Dataset Information

Deep Red Blood Cell Proteome Defines the Band 3 N-Terminus Interactome as a Regulator of Hypoxic Adaptation via BLVRB-Dependent S-Nitroso Transfer

ABSTRACT: Red blood cells (RBCs) are transcriptionally silent yet dynamically remodel metabolism in response to oxygen tension. Using ultra-pure human RBCs, we generated the deepest contamination-free proteome to date (3,775 proteins) and mapped the oxygen-dependent interactome. These datasets reveal an oxygen-responsive metabolon centered on the Band 3 (SLC4A1) N-terminus. We identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that dissociates under hypoxia, coincident with increased Band 3–deoxyhemoglobin contacts. This reversible assembly functions as an oxygen-sensitive switch coordinating redox and glycolytic remodeling. Humanized mice lacking Band 3 N-terminal segments exhibit impaired glycolytic activation, reduced 2,3-bisphosphoglycerate synthesis, and diminished exercise tolerance, demonstrating physiological relevance. Population-scale cis-pQTLs for SLC4A1 and BLVRB suggest functions beyond canonical heme catabolism. Mechanistically, BLVRB serves as a nitric-oxide relay that trans-nitrosates the glycolytic enzyme GAPDH at C152, transiently inhibiting glycolysis. Together, these findings define a Band 3–BLVRB–hemoglobin axis integrating oxygen sensing and metabolic control.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Erythrocyte, Blood

DISEASE(S): Disease Free

SUBMITTER: Shaun Bevers

LAB HEAD: Angelo D'Alessandro

PROVIDER: PXD072059 | Pride | 2026-06-29

REPOSITORIES: Pride

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			Action	DRS
	AIssaian_NorHyp_RBC_NO_xLinks_TMT16_DSSO.mzML	Mzml
	AIssaian_NorHyp_RBC_NO_xLinks_TMT16_DSSO.mzid	Mzid
	AIssaian_NorHyp_RBC_NO_xLinks_TMT16_DSSO_Crosslinks.xlsx	Xlsx
	AIssaian_NorHyp_RBC_NO_xLinks_TMT16_SDA.mzML	Mzml
	AIssaian_NorHyp_RBC_NO_xLinks_TMT16_SDA.mzid	Mzid

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Publications

The red blood cell proteome and interactome identify a Band 3-BLVRB axis regulating hypoxic metabolic adaptation.

Issaian Aaron Vahe AV Dzieciatkowska Monika M Bevers Shaun S Safari Zohreh Z Hay Ariel M AM Cendali Francesca I FI Argabright Amy A Rogers Stephen C SC Saviola Anthony J AJ Redzic Jasmina S JS Wartchow Eric E Reisz Haines Julie J Keele Gregory R GR Haiman Zachary B ZB Nemkov Travis T Stephenson Daniel D Lisk Christina C Vallese Francesca F Palsson Bernhard O BO King S Bruce SB Page Grier P GP Doctor Allan A Hudson Krystalyn E KE Hansen Kirk C KC Irwin David C DC Mohandas Narla N Zimring James C JC Eisenmesser Elan Z EZ D'Alessandro Angelo A

Blood 20260601

Red blood cells (RBCs) are transcriptionally silent yet dynamically remodel metabolism in response to oxygen tension. Using ultra-pure human RBCs, we generated the deepest contamination-free proteome to date (3,775 proteins) and mapped the oxygen-dependent interactome. These datasets reveal an oxygen-responsive metabolon centered on the Band 3 (SLC4A1) N-terminus. We identify biliverdin reductase B (BLVRB) as a previously unrecognized Band 3 interactor that dissociates under hypoxia, coincident ...[more]

PMID: 42224377

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Project description:The metabolic adaptation of eukaryotic cells to hypoxia involves increasing dependence upon glycolytic ATP production, an event with consequences for both cell bioenergetics and cell fate. This response is regulated at the transcriptional level by HIF-1-dependent transcriptional upregulation of all ten glycolytic enzymes. However, this response alone does not account for the levels of ATP produced in hypoxia. Here, we investigated additional mechanisms of regulating glycolysis in hypoxia. We found that both intestinal epithelial cells treated with inhibitors of transcription and translation and human platelets (which lack nuclei) maintained the capacity for hypoxia-induced glycolysis, suggesting the involvement of a non-transcriptional component to the hypoxia-induced metabolic switch to a highly glycolytic phenotype. Mass spectrometric analysis of the interactome of immunoprecipitated rate-limiting glycolytic enzymes identified hypoxia-sensitive complexes comprising multiple glycolytic enzymes and glucose transporters in intestinal epithelial cells. Surprisingly, the formation of glycolytic complexes, though not dependent upon transcription, occurs via a HIF-1?-dependent mechanism, suggesting that HIF-1? may play a moonlighting role in the formation / maintenance of glycolytic complexes. Furthermore, we provide evidence for the presence of HIF-1? in cytosolic fractions of hypoxic cells which physically associated with the glucose transporter GLUT1 and the glycolytic enzyme PFKP in a hypoxia-sensitive manner. In conclusion, we hypothesize that HIF-1? plays a role in initiation and/or maintenance of glycolytic complexes in intestinal epithelial cells under hypoxic conditions in a manner which optimizes catalytic efficiency of the pathway by facilitating substrate channeling of glycolytic intermediates between sequential pathway enzymes. In hypoxia, cells undergo a metabolic switch to increased glycolysis. This has important implications for cell behavior, phenotype, and fate in both healthy and cancerous cells. Here we describe a mechanism by which HIF-1, in addition to increasing glycolytic enzyme expression, promotes glycolysis via the formation of a metabolic complex.