Biology subjects

Hein, J. B.

Publications and source records attributed to Hein, J. B..

4 recordsLinked to original sources

bioRxiv2023

Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation.1 Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases,2 while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55.3 While the role of kinases in mitotic entry is well-established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited.4 For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent)6,7 and FAM122A5 (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.

biochemistry↗

bioRxiv2023

Global substrate identification and high throughput in vitro dephosphorylation reactions uncover PP1 and PP2A-B55 specificity principles

Phosphoprotein phosphatases (PPPs) dephosphorylate Serine (Ser)/Threonine (Thr) residues to regulate major signaling pathways and cellular transitions. Despite the central role of PPPs the substrates in most cellular processes and the determinants of phosphatase specificity are poorly understood. This is because methods to investigate this at scale are lacking. Here we develop a novel in vitro assay, MRBLE:Dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase preferences. Using MRBLE:Dephos, we establish amino acid preferences of the residues surrounding the dephosphorylation site for PP1 and PP2A- B55, which reveals common and unique preferences for the two phosphatases. To compare the MRBLE:Dephos results to cellular substrates, we focused on mitotic exit that requires extensive dephosphorylation by PP1 and PP2A-B55. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with quantitative phosphoproteomics to identify more than 2000 regulated phosphorylation sites. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with the MRBLE:Dephos results. We use these insights to specifically alter INCENP dephosphorylation kinetics at mitotic exit, resulting in defective cytokinesis thus underscoring the biological relevance of our determined specificity principles. Finally, we provide a comprehensive characterization of PP1 binding motifs and demostrate how binding of phosphatases to substrates shapes dephosphorylation specificity. Collectively, we develop novel approaches to advance our ability to investigate protein phosphatases and use these to provide a framework for understanding mitotic exit regulation by dephosphorylation.

cell biology↗

bioRxiv2020

MRBLE-pep measurements reveal accurate binding affinities for B56, a PP2A regulatory subunit

Signal transduction pathways rely on dynamic interactions between protein globular domains and short linear motifs (SLiMs). The weak affinities of these interactions are essential to allow fast rewiring of signaling pathways and downstream responses, but pose technical challenges for interaction detection and measurement. We recently developed a technique (MRBLE-pep) that leverages spectrally encoded hydrogel beads to measure binding affinities between a single protein and 48 different peptide sequences in a single small volume. In prior work, we applied it to map the binding specificity landscape between calcineurin and the PxIxIT SLiM (Nguyen et al. 2019). Here, using peptide sequences known to bind the PP2A regulatory subunit B56, we systematically compare affinities measured by MRBLE-pep or isothermal calorimetry (ITC) and confirm that MRBLE-pep accurately quantifies relative affinity over a wide dynamic range while using a fraction of the material required for traditional methods such as ITC.

bioengineering↗

bioRxiv2020

MRBLEs 2.0: High-throughput generation of chemically functionalized spectrally and magnetically-encoded hydrogel beads using a simple single-layer microfluidic device

Widespread adoption of bead-based multiplexed bioassays requires the ability to easily synthesize encoded microspheres and conjugate analytes of interest to their surface. Here, we present a simple method (MRBLEs 2.0) for efficient high-throughput generation of microspheres with ratiometric barcode lanthanide encoding (MRBLEs) bearing functional groups for downstream bioconjugation. Bead production in MRBLEs 2.0 relies on manual mixing of lanthanide/polymer mixtures (each of which comprises a unique spectral code) followed by droplet generation using single-layer, parallel flow-focusing devices and off-chip batch polymerization of droplets into beads. To streamline downstream analyte coupling, MRBLEs 2.0 crosslinks copolymers bearing functional groups on the bead surface simultaneously during bead generation. Using the MRBLEs 2.0 pipeline, we generate monodisperse MRBLEs containing 48 distinct well-resolved spectral codes in high-throughput (>150,000/min and can be boosted to 450,000/min). We further demonstrate the efficient conjugation of oligonucleotides and entire proteins to carboxyl MRBLEs and biotin to amino MRBLEs. Finally, we show that MRBLEs can also be magnetized via simultaneous incorporation of magnetic nanoparticles with only a minor decrease in the potential code space. We anticipate that MRBLEs 2.0 can be directly applied towards a wide variety of downstream assays from basic biology to diagnostics and other translational research.Competing Interest StatementStanford University and Chan Zuckerberg Biohub have filed a provisional patent application (U.S. Provisional Patent application No. 63/037,804) on the MRBLEs 2.0 methods described here and Y.F., A.K.W., J.B.H., and P.M.F. are named as inventors.View Full Text

bioengineering↗