Biology subjects

Hackenberger, C. P.

Publications and source records attributed to Hackenberger, C. P..

5 recordsLinked to original sources

bioRxiv2026

Cellular protein delivery through membrane potential driven water pores

Providing immediate access for functional proteins inside living cells would unlock unprecedented control over cellular processes; however, commonly used endocytic delivery suffers from endosomal trapping and degradation. One of the most powerful non-endosomal delivery methods uses cell surface anchored cell penetrating peptide (CPP)-additives that allow proteins to enter cells directly. Nevertheless, the underlying molecular mechanism involved in direct entry via crossing the cell membrane (protein translocation through the cell) and the major driving forces remain controversially discussed. Here, we provide a stepwise molecular picture on how CPP-additives enable uptake of protein cargoes through direct membrane translocation. CPP-additives accumulate on the cell surface in nucleation zones, locally hyperpolarizing the membrane, and induce transient water pores that allow selective CPP-protein entry without compromising membrane integrity. These fundamental mechanistic insights provide a firm basis for rationally optimizing delivery strategies using highly cationic CPPs, ultimately resulting in innovative and smart protein delivery strategies to advance therapeutic protein applications. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/707441v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@14c1386org.highwire.dtl.DTLVardef@195f765org.highwire.dtl.DTLVardef@a52258org.highwire.dtl.DTLVardef@171dd7c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

An engineered nanobody inhibitor for molecular-to-circuit control of opioid receptor function

Opioid receptors (ORs) orchestrate pain relief, reward, and dependence, yet their signaling arises from diverse cell types and subcellular compartments that cannot be selectively interrogated with existing pharmacological or genetic approaches. Single-domain antibodies, or nanobodies (Nbs), can probe receptor states, but their potential as tools for controlling native receptor signaling at the system level has remained unexplored. Here, we engineer a suite of high-affinity intracellular Nbs that bind active ORs through structure-guided evolution and in silico design. Iterative optimization yields Nb64, a potent inhibitor that rapidly suppresses transducer engagement, receptor internalization, and downstream signaling, including endogenous pathways in neuronal cells. Organelle targeting highlights Nb64s capacity to control OR activity with subcellular precision, while bio-reversible cell-penetrating peptide (CPP) conjugation enables non-genetic cytosolic delivery. Cell-type-specific expression of Nb64 in VTA interneurons attenuates fentanyl-evoked dopamine release and behavioral responses in mice, demonstrating targeted control of opioid actions in vivo. Nb64 provides a versatile strategy for dissecting OR biology and establishes a generalizable framework for precision inhibition of native GPCR signaling in vivo.

neuroscience↗

bioRxiv2026

Active Site-Directed Probes for targeting Bacterial Phosphoarginine Phosphatases

Canonical protein phosphorylation patterns are a thoroughly studied post-translational modification (PTM) driving distinct regulatory mechanisms in both prokaryotes, and eukaryotes. In contrast, the identification and investigation of essential components that regulate non-canonical phosphorylation has received considerably less attention, although these PTMs are associated with important functions. One notable example is arginine phosphorylation which modulates processes such as protein degradation, transcriptional regulation and spore germination in bacteria. Herein we introduce the first in class covalent activity-based probes to study phosphoarginine-phosphatases. We identify unsaturated phosphonamidic acids as bespoke electrophilic phosphoarginine (pArg) mimics, which allowed to uncover a series of unprecedented pArg-phosphatases, which in part had been previously annotated as low molecular weight tyrosine-phosphatases across phylogenetically distinct microbial species. This work, which serves as the first example of proteome-wide activity-based profiling of pArg phosphatases will help inform the development of new therapeutic modalities and expand our understanding of bacterial signal transduction.

microbiology↗

bioRxiv2025

Quantitative analysis of inhibitor-induced assembly disruption in human UDP-GlcNAc 2-epimerase using mass photometry

UDP-GlcNAc 2-epimerase/N-acetylmannosamine kinase (GNE/MNK) is the rate-limiting enzyme in sialic acid biosynthesis and a promising therapeutic target. We applied interferometric scattering microscopy (iSCAM) to investigate GNE oligomerization and its modulation by three small-molecule inhibitors (C5, C13, C15). Substrate binding (UDP-GlcNAc) stabilized tetramer formation by increasing dimer-dimer affinity 120-fold. All inhibitors destabilized tetramers in a concentration-dependent manner, with IC50 values in the low micromolar range. Using a modified Cheng-Prusoff equation, IC50 values were converted into Ki values. Schild analysis was applied to estimate an apparent KB,app value and assess cooperative inhibition effects. Molecular docking confirmed competitive binding for all inhibitors and helped rationalize observed potency trends. While iSCAM has previously been used to study protein assembly, our work demonstrates its applicability for the label-free, quantitative characterization of small-molecule inhibitors affecting protein oligomerization. These findings provide a foundation for further mechanistic studies and underscore the potential of iSCAM in drug-target interaction profiling.

biophysics↗

bioRxiv2024

A Cell-Permeable Nanobody to Restore F508del Cystic Fibrosis Transmembrane Conductance Regulator Activity

Nanobodies have gained considerable attention as particularly promising biopharmaceuticals. However, nanobody-based modalities are currently limited to extracellular targets due to a lack of efficient delivery methods required to reach targets inside cells. In this study, we introduce cell-permeable nanobodies for targeting a disease-relevant intracellular protein, namely the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel with the most common cystic fibrosis (CF)-causing mutation F508del. We employ cell-penetrating peptides (CPPs) to deliver a CFTR-binding nanobody (NB1) that stabilizes misfolded F508del-CFTR and prevents its degradation to restore its function. Our data show that conjugation of a disulfide-linked CPP in combination with a cell-surface anchored CPP-additive enables intracellular delivery of NB1 into CF bronchial epithelial cells, which promotes maturation and trafficking of F508del-CFTR protein to the apical cell membrane. Furthermore, we demonstrate that the cell-permeable nanobody restores CFTR chloride channel function, which can be further enhanced by the clinically approved small molecule CFTR potentiator ivacaftor. This study highlights the use of cell-permeable nanobodies for modulation of protein function and illustrates their therapeutic potential as next-generation biopharmaceuticals for intracellular delivery and targeting. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/591242v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@45d357org.highwire.dtl.DTLVardef@362e56org.highwire.dtl.DTLVardef@11c4dc2org.highwire.dtl.DTLVardef@13512b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗