Selective enrichment of metal-binding proteins based on magnetic core/shell microspheres functionalized with metal cations

Fang, Changming; Zhang, Lei; Zhang, Xiaoqin; Lu, Haojie

doi:10.1039/c5an00599j

Cited by 9 publications

(7 citation statements)

References 38 publications

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“…The metal-coordinating potential of GRNs has been hypothesized since their initial characterization [55][56] while the recent study by Fang and colleagues strengthened the rationale for such an investigation by indicating potential coordination of metals by GRNs 57 . Our report brings forth a functionality of GRNs that has remained largely unexplored, particularly at the molecular level.…”

Section: Discussionmentioning

confidence: 99%

“…Properties such as the unusually high number of conserved cysteines and disorder within the sequence in the fully reducing conditions 21 make GRNs similar to MTs, which are known to bind Cu(II), Zn (II) and Cd(II) ions, among others 40 . Further motivated by the IUPred2A predictions that show GRNs to be similar to MTs in redox sensitivity, we set out to test whether GRNs show affinity for divalent cations and reveal potential promiscuity or specificity of such metal coordination 57 . The results indicate that both GRNs -3 and -5 have affinity towards Cu(II) with GRN-3 showing greater affinity than GRN-5 at neural pH.…”

Section: Discussionmentioning

confidence: 99%

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Are Granulins Copper Sequestering Proteins?

Bhopatkar

Rangachari

2020

Preprint

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Granulins (GRN 1-7) are short (∼6 kDa), cysteine-rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These modules, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Our previous investigations into GRN-3 and GRN-5 reveal them to be fully disordered in the reduced form and implicate redox sensitive attributes to the proteins. Such redox-dependent modulation has become associated with proteins involved in oxidative stress regulation and maintaining metal-homeostasis within cells. To probe whether GRNs play a contributory role in such functions, we tested the metal binding potential of the reduced form of GRNs -3 and -5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu(II) and no other divalent cations. Binding of Cu(II) also partly triggered the oxidative multimerization of GRNs via uncoordinated cystines at both pH conditions. Furthermore, binding did not induce gain in secondary structure and the protein remained disordered. Overall, the results indicate that GRN-3 and -5 have a surprisingly strong affinity for Cu(II) in the pM range, comparable to known copper sequestering proteins. This data also hints at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu-induced ROS cytotoxicity in cells. Together, this report uncovers a metal-coordinating capability of GRNs for the first time, which could have profound significance in their structure and function.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Are Granulins Copper Sequestering Proteins?

Bhopatkar

Rangachari

2020

Preprint

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“…MTs function as cellular reservoirs and scavengers of metal ions 29‐32 and as heavy metal detoxifiers 33,34 . GRNs have also been hypothesized to bind metals, 19,35 and were identified as a member of metalloproteins isolated from rat liver 36 . Motivated by prior reports and the observation that the cystine‐rich GRNs are present both extracellularly and intracellularly within lysosomes, we hypothesize that GRNs sequester metals to function as redox‐sensitive regulators of cellular toxicity.…”

Section: Introductionmentioning

confidence: 92%

Are granulins copper sequestering proteins?

Bhopatkar

Rangachari

2020

Proteins

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Granulins (GRN 1‐7) are short (~6 kDa), cysteine‐rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These peptides, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Previously we showed that GRN‐3 and GRN‐5 are fully disordered in the reduced form implicating redox sensitive attributes to the proteins. Redox‐based modulations are often carried out by metalloproteins in mitigating oxidative stress and maintaining metal‐homeostasis within cells. To probe whether GRNs play a role in metal sequestration, we tested the metal binding propensity of the reduced forms of GRNs −3 and − 5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu and no other divalent metal cations, with a greater specificity for Cu(I). Binding of Cu did not result in a disorder‐to‐order structural transition but partly triggered the multimerization of GRNs via uncoordinated cystines at both pH conditions. Overall, the results indicate that GRNs −3 and − 5 have surprisingly strong affinity for Cu in the pM range, comparable to other known copper sequestering proteins. The results also hint at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu‐induced ROS cytotoxicity in cells. Together, this report uncovers metal‐coordinating property of GRNs for the first time, which may have profound significance in their structure and pathophysiological functions.

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“…This has been used previously to find Cu 2+ , Zn 2+ , Ni 2+ and Co 2+ binding proteins in Streptococcus pneumonia and Ni 2+ and Bi 2+ in Helicobacter pylori [25,26,27,28]. Recently, magnetic microspheres have been developed to perform IMAC [29]. …”

Section: Metalloproteomic Approachesmentioning

confidence: 99%

Microbial Metalloproteomics

Hagedoorn

2015

Proteomes

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Metalloproteomics is a rapidly developing field of science that involves the comprehensive analysis of all metal-containing or metal-binding proteins in a biological sample. The purpose of this review is to offer a comprehensive overview of the research involving approaches that can be categorized as inductively coupled plasma (ICP)-MS based methods, X-ray absorption/fluorescence, radionuclide based methods and bioinformatics. Important discoveries in microbial proteomics will be reviewed, as well as the outlook to new emerging approaches and research areas.

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Selective enrichment of metal-binding proteins based on magnetic core/shell microspheres functionalized with metal cations

Cited by 9 publications

References 38 publications

Are Granulins Copper Sequestering Proteins?

Are Granulins Copper Sequestering Proteins?

Are granulins copper sequestering proteins?

Microbial Metalloproteomics

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