Biology subjects

Meisenhelder, J.

Publications and source records attributed to Meisenhelder, J..

3 recordsLinked to original sources

bioRxiv2024

Using phage display for rational engineering of a higher affinity humanized 3-phosphohistidine-specific antibody

Histidine phosphorylation (pHis) is a non-canonical post-translational modification (PTM) that is historically understudied due to a lack of robust reagents that are required for its investigation, such as high affinity pHis-specific antibodies. Engineering pHis-specific antibodies is very challenging due to the labile nature of the phosphoramidate (P-N) bond and the stringent requirements for selective recognition of the two isoforms, 1-phosphohistidine (1-pHis) and 3-phosphohistidine (3-pHis). Here, we present a strategy for in vitro engineering of antibodies for detection of native 3-pHis targets. Specifically, we humanized the rabbit SC44-8 anti-3-pTza (a stable 3-pHis mimetic) mAb into a scaffold (herein referred to as hSC44) that was suitable for phage display. We then constructed six unique Fab phage-displayed libraries using the hSC44 scaffold and selected high affinity 3-pHis binders. Our selection strategy was carefully designed to enrich antibodies that bound 3-pHis with high affinity and had specificity for 3-pHis versus 3-pTza. hSC44.20N32FL, the best engineered antibody, has an [~]10-fold higher affinity for 3-pHis than the parental hSC44. Eleven new Fab structures, including the first reported antibody-pHis peptide structures were solved by X-ray crystallography. Structural and quantum mechanical calculations provided molecular insights into 3-pHis and 3-pTza discrimination by different hSC44 variants and their affinity increase obtained through in vitro engineering. Furthermore, we demonstrate the utility of these newly developed high-affinity 3-pHis-specific antibodies for recognition of pHis proteins in mammalian cells by immunoblotting and immunofluorescence staining. Overall, our work describes a general method for engineering PTM-specific antibodies and provides a set of novel antibodies for further investigations of the role of 3-pHis in cell biology. Significance StatementHistidine phosphorylation is an elusive PTM whose role in mammalian cell biology is largely unknown due to the lack of robust tools and methods for its analysis. Here we report the development of antibodies with unprecedented affinity and specificity towards 3-pHis and present the first crystal structures of a pHis peptide in complex with an antibody. Finally, we show how these antibodies can be used in standard molecular biology workflows to investigate pHis-dependent biology.

synthetic biology↗

bioRxiv2021

MAP4K3 inhibits Sirtuin-1 to repress LKB1-AMPK to promote amino acid dependent activation of mTORC1

mTORC1 is the key rheostat controlling cellular metabolic state. Of the various inputs to mTORC1, the most potent effector of intracellular nutrient status is amino acid supply. Despite an established role for MAP4K3 in promoting mTORC1 activation in the presence of amino acids, the signaling pathway by which MAP4K3 controls mTORC1 activation remains unknown. Here we examined the process of MAP4K3 activation of mTORC1 and found that MAP4K3 represses the LKB1-AMPK pathway to prevent TSC1/2 complex inactivation of Rheb. When we sought the regulatory link between MAP4K3 and LKB1 inhibition, we discovered that MAP4K3 physically interacts with the master nutrient regulatory factor Sirtuin-1 and phosphorylates Sirtuin-1 to repress LKB1 activation. Our results reveal the existence of a novel signaling pathway linking amino acid satiety with MAP4K3-dependent suppression of SIRT1 to inactivate the repressive LKB1-AMPK pathway and thereby potently activate the mTORC1 complex to dictate the metabolic disposition of the cell.

cell biology↗

bioRxiv2019

A non-acidic method using hydroxyapatite and phosphohistidine monoclonal antibodies allows enrichment of phosphopeptides containing non-conventional phosphorylations for mass spectrometry analysis.

Four types of phosphate-protein linkage generate nine different phosphoresidues in living organisms. Histidine phosphorylation is a long-time established but largely unexplored post-translational modification, mainly because of the acid-lability of the phosphoramidate bonds. This lability means that standard phosphoproteomic methods used for conventional phosphate esters (phospho-Ser/Thr/Tyr) must be modified to analyze proteins containing the phosphoramidate-amino acids - phospho-His/Arg/Lys. We show that a non-acidic method allows enrichment of non-conventional phosphoresidue-containing peptides from tryptic digests of human cell lines, using hydroxyapatite binding and/or immobilized 1-pHis and 3-pHis monoclonal antibodies for enrichment. 425 unique non-conventional phosphorylation sites (i.e. pHis, pLys and pArg) were detected with a high probability of localization by LC-MS/MS analysis and identified using a customized MaxQuant configuration, contributing to a new era of study in post-translational modification and cell signaling in humans. This is the first fully non-acidic method for phosphopeptide enrichment which uses immunoaffinity purification and remains compatible with mass spectrometry analysis for a wider coverage of potential protein phosphorylation events.

biochemistry↗