Characterizing Substrate Selectivity of Ubiquitin C-Terminal Hydrolase-L3 Using Engineered α-Linked Ubiquitin Substrates

Navarro, Mario Fco; Carmody, Lisa; Romo‐Fewell, Octavio; Lokensgard, Melissa E.; Love, John J.

doi:10.1021/bi5006317

Cited by 8 publications

(10 citation statements)

References 67 publications

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“…Reduced thermal stability was also reported experimentally due to ubiquitination of the β1 domain of streptococcal protein G (Gβ1) and the B domain of staphylococcal protein A (SpAB). Several variants of the Gβ1 and SpA B proteins were linked to a monomeric ubiquitin at the N-terminus . Most of the variants showed lower melting temperatures with an average difference between the free Gb1 (SpA B ) variant and the Gb1 (SpA B ) variant fused to Ub of 7.2 ± 3.7 °C (1.8 ± 1.5 °C).…”

Section: Biological Implications Of Destabilization Following Protein...mentioning

confidence: 99%

Protein Assembly and Building Blocks: Beyond the Limits of the LEGO Brick Metaphor

Levy

2017

Biochemistry

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Proteins, like other biomolecules, have a modular and hierarchical structure. Various building blocks are used to construct proteins of high structural complexity and diverse functionality. In multidomain proteins, for example, domains are fused to each other in different combinations to achieve different functions. Although the LEGO brick metaphor is justified as a means of simplifying the complexity of three-dimensional protein structures, several fundamental properties (such as allostery or the induced-fit mechanism) make deviation from it necessary to respect the plasticity, softness, and cross-talk that are essential to protein function. In this work, we illustrate recently reported protein behavior in multidomain proteins that deviates from the LEGO brick analogy. While earlier studies showed that a protein domain is often unaffected by being fused to another domain or becomes more stable following the formation of a new interface between the tethered domains, destabilization due to tethering has been reported for several systems. We illustrate that tethering may sometimes result in a multidomain protein behaving as "less than the sum of its parts". We survey these cases for which structure additivity does not guarantee thermodynamic additivity. Protein destabilization due to fusion to other domains may be linked in some cases to biological function and should be taken into account when designing large assemblies.

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Section: Biological Implications Of Destabilization Following Protein...mentioning

confidence: 99%

Protein Assembly and Building Blocks: Beyond the Limits of the LEGO Brick Metaphor

Levy

2017

Biochemistry

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“…The dramatic destabilization observed in ubiquitination of Ubc7 at specific sites was mostly attributed to changes in the unfolded state. Furthermore, it was recently shown that in vitro attachment of Ub reduces the thermodynamic stability of the modified protein (Ub C‐terminal hydrolase‐L3) …”

Section: Introductionmentioning

confidence: 99%

“…It was computationally shown that protein tethering may result in intrinsic destabilization . Experimentally, both destabilization and stabilization were observed upon tethering or cross‐linking . These effects may have different origins related to changes in the dynamics and energetics of either the folded or unfolded state.…”

Section: Introductionmentioning

confidence: 99%

Nonspecific yet decisive: Ubiquitination can affect the native‐state dynamics of the modified protein

2015

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Ubiquitination is one of the most common post-translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site-specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48-linked tetra-ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the protein's native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half-life through proteasomal degradation.

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“…Early study showed that UCH-L3 mRNA level was significantly up-regulated in breast cancer tissue compared to adjacent normal breast tissue and had significantly a positive correlation with histological grades and negative correlation with estrogen and progesterone receptor status, also suggesting the possible involvement of UCH-L3 in the pathogenesis and progression of breast cancer [19]. UCH-L3 hydrolysis is significantly influenced by thermal stability [24]. A recent study showed that UCH-L3 might play a role in the regulation of autophagic death of leukemia [25].…”

Section: Discussionmentioning

confidence: 99%

The Effects of UCH-L3 on the Biological Behaviours of Breast Cancer: Correlation with the Expressions of HIF-1α

Zheng¹,

Zhong²,

Ren³

et al. 2019

J Cancer Sci Ther

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Projective: HIF-1α is highly expressed in the triple negative breast cancer cell line (MDA-MB-231), which is lack of the expression of ER,PR and HER2 and exhibits high invasive and metastatic ability. Previous study detected that siRNA targeting HIF-1α in MDA-MB-231 restrained the cell growth and the abilities of immigration and invasion, and promoted apoptosis. UCH-L3 protein was found to be one of the differential proteins detected by Bidirectional gel Electrophoresis and Proteomics in the cells with HIF-1α siRNA cells comparing to no-siRNA cells. The objective of this study is to probe the effect of UCH-L3 on the biological behaviors of the triple negative breast cancer cell line (MDA-MB-231).Methods: Over or blocking expressions of UCH-L3 were established by the transfections with lentiviral constitutive vector and siRNA targeting UCH-L3 respectively. Real time quantitative PCR and Western blot or Co-ip were used to detect the mRNA and proteins. CCK8, and clone formation assays were to evaluate the cell growth and clonality. Matrigel Transwell and Would Scratch assay were used to estimate the cell invasion and mobility. Results:The over-expression of UCH-L3 inhibited the cell growth and clonality, weaken the abilities of cell immigration and invasion, and lowered the expression of free and ubiquitined HIF-1α in MDA-MB-231 cells with lentiviral vector comparing to those in the cells with control vector. Application of proteolytic enzyme inhibitor, MG132 increased the protein level of UCH-L3 but still decreased the protein level of HIF-1α in the cells with UCH-L3 over-expression. Blocking UCH-L3 expression by siRNA technique increased the expression of HIF-1α properly. Conclusion: high expression of UCH-L3showed an inhibitory effect on the biological behaviors of triple negative breast cancer cell and negative effect on HIF-1α expression, implying that UCH-L3 likely to be a therapeutic strategy for triple negative breast cancer.

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Characterizing Substrate Selectivity of Ubiquitin C-Terminal Hydrolase-L3 Using Engineered α-Linked Ubiquitin Substrates

Cited by 8 publications

References 67 publications

Protein Assembly and Building Blocks: Beyond the Limits of the LEGO Brick Metaphor

Protein Assembly and Building Blocks: Beyond the Limits of the LEGO Brick Metaphor

Nonspecific yet decisive: Ubiquitination can affect the native‐state dynamics of the modified protein

The Effects of UCH-L3 on the Biological Behaviours of Breast Cancer: Correlation with the Expressions of HIF-1α

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