In Vivo Fluorescent Detection of Fe-S Clusters Coordinated by Human GRX2

Hoff, Kevin G.; Culler, Stephanie J.; Nguyen, Peter Q.; McGuire, Ryan M.; Silberg, Jonathan J.; Smolke, Christina D.

doi:10.1016/j.chembiol.2009.11.011

Cited by 31 publications

(36 citation statements)

References 48 publications

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“…48 Other examples of genes that have been split and reconstituted in vivo include the genes encoding ubiquitin, 49 PurN, 50 adenylate kinase 51 and the yellow fluorescent protein, Venus. 52 Because they are nearly devoid of bacterial sequences, almost all of the Minivector is available to encode a useful sequence. Even at 2000 bp, Minivectors can encode a promoter and ∼1500 bp of gene sequence (either an intact small gene or gene fragment), significantly increasing the arsenal of useful therapeutic sequences.…”

Section: Discussionmentioning

confidence: 99%

Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery

et al. 2011

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Supercoiled DNAs varying from 281 to 5302 bp were subjected to shear forces generated by aerosolization or sonication. DNA shearing strongly correlated with length. Typical sized plasmids (⩾3000 bp) degraded rapidly. DNAs 2000–3000 bp persisted ∼10 min. Even in the absence of condensing agents, supercoiled DNA <1200 bp survived nebulization, and increased forces of sonication were necessary to shear it. Circular vectors were considerably more resistant to shearing than linear vectors of the same length. DNA supercoiling afforded additional protection. These results show the potential of shear-resistant Minivector DNAs to overcome one of the major challenges associated with gene therapy delivery.

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Section: Discussionmentioning

confidence: 99%

Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery

et al. 2011

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“…However, the measurements for sT were only slightly above the negative control (data not shown), an observation likely due to the technical limitation of the assay to detect very low iron levels in sT from mammalian cells. Similar technical issues may help explain why it has not been possible to directly detect iron in any of the known cellular Fe/S cluster proteins purified from mammalian cells (48). However, after 55 Fe labeling of HEK293 cells transfected with a plasmid encoding MCPyV sT, we were able to detect 55 Fe in the immunoprecipitated MCPyV sT sample.…”

Section: Discussionmentioning

confidence: 75%

“…Due to the low level of protein expression in mammalian cells, it is not possible to use the EPR technique to analyze MCPyV sT expressed in cultured cells. This is a common technical limitation in the Fe/S cluster protein field (48,49), and researchers tend to overexpress their protein of interest in bacteria or yeast to detect the presence of Fe/S clusters. In order to obtain supportive evidence in a mammalian cell culture system, we transfected HEK293 cells with either the vector control or a plasmid encoding MCPyV sT fused to two IgG binding domains of S. aureus protein A.…”

Section: Mcpyv St Contains a [2fe-2s] And A [4fe-4s] Clustermentioning

confidence: 99%

The Oncogenic Small Tumor Antigen of Merkel Cell Polyomavirus Is an Iron-Sulfur Cluster Protein That Enhances Viral DNA Replication

Tsang

Wang

Nakamaru‐Ogiso

et al. 2016

J Virol

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Merkel cell polyomavirus (MCPyV) plays an important role in Merkel cell carcinoma (MCC).A ccumulating evidence has suggested a role for Merkel cell polyomavirus (MCPyV) in the development of a lethal skin cancer, Merkel cell carcinoma (MCC), making it the first polyomavirus to be conclusively associated with human cancer (1). MCC tumors develop rapidly and are highly metastatic. It is one of the most aggressive skin cancers with a high mortality rate of 33% (which exceeds the rate of melanoma) (2), and a 5-year observed survival rate of less than 45% (3). High seroprevalence for MCPyV in the adult human population and analyses of healthy human skin suggest that MCPyV is a common component of the normal skin flora (4, 5).MCPyV has a circular, double-stranded DNA genome of ϳ5 kb (6). A regulatory region (RR) separates the early and late regions of the viral genome (6). The RR contains the viral origin of replication (Ori) and bidirectional promoters for viral transcription. The early region encodes large T (LT) and small T (sT) antigens, the 57kT antigen, and a recently discovered protein called alternative LT open reading frame (ORF) (ALTO) (6, 7). The late region encodes the capsid proteins, VP1 and VP2 (8,9). It is well established that clonal integration of the MCPyV genome into the host genome is a key event in the development of MCPyV-associated MCC tumors (10). Integration or other mutagenic events almost invariably result in truncation of LT upstream of its C-terminal helicase domain, rendering the mutant protein defective for mediating viral replication (10). Although both LT and sT antigens are often required for MCPyV-positive MCC cell survival and proliferation (11,12), sT has emerged as the key oncogenic driver in MCC carcinogenesis. This is supported by the observation that sT expression can transform rodent fibroblasts, whereas the expression of LT, or truncated LT found in MCC tumors, cannot (12). MCPyV sT also demonstrates robust transforming activity in transgenic mouse model systems (13).Due to differential splicing, LT and sT share an N-terminal domain with homology to cellular DnaJ chaperone proteins. In sT, the DnaJ motif is followed by an sT-unique C-terminal do- Citation Tsang SH, Wang R, Nakamaru-Ogiso E, Knight SAB, Buck CB, You J. 2016. The oncogenic small tumor antigen of Merkel cell polyomavirus is an iron-sulfur cluster protein that enhances viral DNA replication. J Virol 90:1544 -1556.

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“…These interesting links remain largely unexplored due to the lack of suitable technology. The activities of natural (e.g., mitochondrial aconitase or succinate dehydrogenase; Foury, 1999; Mochel et al, 2008) or artificial (Hoff et al, 2009a,b) ISC-containing enzymes are currently used as measures of ISC synthesis. One limitation is that these activities lay downstream of the initial and rate-limiting step in ISC synthesis (i.e., the assembly of [2Fe–2S] clusters on ISC scaffolds).…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial iron-sulfur cluster dysfunction in neurodegenerative disease

Isaya

2014

Front. Pharmacol.

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Growing evidence supports a role for mitochondrial iron metabolism in the pathophysiology of neurodegenerative disorders such as Friedreich ataxia (FRDA) and Parkinson disease (PD) as well as in the motor and cognitive decline associated with the aging process. Ironsulfur enzyme deficits and regional iron accumulation have been observed in each of these conditions. In spite of significant etiological, clinical and pathological differences that exist between FRDA and PD, it is possible that defects in mitochondrial iron-sulfur clusters (ISCs) biogenesis represent a common underlying mechanism leading to abnormal intracellular iron distribution with mitochondrial iron accumulation, oxidative phosphorylation deficits and oxidative stress in susceptible cells and specific regions of the nervous system. Moreover, a similar mechanism may contribute to the age-dependent iron accumulation that occurs in certain brain regions such as the globus pallidus and the substantia nigra. Targeting chelatable iron and reactive oxygen species appear as possible therapeutic options for FRDA and PD, and possibly other age-related neurodegenerative conditions. However, new technology to interrogate ISC synthesis in humans is needed to (i) assess how defects in this pathway contribute to the natural history of neurodegenerative disorders and (ii) develop treatments to correct those defects early in the disease process, before they cause irreversible neuronal cell damage.

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In Vivo Fluorescent Detection of Fe-S Clusters Coordinated by Human GRX2

Cited by 31 publications

References 48 publications

Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery

Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery

The Oncogenic Small Tumor Antigen of Merkel Cell Polyomavirus Is an Iron-Sulfur Cluster Protein That Enhances Viral DNA Replication

Mitochondrial iron-sulfur cluster dysfunction in neurodegenerative disease

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