Biology subjects

Herberg, F. W.

Publications and source records attributed to Herberg, F. W..

12 recordsLinked to original sources

bioRxiv2025

Kinetic and thermodynamic analysis of PKA-R-cAMP interactions in crude media using focal molography

Quantifying biomolecular interactions under near-physiological conditions is essential for understanding biological processes. However, this remains a challenge in established techniques: for surface plasmon resonance, bulk refractive-index shifts and non-specific adsorption in hydrogel matrices can dominate the signal in serum, whereas for isothermal titration calorimetry, complex media primarily introduce large, composition-dependent background heats and run-to-run variability that obscure the binding enthalpy. Here, we demonstrate that focal molography, a label-free optical biosensing method, can reliably measure the kinetics and thermodynamics of the protein kinase A regulatory subunit binding to cyclic AMP derivatives in both buffer and 50% human serum. By performing the kinetic measurement over a range of temperatures, we were able to reveal that binding remains highly favorable in both environments, yet the results suggest that the balance of driving forces shifts toward enthalpic contributions in serum. This hints at the substantial difference of interaction mechanisms in a complex biological media compared to buffer systems. Our findings show that focal molography reliably quantifies protein-ligand interactions in complex media, providing consistent kinetic and thermodynamic data while overcoming limitations of existing methods. This highlights the potential of focal molography as a valuable tool for studying interactions under near-native conditions.

biophysics↗

bioRxiv2024

A Structural Mechanism for Noncanonical GPCRSignal Transduction in the Hedgehog Pathway

SUMMARY PARAGRAPHThe Hedgehog (Hh) signaling pathway is fundamental to embryogenesis, tissue homeostasis, and cancer. Hh signals are transduced via an unusual mechanism: upon agonist-induced phosphorylation, the noncanonical G protein-coupled receptor SMOOTHENED (SMO) binds the catalytic subunit of protein kinase A (PKA-C) and physically blocks its enzymatic activity. By combining computational structural approaches with biochemical and functional studies, we show that SMO mimics strategies prevalent in canonical GPCR and PKA signaling complexes, despite little sequence or secondary structural homology. An intrinsically disordered region of SMO binds the PKA-C active site, resembling the PKA regulatory subunit (PKA-R) / PKA-C holoenzyme, while the SMO transmembrane domain binds a conserved PKA-C interaction hub, similar to other GPCR-effector complexes. In contrast with prevailing GPCR signal transduction models, phosphorylation of SMO promotes intramolecular electrostatic interactions that stabilize key structural elements within the SMO cytoplasmic domain, thereby remodeling it into a PKA-inhibiting conformation. Our work provides a structural mechanism for a central step in the Hh cascade and defines a paradigm for disordered GPCR domains to transmit signals intracellularly.

biochemistry↗

bioRxiv2024

Pigment-dispersing factor neuropeptides act as multifunctional hormones and modulators in tardigrades

Pigment-dispersing factors (PDFs) are neuropeptides that play key roles in controlling the circadian rhythms in various insects, whereas their function remains elusive in other protostomes including tardigrades (water bears). Here we show that the three PDFs of the tardigrade Hypsibius exemplaris are co-localized in two pairs of inner lobe cells in the brain, whereas only one PDF occurs in four additional cerebral and two extracerebral cells. The axons of the inner lobe cells pass through the contralateral brain hemisphere, descend to the ventral nerve cord and terminate in two pairs of potential release sites in the posteriormost trunk ganglion. Using in vitro assays, we demonstrate that all three PDFs and their deorphanized receptor (PDFR) are functional. Widespread localization of PDFR suggests that tardigrade PDFs may act as multifunctional hormones and neuromodulators that control major functions including light detection, neural processing, locomotion, feeding, digestion, osmoregulation, growth, embryonic development, and oogenesis/reproduction.

zoology↗

bioRxiv2024

Allosteric modulation of protein kinase A in individuals affected by NLPD-PKA , a neurodegenerative disease in which the RIβ-L50R variant is expressed

Protein kinase A (PKA) is a crucial signaling enzyme in neurons, with its dysregulation being implicated in neurodegenerative diseases. Assembly of the PKA holoenzyme, comprising a dimer of heterodimers of regulatory (R) and catalytic (C) subunits, ensures allosteric regulation and functional specificity. Recently, we defined the RI{beta}-L50R variant as a causative mutation that triggers protein aggregation in a rare neurodegenerative disease. However, the mechanism underlying uncontrolled PKA allosteric regulation and its connection to the functional outcomes leading to clinical symptoms remain elusive. In this study, we established an in vitro model using patient-derived cells for a personalized approach and employed direct measurements of purified proteins to investigate disease mechanisms in a controlled environment. Structural analysis and circular dichroism spectroscopy revealed that cellular proteins aggregation resulted from misfolded RI{beta}-subunits, preventing holoenzyme assembly and anchoring through A Kinase Anchoring Proteins (AKAPs). While maintaining high affinity to the C subunit, the resulting RI{beta}-L50R:C heterodimer exhibits reduced cooperativity, requiring lower cAMP concentrations for dissociation. Consequently, there was an increased translocation of C-subunit into the nucleus, impacting gene expression. We successfully controlled C subunit translocation by introducing a mutation that decreased RI{beta}:C dissociation in response to elevated cAMP levels. This research thus sets the stage for developing therapeutic strategies that modulate PKA assembly and allostery, thus exerting control over the unique molecular signatures identified in the disease-associated transcriptome profile.

cell biology↗

bioRxiv2023

Tripartite interactions of PKA catalytic subunit and C-terminal domains of cardiac Ca2+ channel modulate its β-adrenergic regulation

The adrenergic nervous system augments cardiac contraction by increasing the activity of L-type voltage-gated CaV1.2 channels. Dysregulation of this process is linked to severe cardiac dysfunctions. The signaling cascade involves activation of {beta}-adrenergic receptors, elevation of cAMP levels, separation of protein kinase A (PKA) regulatory subunit (PKAR) from catalytic subunit (PKAC), and phosphorylation of the inhibitory protein Rad leading to increased Ca2+ influx. In cardiomyocytes, the core subunit of CaV1.2 (1C) exists in two forms: full-length (FL) or proteolytically processed (truncated), lacking the distal C-terminus (dCT). Specificity and efficiency in the cascade are believed to emanate from unique protein-protein interactions, such as anchoring PKA (via PKAR) to 1C by A-kinase anchoring proteins (AKAPs). However, most AKAPs do not interact with the truncated 1C, and their role in {beta}AR regulation of cardiac CaV1.2 remains unclear. Here we show that PKAC, independently of PKAR or AKAPs, directly interacts with 1C at two domains in 1C-CT: the proximal and distal C-terminal regulatory domains (PCRD and DCRD), which also interact with each other. Furthermore, we find that DCRD competes with PCRD and reduces its interaction with PKAC. The physiological consequences of these complex interactions are incompletely understood; our data suggest that they may fine-tune the {beta}AR regulation of CaV1.2. We propose that the newly discovered interactions take part in governing colocalization of regulatory proteins within the {beta}AR-CaV1.2 multimolecular signaling complexes in cardiomyocytes.

biochemistry↗

bioRxiv2022

Retinal energy metabolism: Photoreceptors switch between Cori, Cahill, and mini-Krebs cycles to uncouple glycolysis from mitochondrial respiration

The retina consumes massive amounts of energy, yet its metabolism and substrate exploitation remain poorly understood. Here, we used a murine explant model to manipulate retinal energy metabolism under entirely controlled conditions and utilized 1H-NMR spectroscopy-based metabolomics, in situenzyme detection, and cell viability readouts to uncover the pathways of retinal energy production. Our experimental manipulations resulted in varying degrees of photoreceptor degeneration, while the inner retina and retinal pigment epithelium were essentially unaffected. This selective vulnerability of photoreceptors suggested very specific adaptations in their energy metabolism. Rod photoreceptors were found to rely strongly on oxidative phosphorylation, but only mildly on glycolysis. Conversely, cone photoreceptors were dependent on glycolysis but insensitive to electron transport chain decoupling. Importantly, photoreceptors appeared to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: 1) the mini-Krebs-cycle, fueled by glutamine and branched-chain amino acids, generating N-acetylaspartate; 2) the alanine-generating Cahill-cycle; 3) the lactate-releasing Cori-cycle. Moreover, the metabolomic data indicated a shuttling of taurine and hypotaurine between the retinal pigment epithelium and photoreceptors, likely resulting in an additional net transfer of reducing power to photoreceptors. These findings expand our understanding of retinal physiology and pathology and shed new light on neuronal energy homeostasis and the pathogenesis of neurodegenerative diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/496788v3_figa1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1ef3692org.highwire.dtl.DTLVardef@1e7e193org.highwire.dtl.DTLVardef@8aa6fborg.highwire.dtl.DTLVardef@d8c5bd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Retinal photoreceptors employ both glucose and glutamate as fuels. While rod photoreceptors rely strongly on oxidative phosphorylation and the N-acetylaspartate producing mini-Krebs-cycle, cone photoreceptors rely on the lactate-producing Cori cycle and the oxidative, alanine producing Cahill cycle. C_FIG HighlightsO_LIThe retina utilizes a complex energy switchboard consisting of the Krebs cycle, mini-Krebs cycle, Cahill cycle, and Cori cycle. C_LIO_LIMini-Krebs cycle runs more efficiently than full Krebs cycle. C_LIO_LIAlanine transaminase decouples glycolysis from the Krebs cycle. C_LIO_LILactate, alanine, and N-acetylaspartate are distinctive energetic pathway signatures. C_LI

cell biology↗

bioRxiv2022

An auto-inhibited state of protein kinase G and implications for selective activation

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Campaigns targeting nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis suggest that understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG I and I{beta} that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is still not well understood. Here we present a crystal structure of PKG I{beta} in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG I{beta} auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG I{beta} auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and activation of PKGs.

biochemistry↗

bioRxiv2021

Two helices control the dynamic crosstalk between the catalytic domains of LRRK2

The two major molecular switches in biology, kinases and GTPases, are both contained in the Parkinsons Disease-related Leucine-rich repeat kinase 2 (LRRK2). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Molecular Dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2RCKW). We identified two helices that shield the kinase domain and regulate LRRK2 conformation and function. One docking helix in COR-B (Dk-Helix) tethers the COR-B domain to the C helix of the kinase domain and faces its Activation Loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the Dk-Helix create a "cap" that regulates the N-Lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

biochemistry↗

bioRxiv2021

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2

Mutations in the gene coding for Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinsons disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multi-domain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for inhibitory drug design. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of Nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 activity in cells and in vitro. Importantly, diverse groups of Nanobodies were identified that inhibit LRRK2 kinase activity through a mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain Nanobodies completely inhibit the LRRK2 kinase activity, we also identified Nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type-I kinase inhibitors, the studied kinase-inhibitory Nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized Nanobodies represent versatile tools to study the LRRK2 function and mechanism, and can pave the way toward novel diagnostic and therapeutic strategies for PD.

biochemistry↗

bioRxiv2021

Retinal degeneration: Multilevel protection of photoreceptor and ganglion cell viability and function with the novel PKG inhibitor CN238.

Hereditary retinal degeneration (RD) is often associated with excessive cGMP-signaling in photoreceptors. Previous research has shown that inhibition of cGMP-dependent protein kinase G (PKG) can slow down the loss of photoreceptors in different RD animal models. In this study, we identified a novel PKG inhibitor, the cGMP analogue CN238, with strong protective effects on photoreceptors in retinal degeneration rd1 and rd10 mutant mice. In long-term organotypic retinal explants, CN238 preserved rd1 and rd10 photoreceptor viability and function. Surprisingly, in explanted retinae CN238 also protected retinal ganglion cells from axotomy induced retrograde degeneration and preserved their functionality. Together, these results confirm the strong neuroprotective capacity of PKG inhibitors for both photoreceptors and retinal ganglion cells, thereby significantly broadening their potential applications for the treatment of retinal diseases and possibly neurodegenerative diseases in general.

neuroscience↗

bioRxiv2021

A PKA Inhibitor Motif within Smoothened Controls Hedgehog Signal Transduction

The Hedgehog (Hh) cascade is central to development, tissue homeostasis, and cancer. A pivotal step in Hh signal transduction is the activation of GLI transcription factors by the atypical G protein-coupled receptor (GPCR) Smoothened (SMO). How SMO activates GLI has remained unclear for decades. Here we show that SMO employs a decoy substrate sequence to physically block the active site of the PKA catalytic subunit (PKA-C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation-induced inhibition. Using a combination of in vitro, cellular, and organismal models, we demonstrate that interfering with SMO / PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism we uncovered echoes one utilized by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly. More broadly, our findings define a new mode of GPCR-PKA communication that may be harnessed by a range of membrane receptors and kinases.

biochemistry↗

bioRxiv2020

Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

In a multi-tiered approach, we explored how Parkinsons Disease-related mutations hijack the finely tuned activation process of Leucine-Rich Repeat Kinase 2 (LRRK2) using a construct containing the ROC, Cor, Kinase and WD40 domains (LRRK2RCKW). We hypothesized that the N-terminal domains shield the catalytic domains in an inactive state. PD mutations, type-I LRRK2 inhibitors, or physiological Rab GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase activity, is essential for docking onto microtubules. Mapping solvent accessible regions of LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how inhibitor binding is sensed by the entire protein. Molecular Dynamics simulations of the kinase domain elucidated differences in conformational dynamics between wt and mutants of the DYG{psi} motif. While all domains contribute to regulating kinase activity and spatial distribution, the kinase domain, driven by the DYG{psi} motif, coordinates domain crosstalk and serves as an intrinsic hub for LRRK2 regulation.

biochemistry↗