The Dark Side of EGFP: Defective Polyubiquitination

Baens, Mathijs; Noels, Heidi; Broeckx, Vicky; Hagens, Sofie; Fevery, Sabine; Billiau, An; Vankelecom, Hugo; Marynen, Peter

doi:10.1371/journal.pone.0000054

Cited by 132 publications

(98 citation statements)

References 28 publications

(36 reference statements)

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“…This vector Ubiquilin-1 Is a Molecular Chaperone has been described elsewhere, and this particular GFP variant has been shown to have no effect on proteasomal degradation (35). PCR primers were 5Ј-TTC AAG CTT CCA TGC TGC CCG GTT TGG CAC TG-3Ј and 5Ј-TCC CCG CGG GTT CTG CAT CTG CTC AAA GAA CTT GTA G-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Ubiquilin-1 Is a Molecular Chaperone for the Amyloid Precursor Protein

Stieren

Ayadi

Xiao

et al. 2011

Journal of Biological Chemistry

109

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Alzheimer disease (AD) is associated with extracellular deposition of proteolytic fragments of amyloid precursor protein (APP). Although mutations in APP and proteases that mediate its processing are known to result in familial, early onset forms of AD, the mechanisms underlying the more common sporadic, yet genetically complex forms of the disease are still unclear. Four single-nucleotide polymorphisms within the ubiquilin-1 gene have been shown to be genetically associated with AD, implicating its gene product in the pathogenesis of late onset AD. However, genetic linkage between ubiquilin-1 and AD has not been confirmed in studies examining different populations. Here we show that regardless of genotype, ubiquilin-1 protein levels are significantly decreased in late onset AD patient brains, suggesting that diminished ubiquilin function may be a common denominator in AD progression. Our interrogation of putative ubiquilin-1 activities based on sequence similarities to proteins involved in cellular quality control showed that ubiquilin-1 can be biochemically defined as a bona fide molecular chaperone and that this activity is capable of preventing the aggregation of amyloid precursor protein both in vitro and in live neurons. Furthermore, we show that reduced activity of ubiquilin-1 results in augmented production of pathogenic amyloid precursor protein fragments as well as increased neuronal death. Our results support the notion that ubiquilin-1 chaperone activity is necessary to regulate the production of APP and its fragments and that diminished ubiquilin-1 levels may contribute to AD pathogenesis.

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Section: Methodsmentioning

confidence: 99%

Ubiquilin-1 Is a Molecular Chaperone for the Amyloid Precursor Protein

Stieren

Ayadi

Xiao

et al. 2011

Journal of Biological Chemistry

109

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“…Finally, the fusions to MotB induce a novel BFM dynamic that involves an asymmetry in the speeds attained in opposing rotational directions, depending on the motor's bias. These effects of the fluorescent fusion proteins on the BFM could be partially restored by a rigid linker (EAAAK) 3 inserted at the fusion point. Together, these findings reveal how a fluorescent protein fusion that does not abolish function completely can modulate biologically relevant dynamics of a protein complex and even induce new behavior, all of which can be partially relieved by the incorporation of a linker.…”

Section: Discussionmentioning

confidence: 99%

“…In order to choose the best linker, the population motility was measured for Dendra2 stators joined to four different linkers of the following sequences (see Fig. 5D): (GGGGS), (EAAAK), (GGGGS) 3 , and (EAAAK) 3 .…”

Section: Methodsmentioning

confidence: 99%

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Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

Heo

Nord

Chamousset

et al. 2018

Biophysical Journal

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Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation-new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Fusion of a fluorescent protein to a protein of interest is an invaluable tool for the study of protein function in a broad, and still expanding range of in vivo and in vitro systems. However, it is widely recognized that the presence of fluorescent proteins can alter functional properties of the native protein [1][2][3][4][5] . Careful assessment of these side effects and strategies to minimize them have therefore been critical to the success of fluorescent protein fusions (FPFs) [6][7][8][9] . The continuing development of FPF-based approaches to explore novel phenomena also calls for new insight into the associated side effects and methods to alleviate them [10][11][12] . One direction of research for which this holds is the study of the internal dynamics of proteins complexes 13,14 . Recently, several studies have made use of FPFs to study the internal subunit dynamics of the bacterial flagellar motor (BFM) [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] . Although FPFs were shown to affect BFM function by decreasing the chemotactic motility of cells and average speed of motors 15 , a quantitative characterization of the impact on the BFM mechanical behavior is lacking. Such perturbations limit the depth to which the internal dynamics and function of the BFM, and protein complexes in general, can be probed with FPFs. Linker peptides inserted at the fusion point have been instrumental in minimizing non-native behaviour of FPFs in many biological systems [8][9][10][11] , but their use in the study of protein-complex dynamics is limited in the BFM has thus far been limited.The BFM is located at the base of each flagellum in the membrane of many motile bacteria (Fig. 1A). The torque gene...

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“…In general, green fluorescent protein (GFP) is considered nontoxic both in in vitro and in vivo systems. However, there are some reports showing side effects of GFP in in vitro and in vivo studies, but the details of molecular mechanism of these interactions still remain unclear (Agbulut et al, 2006(Agbulut et al, , 2007Baens et al, 2006;Huang et al, 2000;Liu et al, 1999). Recently, it has been shown that Ku80 attenuates cytotoxicity induced by green fluorescent protein transduction independently of non-homologous end joining (Koike et al, 2013).…”

Section: Introductionmentioning

confidence: 99%