Dissecting FOXP2 Oligomerization and DNA Binding

Häußermann, Katharina; Young, Gavin; Kukura, Philipp; Dietz, Hendrik

doi:10.1002/ange.201901734

Cited by 11 publications

(11 citation statements)

References 43 publications

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“…While a number of inferences have been made as to the purpose of the various FOXP domains through studies conducted both in vivo [5,6] and in vitro [25], the significance of the large proportion of undefined interlinking sequence in these proteins has not been considered. Bearing in mind that the undefined sequence constitutes well over 50% of the protein sequence, it is highly probable that these regions of the sequence cooperate with the defined domains so as to enable the protein to perform its function.…”

Section: Resultsmentioning

confidence: 99%

“…How exactly does such a large superfamily of ubiquitously expressed proteins have such specific non-overlapping functions, and do all parts of the sequence have a unique role to play in ensuring that the proteins perform their specific functions? While there is some evidence already available to suggest putative roles for certain parts of the sequence, (for example the polyglutamine tract and zinc finger motif are believed to be involved in protein-protein associations, the leucine zipper has been shown to cause homo and heterotypic multimerisation and the forkhead domain is responsible for DNA binding specificity [5,19,25]) it is not known how these domains, along with the remaining undefined regions of the sequence including the acid rich tail, function together to support a cohesive mechanism of transcriptional regulation. The objective of this study, therefore, is to provide more clarity on how the two oligomerisation interfaces, the leucine zipper and the forkhead domain, as well as the interlinking disordered sequence including the acid rich tail, co-operate so as to contribute to the structure and DNA binding of the FOXP proteins.…”

Section: Introductionmentioning

confidence: 99%

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The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function

et al. 2021

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FOX proteins are a superfamily of transcription factors which share a DNA-binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In the present study, we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper, and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding, and therefore the acid rich tail may play an important regulatory role in FOXP transcription.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function

et al. 2021

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“…Cooled non-reductive SDS-PAGE followed by peptide profiling supported the presence of a maturely processed low-abundance nMPO monoprotomer, Supplementary Figure S4. Single-molecule mass photometry, a method capable of quantifying the assembly, binding affinities and kinetics of protein complexes (41)(42)(43) confirmed that nMPO exists as a lowabundance monoprotomer (αβ, 7%) and highabundance diprotomer (ααββ, 93%) with an apparent mono-/diprotomer (αβ/ααββ) K d of ~50 pM, Figure 1G. The biological role(s) of the maturely processed monoprotomeric MPO, which differs from the secreted monoprotomeric proMPO reportedly elevated in blood in cardiovascular disease (44,45), remains unknown.…”

Section: Comprehensive Characterisation Of Neutrophilderived Myelopermentioning

confidence: 99%

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase

Tjondro

Ugonotti

Kawahara

et al. 2021

Journal of Biological Chemistry

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Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure–biosynthesis–activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.

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“…Cooled non-reductive SDS-PAGE followed by peptide profiling supported the presence of a maturely processed low-abundance nMPO monomer, Supplementary Figure S4. Single-molecule mass photometry, a method capable of quantifying the assembly, binding affinities and kinetics of protein complexes (Haussermann et al, 2019;Soltermann et al, 2020;Young et al, 2018) confirmed that nMPO exists as a lowabundance monoprotomer (αβ, 7%) and high-abundance diprotomer (ααββ, 93%) with an apparent mono-/diprotomer (αβ/ααββ) Kd of ~50 pM, Figure 1G. The biological role(s) of the maturely processed monoprotomeric MPO, which differs from the secreted monoprotomeric proMPO reportedly elevated in blood in cardiovascular disease (Gorudko et al, 2018;Khalilova et al, 2018), remains unknown.…”

Section: Comprehensive Characterisation Of Neutrophil-derived Myelopementioning

confidence: 99%

Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase

Tjondro

Ugonotti

Kawahara

et al. 2020

Preprint

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Myeloperoxidase (MPO), an important glycoprotein in neutrophil-mediated immunity, produces microbicidal hypohalous acids, but the underpinning glycobiology remains elusive. Deep characterisation of neutrophil-derived MPO (nMPO) using advanced mass spectrometry demonstrated that under-processed oligomannosidic-, truncated paucimannosidic- and hyper-truncated N-acetyl-β-D-glucosamine (GlcNAc) core-type asparagine-linked glycans decorate the protein. Occlusion of Asn355 and Asn391 and sterical hindrance of Asn323- and Asn483-glycans located in the MPO dimerisation zone were found to shape the local glycan processing thereby providing a molecular basis for the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry, and glycopeptide profiling revealed extreme molecular complexity of dimeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants, and a lower-abundance monomer. Longitudinal profiling of maturing, mature, granule-separated, and pathogen-activated neutrophils demonstrated that MPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and rapidly degranulated, but surprisingly carries uniform glycosylation across conditions. Complete proMPO-to-MPO maturation evidently occur during early/mid-stage granulopoiesis. The conserved Asn355- and Asn391-sequons displayed elevated GlcNAc signatures and higher oxidation and chlorination activity of the secretory vesicle/plasma membrane-resident MPO relative to MPO from other granules. Endoglycosidase H-treated nMPO displaying Asn355-/Asn391-GlcNAcylation recapitulated the activity gain and showed increased thermal stability and enhanced polypeptide flexibility relative to untreated nMPO as measured by activity assays, circular dichroism and molecular dynamics. Endoglycosidase H-treated nMPO also demonstrated an elevated ceruloplasmin-mediated inhibition relative to nMPO. Modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO:ceruloplasmin interface are critical for contact-free inhibition. We report on novel roles of the peculiar MPO glycosylation providing new insight into neutrophil glycobiology.

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Dissecting FOXP2 Oligomerization and DNA Binding

Cited by 11 publications

References 43 publications

The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function

The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function

Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase

Hyper-Truncated Glycans Augment the Activity of Neutrophil Granule Myeloperoxidase

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