontko ◽  
...  
Keyword(s):  
Primary Tumor ◽  
Hypoxia Inducible Factor ◽  
Metastatic Tumor ◽  
Breast Tumors ◽  
Tumor Burden ◽  
Site Specific ◽  
Tumor Dissemination ◽  
Specific Manner ◽  
A Site ◽  
The Impact

AbstractHypoxia is a common feature in tumors and induces signaling that promotes tumor cell survival, invasion, and metastasis, but the impact of hypoxia inducible factor (HIF) signaling in the primary tumor on dissemination to bone in particular remains unclear. To better understand the contributions of hypoxia inducible factor 1 alpha (HIF1α), HIF2α, and general HIF pathway activation in metastasis, we employ a PyMT-driven spontaneous murine mammary carcinoma model with mammary specific deletion of Hif1α, Hif2α, or von Hippel-Lindau factor (Vhl) using the Cre-lox system. Here we show that Hif1α or Hif2α deletion in the primary tumor decreases metastatic tumor burden in the bone marrow, while Vhl deletion increases bone tumor burden, as hypothesized. Unexpectedly, Hif1α deletion increases metastatic tumor burden in the lung, while deletion of Hif2α or Vhl does not affect pulmonary metastasis. Mice with Hif1α deleted tumors also exhibit reduced bone volume as measured by micro computed tomography, suggesting that disruption of the osteogenic niche may be involved in the preference for lung dissemination observed in this group. Thus, we reveal that HIF signaling in breast tumors controls tumor dissemination in a site-specific manner.

scholarly journals Site-specific monoubiquitination downregulates Rab5 by disrupting effector binding and guanine nucleotide conversion

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Donghyuk Shin ◽  
Wooju Na ◽  
Ji-Hyung Lee ◽  
Gyuhee Kim ◽  
Jiseok Baek ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Regulatory Mechanism ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Site Specific ◽  
Downstream Effectors ◽  
Specific Manner ◽  
A Site ◽  
Effector Binding

Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5K140, whereas GDP release and GTP loading activities were altered in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.

scholarly journals Recent advances in covalent, site-specific protein immobilization

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 2303 ◽  
Author(s):  
Morten Meldal ◽  
Sanne Schoffelen
Keyword(s):  
Selection Process ◽  
Specific Protein ◽  
Sortase A ◽  
Site Specific ◽  
Recent Advances ◽  
Determining Factors ◽  
Protein Molecules ◽  
Specific Manner ◽  
Immobilized Proteins ◽  
A Site

The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard.

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