HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation
Yanxin Fan, Marta Murgia, Monika I. Linder, Yoko Mizoguchi, Cong Wang, Marcin Łyszkiewicz, Natalia Ziȩtara, Yanshan Liu, Stephanie Frenz, Gabriela Sciuccati, Armando Partida-Gaytan, Zahra Alizadeh, Nima Rezaei, Peter Rehling, Sven Dennerlein, Matthias Mann, Christoph Klein
Yanxin Fan, Marta Murgia, Monika I. Linder, Yoko Mizoguchi, Cong Wang, Marcin Łyszkiewicz, Natalia Ziȩtara, Yanshan Liu, Stephanie Frenz, Gabriela Sciuccati, Armando Partida-Gaytan, Zahra Alizadeh, Nima Rezaei, Peter Rehling, Sven Dennerlein, Matthias Mann, Christoph Klein
View: Text | PDF
Research Article Cell biology Immunology

HAX1-dependent control of mitochondrial proteostasis governs neutrophil granulocyte differentiation

Abstract

The relevance of molecular mechanisms governing mitochondrial proteostasis to the differentiation and function of hematopoietic and immune cells is largely elusive. Through dissection of the network of proteins related to HCLS1-associated protein X-1, we defined a potentially novel functional CLPB/HAX1/(PRKD2)/HSP27 axis with critical importance for the differentiation of neutrophil granulocytes and, thus, elucidated molecular and metabolic mechanisms underlying congenital neutropenia in patients with HAX1 deficiency as well as bi- and monoallelic mutations in CLPB. As shown by stable isotope labeling by amino acids in cell culture (SILAC) proteomics, CLPB and HAX1 control the balance of mitochondrial protein synthesis and persistence crucial for proper mitochondrial function. Impaired mitochondrial protein dynamics are associated with decreased abundance of the serine-threonine kinase PRKD2 and HSP27 phosphorylated on serines 78 and 82. Cellular defects in HAX1–/– cells can be functionally reconstituted by HSP27. Thus, mitochondrial proteostasis emerges as a critical molecular and metabolic mechanism governing the differentiation and function of neutrophil granulocytes.

Authors

Yanxin Fan, Marta Murgia, Monika I. Linder, Yoko Mizoguchi, Cong Wang, Marcin Łyszkiewicz, Natalia Ziȩtara, Yanshan Liu, Stephanie Frenz, Gabriela Sciuccati, Armando Partida-Gaytan, Zahra Alizadeh, Nima Rezaei, Peter Rehling, Sven Dennerlein, Matthias Mann, Christoph Klein

×

Figure 6

PRKD2 is a mitochondrial kinase involved in HSP27 phosphorylation.

Options: View larger image (or click on image) Download as PowerPoint
PRKD2 is a mitochondrial kinase involved in HSP27 phosphorylation. (A) V...
(A) Volcano plot comparing protein expression (LFQ intensity, SILAC light) of kinases (black) and phosphatases (blue, both keyword annotation) in WT and in HAX1–/– clones (n = 18 each, 2 replicates). Data were filtered for 14 valid values in at least 1 group (106 proteins in total). (B) Isolated mitochondria from indicated genotypes were solubilized with Laemmli buffer and analyzed by immunoblotting. (C) Isolated mitochondria were swollen or sonicated and/or treated with PK and analyzed by immunoblotting (EM: EDTA, MOPS; SEM: sucrose, EDTA, MOPS). (D) Lysates of PLB-985 cells treated with the protein kinase D inhibitor CRT0066101 were analyzed by immunoblotting with the indicated antibodies. (E) Lysates of PLB-985 cells expressing doxycycline-inducible shRNA targeting either control or PRKD2 were analyzed by immunoblotting with the indicated antibodies. (F) Volcano plot illustrating the mitochondrial interactome of HSP27 (n = 6) versus control (non-bait) (n = 6). The analysis is based on 724 proteins that were commonly identified in 2 biological replicates. The bait (HSP27/HSPB1) and the interactors with the highest P values are marked in black. Significant interactors annotated as mitochondrial translation (Gene Ontology) are marked in red and blue and listed. (G) Gene Ontology Biological Process pathway enrichment analysis of the HSP27 interactome (F, right), color-coded by enrichment P value as indicated. (H) Quantification of mitochondrial ROS production in WT, HAX1–/–, and HAX1–/– cells reconstituted with either HAX1 or HSP27FLAG cells using MitoSOX (n = 4, ***P < 0.001, 1-way ANOVA followed by Tukey’s test). (I) MMP in WT, HAX1–/–, HAX1–/– + HAX1, or HAX1–/– + HSP27 PLB-985 cells in the absence or presence of CCCP (2.5 μM) by TMRM (2.5 nM). Data represent 3 independent experiments.