Recent Research Highlights

Drees C, Rühl P, Czerny J, Chandra G, Bajorath J, Haase M, Heinemann SH, Piehler J

Diffraction-unlimited photomanipulation at the plasma membrane via specifically targeted upconversion nanoparticles

Nano Letters (2021) 21:8025-8034  (PubMedExternal link)

Frequency upconverting nanoparticles (UCNPs) utilize long-lived excited states of lanthanides to populate high-energy states of dopant ions (e.g. Tm³⁺ or Er³⁺) by subsequent absorption of near infrared (NIR) photons. UCNPs therefore have the potential to serve as local light sources when targeted to specific sites in biological environments. Here we demonstrate that UCNPs are suited to activate the photoconvertible fluorescence protein Dendra2 with a resolution below the optical diffraction limit. UCNPs were coated with non-fluorescent GFP to target them specifically to plasma membrane proteins tagged with antiGFP nanobodies. We showed that Dendra2 is also activated across the membrane when extracellularly bound UCNPs are excited with an NIR laser. These results identify UCNPs as exciting tools for bioanalytics, such as quantitative investigation of protein interaction kinetics and spatiotemporal organization at the micro- and the nanoscale, as well as the spatially confined, minimally invasive release of caged compounds.

Gessner G, Rühl P, Westerhausen M, Hoshi T, Heinemann SH

Fe²⁺-mediated activation of BK_Ca channels by rapid photolysis of CORM-S1 releasing CO and Fe²⁺

ACS - Chemical Biology (2020)15(8): 2098-2106 (PubMedExternal link)

Catabolism of heme by heme oxygenase results in the liberation of carbon monoxide (CO) and iron ions (Fe²⁺). Studying the impact of both mediators is not straightforward because of rapid diffusion and limited stability. Here we demonstrate that CORM-S1 (dicarbonyl-bis(cysteamine)iron(II)) is a useful chemical that precisely delivers CO and Fe²⁺ when illuminated with blue light. Fe²⁺, rapidly released from CORM-S1 in a flash-photolysis setting, activates BK_Ca channels with an efficacy much higher than CO. BK_Ca channels are therefore downstream targets of heme oxygenase and may connect heme signaling with electrical cell signaling. Physiologically relevant micromolar Fe²⁺ concentrations activate BK_Ca channels as much as unphysiologically high (> 2 mM) Mg²⁺ concentrations.

Kofuji S, Hirayama A, Eberhardt AO, Kawaguchi R, Sugiura Y, Sampetrean O, Ikeda Y, Warren M, Sakamoto N, Kitahara S, Yoshino H, Yamashita D, Sumita K, Wolfe K, Lange L, Ikeda S, Shimada H, Minami N, Malhotra A, Morioka S, Ban Y, Asano M, Flanary V,L, Ramkissoon A, Chow LML, Kiyokawa J, Mashimo T, Lucey G, Mareninov S, Ozawa T, Onishi N, Okumura K, Terakawa J, Daikoku T, Wise-Draper T, Majd N, Kofuji K, Sasaki M, Mori M, Kanemura Y, Smith EP, Anastasiou D, Wakimoto H, Holland EC, Yong WH, Horbinski C, Nakano I, DeBerardinis RJ, Bachoo RM, Mischel PS, Yasui W, Suematsu M, Saya H, Soga T, Grummt I, Bierhoff H, Sasaki AT

IMP dehydrogenase-2 drives aberrant nucleolar activity and promotes tumorigenesis in glioblastoma

Nature Cell Biology (2019) 21 1003-1014 (PubMedExternal link)

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer glioblastoma. This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in glioblastoma reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of glioblastoma cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for the occurrence of aberrant nucleolar function and increased anabolic processes in glioblastoma, which constitutes a primary event in gliomagenesis.

Erdogmus S, Storch U, Danner L, Becker J, Winter M, Ziegler M, Wirth A, Offermanns S, Hoffmann C, Gudermann T, Mederos y Schnitzler M

Helix 8 is the essential structural motif of mechanosensitive GPCRs

Nature Communications (2019) 10 5784 (PubMedExternal link)

G-protein coupled receptors (GPCRs) are versatile cellular sensors for chemical stimuli, but also serve as mechanosensors involved in various (patho)physiological settings like vascular regulation, cardiac hypertrophy and preeclampsia. However, the molecular mechanisms underlying mechanically induced GPCR activation have remained elusive. Here we show that mechanosensitive histamine H1 receptors (H1Rs) are endothelial sensors of fluid shear stress and contribute to flow-induced vasodilation. At the molecular level, we observe that H1Rs undergo stimulus-specific patterns of conformational changes suggesting that mechanical forces and agonists induce distinct active receptor conformations. GPCRs lacking C-terminal helix 8 (H8) are not mechanosensitive, and transfer of H8 to non-responsive GPCRs confers, while removal of H8 precludes, mechanosensitivity. Moreover, disrupting H8 structural integrity by amino acid exchanges impairs mechanosensitivity. Altogether, H8 is the essential structural motif endowing GPCRs with mechanosensitivity. These findings provide a mechanistic basis for a better understanding of the roles of mechanosensitive GPCRs in (patho)-physiology.

Yang K, Coburger I, Langner JM, Peter N, Hoshi T, Schönherr R, Heinemann SH

Modulation of K⁺ channel N-type inactivation by sulfhydration through hydrogen sulfide and polysulfides

Pflügers Archiv (2019) 471 557-571 (PubMedExternal link)

Hydrogen sulfide (H₂S), which is enzymatically produced in various tissues, is increasingly recognized as an important signaling molecule in the cardiovascular and nervous systems. Here we show that H₂S and its oxidized sulfur species polysulfides potently remove A-type potassium channel inactivation. Such channels determine action potential frequency in excitable cells. Polysulfides at nanomolar concentrations impair A-type channel inactivation by sulfhydrating cysteine residues in their N-terminal ball-and-chain domains. H₂S and polysulfides therefore could be potent modulators of cellular electrical excitability by altering the availability of A-type channels and their kinetics of inactivation.

Gessner G, Sahoo N, Swain SM, Hirth G, Schönherr R, Mede R, Westerhausen M, Brewitz HH, Heimer P, Imhof D, Hoshi T, Heinemann SH

CO-independent modification of K⁺ channels by tricarbonyldichlororuthenium(II) dimer (CORM-2)

European Journal of Pharmacology (2017) 815 33-41 (PubMedExternal link)

Carbon monoxide (CO), despite its toxicity, may serve as physiological messenger. To study the underlying molecular mechanism and for potential medicinal application, so-called CORMs (CO-releasing molecules), like tricarbonyldichlororuthenium(II) dimer (CORM-2), have been developed and frequently used. Using the patch-clamp technique, we show that for several K⁺ channels most of the functional impacts of CORM-2 are not mediated by CO but result from intermediate breakdown products: CORM-2 has the propensity of forming Ru(CO)₂ adducts to histidine residues, as demonstrated using mass spectrometry. This off-site effect is completely abolished by excess free histidine or human serum albumin. Such adduct-formations may have been misinterpreted as CO gas effects in many previous and still ongoing studies, necessitating a re-assessment of derived interpretations thereof.

Ojha NK, Leipold E, Schönherr R, Hoshi T, Heinemann SH

Non-photonic sensing of membrane-delimited reactive species with a Na⁺ channel protein containing selenocysteine

Scientific Reports (2017) 7 46003 (PubMedExternal link)

The detection of cellular reactive species (RS), either endogenously produced or generated by illuminating cells during life-cell fluorescence imaging, is a demanding task because of the labile nature of RS. To measure the influence of RS and the impact of blue light close to the plasma membrane we introduce here a voltage-gated Na⁺ channel harboring a selenocysteine in its inactivation motif: roNa_V2. Current measurements of roNa_V2 yield ratiometric signals that report on local occurrences of RS without the need for confounding cell illumination. In addition, roNa_V2 is reversible and its sensitivity towards light can be switched on and off by means of the electrical membrane voltage.

Swain SM, Sahoo N, Dennhardt S, Schönherr R, Heinemann SH

Ca²⁺/calmodulin regulates Kvβ1.1-mediated inactivation of voltage-gated K⁺ channels

Scientific Reports (2015) 5 15509 (PubMedExternal link)

Some auxiliary β subunits of voltage-gated K⁺ channels equip them with a rapid N-type inactivation mechanism, thus limiting K⁺ outward flow upon stimulation. Here we show that an elevation of the intracellular Ca²⁺ concentration removes Kvβ1.1-induced inactivation. This process is mediated by the Ca²⁺-binding protein calmodulin (CaM) binding to the “chain” element of the Kvβ1.1 N terminus, which normally operates as a “ball-and-chain” inactivation structure. By means of Kvβ1.1 subunits, voltage-gated K⁺ channels in neurons therefore are capable of functioning as negative feedback regulators to limit neuronal excitation and Ca²⁺ overload.

Goral RO, Leipold E, Nematian-Ardestani E, Heinemann SH

Heterologous expression of Na_V1.9 chimeras in various cell systems

Pflügers Archiv – European Journal of Physiology (2015) 467 2423-2435 (PubMedExternal link)

Voltage-gated sodium channel Na_V1.9 is expressed in nociceptive neurons and thus plays an important role in the processing of neuronal signals leading to the sensation of pain. However, systematic investigations of Na_V1.9 in isolation have been hampered because of unsuccessful expression in non-neuronal host cells. Here we show that a chimera of Na_V1.9, harboring the C-terminal domain of skeletal muscle Na_V1.4, yields functional channels when expressed in Xenopus oocytes and HEK 293T cells. This chimera largely conserves the functional properties of Na_V1.9 as far as channel activation and pore pharmacology are concerned, thus providing a model system for research on Na_V1.9 channels.

Hennig D, Schubert S, Dargatz H, Kostenis E, Fahr A, Schubert US, Heinzel T, Imhof D

Novel insights into appropriate encapsulation methods for bioactive compounds into polymers: a study with peptides and HDAC inhibitors

Macromolecular Bioscience (2014) 14 69-80 (PubMedExternal link)

The use of different nanoparticles (NPs) for successful encapsulation of bioactive substances is discussed. The inclusion efficiency into liposomes, acetalated dextran (Ac-Dex), and variants of poly[(lactic acid)-co-(glycolic acid)] (PLGA) NPs is analyzed after chemical degradation. Efficient inclusion of SIRT1 inhibitor Ex527 in liposomes, Ac-Dex- and PLGA-NPs is observed for all procedures used. Activity of Ex527 is demonstrated by monitoring the acetylation status of SIRT1-target p53. In contrast, small peptides are only incorporated into acid-terminated PLGA-NPs and marginally into Ac-Dex-NPs. The yield depends on peptide sequence and terminal modifications. Activity is exemplified for angiotensin II using the dynamic mass redistribution technology.

Frister A, Schmidt C, Schneble N, Brodhun M, Gonnert FA, Bauer M, Hirsch E, Müller JP, Wetzker R, Bauer R

Phosphoinositide 3-kinase gamma affects LPS-induced disturbance of blood-brain barrier via lipid kinase-independent control of cAMP in microglial cells

Neuromolecular Medicine (2014) 16 704-713 (PubMedExternal link)

The breakdown of the blood-brain barrier (BBB) is a key event in the development of sepsis-induced brain damage. BBB opening allows blood-born immune cells to enter the CNS to provoke a neuroinflammatory response. Abnormal expression and activation of matrix metalloproteinases (MMP) was shown to contribute to BBB opening. Using different mouse genotypes in a model of LPS-induced systemic inflammation, our present report reveals phosphoinositide 3-kinase γ (PI3Kγ) as a mediator of BBB deterioration and concomitant generation of MMP by microglia. Unexpectedly, microglia expressing lipid kinase-deficient mutant PI3Kγ exhibited similar MMP regulation as wild-type cells. Our data suggest kinase-independent control of cAMP phosphodiesterase activity by PI3Kγ as a crucial mediator of microglial cell activation, MMP expression and subsequent BBB deterioration. The results identify the suppressive effect of PI3Kγ on cAMP as a critical mediator of immune cell functions.

Kosan C, Rashkovan M, Ross J, Schaffer AM, Saba I, Lemsaddek W, Trudel M, Möröy T

The transcription factor Miz-1 is required for embryonic and stress-induced erythropoiesis but dispensable for adult erythropoiesis

American Journal of Blood Research (2014) 4 7-19 (PubMedExternal link)

Myc-interacting zinc finger protein 1 (Miz-1) is a BTB/POZ domain transcription factor that regulates complex cellular processes such as proliferation and apoptosis. Constitutively Miz-1-deficient animals arrest embryonic development at E14.5 due to severe anemia and fetal liver cells lacking Miz-1 show a high cell death rate and a significant reduction of mature erythroid cells. Mice with conditional Miz-1 alleles deleted around E14.5 were born at normal ratios, but had reduced numbers of erythrocytes, and showed an increase in reticulocytes in the peripheral blood. When challenged with the hemolytic agent phenylhydrazine (PHZ), Miz-1-deficient mice responded with a severe anemia. In addition, an accumulation of immature erythroid cells occurred in the bone marrow and spleen. Epo-signaling is the essential pathway for erythrocyte maturation and we could show that Miz-1 regulates Epo-signaling through Stat5 phosphorylation.
Taken together, Miz-1 is an important factor for erythroid differentiation and maturation. Moreover, Miz-1 is necessary to maintain a peripheral red blood cell homeostasis in particular in response to oxidative stress.