Parsing the functional specificity of Siderocalin/Lipocalin 2/NGAL for siderophores and related small-molecule ligands

Clifton, M.C.; Rupert, Peter B.; Hoette, Trisha M.; Raymond, Kenneth N.; Abergel, Rebecca J.; Strong, Roland K.

doi:10.1016/j.yjsbx.2019.100008

Cited by 20 publications

(31 citation statements)

References 59 publications

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“…In addition to intrinsic factors controlling the uptake of siderophores, it is also known that during infection neutrophils release siderocalin (also known as neutrophil gelatinase associated lipocalin (NGAL), 24p3, and lipocalin-2), an acute phase protein that is capable of binding and sequestering extracellular ferric catecholate and carboxymycobactin-type siderophores, thus preventing their capture by invading pathogens [ 70 – 72 ]. To circumvent the effects of siderocalin, some bacteria secrete structurally modified siderophores that are incompatible with the binding site of the protein, and thus are referred to as “stealth siderophores” [ 73 ].…”

Section: Discussionmentioning

confidence: 99%

Acinetobacter baumannii can use multiple siderophores for iron acquisition, but only acinetobactin is required for virulence

2020

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Acinetobacter baumannii is an emerging pathogen that poses a global health threat due to a lack of therapeutic options for treating drug-resistant strains. In addition to acquiring resistance to last-resort antibiotics, the success of A . baumannii is partially due to its ability to effectively compete with the host for essential metals. Iron is fundamental in shaping host-pathogen interactions, where the host restricts availability of this nutrient in an effort to curtail bacterial proliferation. To circumvent restriction, pathogens possess numerous mechanisms to obtain iron, including through the use of iron-scavenging siderophores. A . baumannii elaborates up to ten distinct siderophores, encoded from three different loci: acinetobactin and pre-acinetobactin (collectively, acinetobactin), baumannoferrins A and B, and fimsbactins A-F. The expression of multiple siderophores is common amongst bacterial pathogens and often linked to virulence, yet the collective contribution of these siderophores to A . baumannii survival and pathogenesis has not been investigated. Here we begin dissecting functional redundancy in the siderophore-based iron acquisition pathways of A . baumannii . Excess iron inhibits overall siderophore production by the bacterium, and the siderophore-associated loci are uniformly upregulated during iron restriction in vitro and in vivo . Further, disrupting all of the siderophore biosynthetic pathways is necessary to drastically reduce total siderophore production by A . baumannii , together suggesting a high degree of functional redundancy between the metabolites. By contrast, inactivation of acinetobactin biosynthesis alone impairs growth on human serum, transferrin, and lactoferrin, and severely attenuates survival of A . baumannii in a murine bacteremia model. These results suggest that whilst A . baumannii synthesizes multiple iron chelators, acinetobactin is critical to supporting growth of the pathogen on host iron sources. Given the acinetobactin locus is highly conserved and required for virulence of A . baumannii , designing therapeutics targeting the biosynthesis and/or transport of this siderophore may represent an effective means of combating this pathogen.

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Section: Discussionmentioning

confidence: 99%

Acinetobacter baumannii can use multiple siderophores for iron acquisition, but only acinetobactin is required for virulence

2020

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“…Binding of aferric Ent to Scn was previously reported by cocrystallization during which the Ent was hydrolyzed. 5 Fluorescence studies of Scn ligand binding rely on tryptophan quenching, which is likely due to a heavy-metal effect and therefore insensitive to aferric complexes. To our knowledge, this is the first convincing solution phase data of the aferric lin-Ent-Scn complex with intact lin-Ent.…”

Section: Results and Discussionmentioning

confidence: 99%

“…As a reference, we used a crystal structure of Scn bound with TrenCam, an Ent analog with a nonhydrolyzable, tertiary amine backbone (PDB 3HWG). 5 …”

Section: Results and Discussionmentioning

confidence: 99%

“…The ferric lin-Ent interface is consistent with the model developed by the laboratory of Roland Strong. 5 It is notable that the three pockets of the Arg and Lys-rich Scn calyx bind both ferric and aferric lin-Ent, because the latter molecule lacks the rigidity and anionic character of ferric Ent complexes. Ent fully occupies the first coordination sphere of Fe(III) with its six deprotonated catechol rings, making the Ent structure more rigid and sterically matched to the Scn calyx.…”

Section: Discussionmentioning

confidence: 99%

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Site-Specific Siderocalin Binding to Ferric and Ferric-Free Enterobactin As Revealed by Mass Spectrometry

et al. 2019

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Both host and pathogen competitively manipulate coordination environments during bacterial infections. Human cells release the innate immune protein siderocalin (Scn, also known as lipocalin-2/Lcn2, neutrophil gelatinase-associated lipocalin/NGAL) that can inhibit bacterial growth by sequestering iron in a ferric complex with enterobactin (Ent), the ubiquitous Escherichia coli siderophore. Pathogenic E. coli use the virulence-associated esterase IroE to linearize the Ent cyclic trilactone to linear enterobactin (lin-Ent). We characterized lin-Ent interactions with Scn by using native mass spectrometry (MS) with hydrogen–deuterium exchange (HDX) and Lys/Arg specific covalent footprinting. These approaches support 1:1 binding of both Fe(III)-lin-Ent to Scn and iron-free lin-Ent to Scn. Both ferric and nonferric lin-Ent localize to all three pockets of the Scn calyx, consistent with Scn capture of lin-Ent both before and after Fe(III) chelation. These findings raise the possibility that Scn neutralizes both siderophores and siderophore-bound iron during infections. This integrated, MS-based approach circumvents the limitations that frustrate traditional structural approaches to examining Scn interactions with enterobactin-based ligands.

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“…To identify lead compounds that potentially target LCN2, we analyzed the structural properties of the crystal structure of the LCN2-calyx pocket and ligand-bound structures (28). The LCN2-calyx comprises three pockets (Pockets #1, #2, and #3 of Figure 4A) that accommodate critical functional groups for siderophores, which creates specificity for ligand recognition [25,26]. The key siderophore-contacting residues are Trp79, Arg81, Tyr106, Lys125, and Lys134.…”

Section: Identification Of Lcn2 Small Molecule Inhibitors By In-silico Analysismentioning

confidence: 99%

Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

Santiago-Sánchez

Noriega-Rivera

O'Farril

et al. 2021

IJMS

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Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer, highly metastatic, representing 2–4% of all breast cancer cases in the United States. Despite its rare nature, IBC is responsible for 7–10% of all breast cancer deaths, with a 5-year survival rate of 40%. Thus, targeted and effective therapies against IBC are needed. Here, we proposed Lipocalin-2 (LCN2)—a secreted glycoprotein aberrantly abundant in different cancers—as a plausible target for IBC. In immunoblotting, we observed higher LCN2 protein levels in IBC cells than non-IBC cells, where the LCN2 levels were almost undetectable. We assessed the biological effects of targeting LCN2 in IBC cells with small interference RNAs (siRNAs) and small molecule inhibitors. siRNA-mediated LCN2 silencing in IBC cells significantly reduced cell proliferation, viability, migration, and invasion. Furthermore, LCN2 silencing promoted apoptosis and arrested the cell cycle progression in the G0/G1 to S phase transition. We used in silico analysis with a library of 25,000 compounds to identify potential LCN2 inhibitors, and four out of sixteen selected compounds significantly decreased cell proliferation, cell viability, and the AKT phosphorylation levels in SUM149 cells. Moreover, ectopically expressing LCN2 MCF7 cells, treated with two potential LCN2 inhibitors (ZINC00784494 and ZINC00640089) showed a significant decrease in cell proliferation. Our findings suggest LCN2 as a promising target for IBC treatment using siRNA and small molecule inhibitors.

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Parsing the functional specificity of Siderocalin/Lipocalin 2/NGAL for siderophores and related small-molecule ligands

Cited by 20 publications

References 59 publications

Acinetobacter baumannii can use multiple siderophores for iron acquisition, but only acinetobactin is required for virulence

Acinetobacter baumannii can use multiple siderophores for iron acquisition, but only acinetobactin is required for virulence

Site-Specific Siderocalin Binding to Ferric and Ferric-Free Enterobactin As Revealed by Mass Spectrometry

Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors

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