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In red-light sensing phytochromes, isomerization of the bilin chromophore triggers structural and dynamic changes across multiple domains, ultimately leading to control of the output module (OPM) activity. In between, a hairpin structure, "arm", extends from an interconnecting domain to the chromophore region. Here, by removing this protein segment in a bacteriophytochrome from Deinococcus radiodurans (DrBphP), we show that the arm is crucial for signal transduction. Crystallographic, spectroscopic, and biochemical data indicate that this variant maintains the properties of DrBphP in the resting state. Spectroscopic data also reveal that the armless systems maintain the ability to respond to light. However, there is no subsequent regulation of OPM activity without the arms. Thermal denaturation reveals that the arms stabilize the DrBphP structure. Our results underline the importance of the structurally flexible interconnecting hairpin extensions and describe their central role in the allosteric coupling of phytochromes. peerReviewed

The NRF2 pathway is frequently activated in various cancer types, yet a comprehensive analysis of its effects across different malignancies is currently lacking. We developed a NRF2 activity metric and utilized it to conduct a pan-cancer analysis of oncogenic NRF2 signaling. We identified an immunoevasive phenotype where high NRF2 activity is associated with low interferon-gamma (IFNγ), HLA-I expression and T cell and macrophage infiltration in squamous malignancies of the lung, head and neck area, cervix and esophagus. Squamous NRF2 overactive tumors comprise a molecular phenotype with SOX2/TP63 amplification, TP53 mutation and CDKN2A loss. These immune cold NRF2 hyperactive diseases are associated with upregulation of immunomodulatory NAMPT, WNT5A, SPP1, SLC7A11, SLC2A1 and PD-L1. Based on our functional genomics analyses, these genes represent candidate NRF2 targets, suggesting direct modulation of the tumor immune milieu. Single-cell mRNA data shows that cancer cells of this subtype exhibit decreased expression of IFNγ responsive ligands, and increased expression of immunosuppressive ligands NAMPT, SPP1 and WNT5A that mediate signaling in intercellular crosstalk. In addition, we discovered that the negative relationship of NRF2 and immune cells are explained by stromal populations of lung squamous cell carcinoma, and this effect spans multiple squamous malignancies based on our molecular subtyping and deconvolution data. peerReviewed

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]x0PxxP, the hydrophobic residue 0 being proline or leucine. We have studied the unusual 0 = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The RxAPxxP motif was also found in human T cell leukemia virus-1 (HTLV-1) Gag polyprotein. We found that this motif was required for efficient HTLV-1 infection, and that the specificity of SNX9 SH3 for the RxAPxxP core binding motif was importantly involved in this process.

Protein Phosphatase 2A, PP2A, the principal Serine/threonine phosphatase, has major roles in broad range of signaling pathways that include regulation of cell cycle, cell proliferation and neuronal signaling. The loss of function of PP2A is linked with many human diseases, like cancer and neurodegenerative disorders. Protein phosphatase 2A (PP2A) functions as tumor suppressor and its tumor suppressor activity is inhibited by the overexpression of PP2A inhibitor proteins in most of the cancers. ARPP-19/ARPP-16 has been identified as one of the potential PP2A inhibitor proteins. Here, we report the resonance assignment of backbone 1H, 13C and 15N atoms of human ARPP-19 and ARPP-16 proteins. These chemical shift values can provide valuable information for the further study of the dynamics and interaction of ARPP-proteins to PP2A using NMR spectroscopy. Electronic supplementary material The online version of this article (doi:10.1007/s12104-020-09951-w) contains supplementary material, which is available to authorized users.

Paxillin is a potential participant in the direct intracellular force transmission which is considered as the foundation of cells sensing and responding to extracellular environment. However, the detection of tension across paxillin has not been achieved due to lacking microsized tools. Herein, a paxillin tension sensor (PaxTs) based on Fluorescence Resonance Energy Transfer (FRET) technique was constructed. PaxTs can be expressed and assembled to FA sites spontaneously to visualize the tension across paxillin with FRET efficiency of ~62.4% in living cells. The tension across paxillin was found to decrease upon shear stress, in which the membrane fluidity and contractility of actin acted as cushions. It is observed that paxillin participates in the pathway of cell membrane-cytoskeleton-FAs for force transmission upon mechanical force in real time visualization, which provides a promising new method to investigate the direct intracellular force transmission in biology and technology. peerReviewed

Denervation of skeletal muscles induces severe muscle atrophy, which is preceded by cellular alterations such as increased plasma membrane permeability, reduced resting membrane potential and accelerated protein catabolism. The factors that induce these changes remain unknown. Conversely, functional recovery following denervation depends on successful reinnervation. Here, we show that activation of nicotinic acetylcholine receptors (nAChRs) by quantal release of acetylcholine (ACh) from motoneurons is sufficient to prevent changes induced by denervation. Using in vitro assays, ACh and non-hydrolysable ACh analogs repressed the expression of connexin43 and connexin45 hemichannels, which promote muscle atrophy. In co-culture studies, connexin43/45 hemichannel knockout or knockdown increased innervation of muscle fibers by dorsal root ganglion neurons. Our results show that ACh released by motoneurons exerts a hitherto unknown function independent of myofiber contraction. nAChRs and connexin hemichannels are potential molecular targets for therapeutic intervention in a variety of pathological conditions with reduced synaptic neuromuscular transmission., Denervation of muscle fibres induces muscle atrophy, via mechanisms that remain unclear. Here, the authors show that binding of acetylcoline to its receptor at the neuromuscular junction represses the expression of connexins 43 and 45, which promote atrophy, and is sufficient to prevent denervation-induced loss of myofibre mass.

Muscle wasting, i.e., cachexia, frequently occurs in cancer and associates with poor prognosis and increased morbidity and mortality. Anticancer treatments have also been shown to contribute to sustainment or exacerbation of cachexia, thus affecting quality of life and overall survival in cancer patients. Pre-clinical studies have shown that blocking activin receptor type 2 (ACVR2) or its ligands and their downstream signaling can preserve muscle mass in rodents bearing experimental cancers, as well as in chemotherapy-treated animals. In tumor-bearing mice, the prevention of skeletal and respiratory muscle wasting was also associated with improved survival. However, the definitive proof that improved survival directly results from muscle preservation following blockade of ACVR2 signaling is still lacking, especially considering that concurrent beneficial effects in organs other than skeletal muscle have also been described in the presence of cancer or following chemotherapy treatments paired with counteraction of ACVR2 signaling. Hence, here, we aim to provide an up-to-date literature review on the multifaceted anti-cachectic effects of ACVR2 blockade in preclinical models of cancer, as well as in combination with anticancer treatments.

Protein phosphatase 2A (PP2A) activity is critical for maintaining normal physiological cellular functions. PP2A is inhibited by endogenous inhibitor proteins in several pathological conditions including cancer. A PP2A inhibitor protein, ARPP-19, has recently been connected to several human cancer types. Accordingly, the knowledge about ARPP-19—PP2A inhibition mechanism is crucial for the understanding the disease development and the therapeutic targeting of ARPP-19—PP2A. Here, we show the first structural characterization of ARPP-19, and its splice variant ARPP-16 using NMR spectroscopy, and SAXS. The results reveal that both ARPP proteins are intrinsically disordered but contain transient secondary structure elements. The interaction mechanism of ARPP-16/19 with PP2A was investigated using microscale thermophoresis and NMR spectroscopy. Our results suggest that ARPP—PP2A A-subunit interaction is mediated by linear motif and has modest affinity whereas, the interaction of ARPPs with B56-subunit is weak and transient. Like many IDPs, ARPPs are promiscuous binders that transiently interact with PP2A A- and B56 subunits using multiple interaction motifs. In summary, our results provide a good starting point for future studies and development of therapeutics that block ARPP-PP2A interactions.

Alongside in vivo models, a simpler and more mechanistic approach is required to study the effects of myostatin on skeletal muscle because myostatin is an important negative regulator of muscle size. In this study, myostatin was administered to murine (C2C12) and human (CHQ) myoblasts and myotubes. Canonical and noncanonical signaling downstream to myostatin, related ligands, and their receptor were analyzed. The effects of tumorkines were analyzed after coculture of C2C12 and colon cancer-C26 cells. The effects of myostatin on canonical and noncanonical signaling were strongly reduced in C2C12 cells after differentiation. This may be explained by increased follistatin, an endogenous blocker of myostatin and altered expression of activin receptor ligands. In contrast, CHQ cells were equally responsive to myostatin, and follistatin remained unaltered. Both myostatin administration and the coculture stimulated pathways associated with inflammation, especially in C2C12 cells. In conclusion, the effects of myostatin on intracellular signaling may be cell line- or organism-specific, and C2C12 myotubes seem to be a nonoptimal in vitro model for investigating the effects of myostatin on canonical and noncanonical signaling in skeletal muscle. This may be due to altered expression of activin receptor ligands and their regulators during muscle cell differentiation.

Signaling through activin receptors regulates skeletal muscle mass and activin receptor 2B (ACVR2B) ligands are also suggested to participate in myocardial infarction (MI) pathology in the heart. In this study, we determined the effect of systemic blockade of ACVR2B ligands on cardiac function in experimental MI, and defined its efficacy to revert muscle wasting in ischemic heart failure (HF). Mice were treated with soluble ACVR2B decoy receptor (ACVR2B-Fc) to study its effect on post-MI cardiac remodeling and on later HF. Cardiac function was determined with echocardiography, and myocardium analyzed with histological and biochemical methods for hypertrophy and fibrosis. Pharmacological blockade of ACVR2B ligands did not rescue the heart from ischemic injury or alleviate post-MI remodeling and ischemic HF. Collectively, ACVR2B-Fc did not affect cardiomyocyte hypertrophy, fibrosis, angiogenesis, nor factors associated with cardiac regeneration except modification of certain genes involved in metabolism or cell growth/survival. ACVR2B-Fc, however, was able to reduce skeletal muscle wasting in chronic ischemic HF, accompanied by reduced LC3II as a marker of autophagy and increased mTOR signaling and Cited4 expression as markers of physiological hypertrophy in quadriceps muscle. Our results ascertain pharmacological blockade of ACVR2B ligands as a possible therapy for skeletal muscle wasting in ischemic HF. Pharmacological blockade of ACVR2B ligands preserved myofiber size in ischemic HF, but did not compromise cardiac function nor exacerbate cardiac remodeling after ischemic injury. peerReviewed

Background Voltage-gated sodium (Nav) channels have traditionally been considered a trademark of excitable cells. However, recent studies have shown the presence of Nav channels in several non-excitable cells, such as astrocytes and macrophages, demonstrating that the roles of these channels are more diverse than was previously thought. Despite the earlier discoveries, the presence of Nav channel-mediated currents in the cells of retinal pigment epithelium (RPE) has been dismissed as a cell culture artifact. We challenge this notion by investigating the presence and possible role of Nav channels in RPE both ex vivo and in vitro. Results Our work demonstrates that several subtypes of Nav channels are found in human embryonic stem cell (hESC)-derived and mouse RPE, most prominently subtypes Nav1.4, Nav1.6, and Nav1.8. Whole cell patch clamp recordings from the hESC-derived RPE monolayers showed that the current was inhibited by TTX and QX-314 and was sensitive to the selective blockers of the main Nav subtypes. Importantly, we show that the Nav channels are involved in photoreceptor outer segment phagocytosis since blocking their activity significantly reduces the efficiency of particle internalization. Consistent with this role, our electron microscopy results and immunocytochemical analysis show that Nav1.4 and Nav1.8 accumulate on phagosomes and that pharmacological inhibition of Nav channels as well as silencing the expression of Nav1.4 with shRNA impairs the phagocytosis process. Conclusions Taken together, our study shows that Nav channels are present in RPE, giving this tissue the capacity of fast electrical signaling. The channels are critical for the physiology of RPE with an important role in photoreceptor outer segment phagocytosis. Electronic supplementary material The online version of this article (10.1186/s12915-019-0681-1) contains supplementary material, which is available to authorized users.

Phytochromes are photoreceptors in plants, fungi, and various microorganisms and cycle between metastable red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Their light responses are thought to follow a conserved structural mechanism that is triggered by isomerization of the chromophore. Downstream structural changes involve refolding of the so-called tongue extension of the phytochrome-specific GAF-related (PHY) domain of the photoreceptor. The tongue is connected to the chromophore by conserved DIP and PRXSF motifs and a conserved tyrosine, but the role of these residues in signal transduction is not clear. Here, we examine the tongue interactions and their interplay with the chromophore by substituting the conserved tyrosine (Tyr(263)) in the phytochrome from the extremophile bacterium Deinococcus radiodurans with phenylalanine. Using optical and FTIR spectroscopy, X-ray solution scattering, and crystallography of chromophore-binding domain (CBD) and CBD-PHY fragments, we show that the absence of the Tyr(263) hydroxyl destabilizes the -sheet conformation of the tongue. This allowed the phytochrome to adopt an -helical tongue conformation regardless of the chromophore state, hence distorting the activity state of the protein. Our crystal structures further revealed that water interactions are missing in the Y263F mutant, correlating with a decrease of the photoconversion yield and underpinning the functional role of Tyr(263) in phytochrome conformational changes. We propose a model in which isomerization of the chromophore, refolding of the tongue, and globular conformational changes are represented as weakly coupled equilibria. The results also suggest that the phytochromes have several redundant signaling routes.

Insulin-like growth factor-I (IGF-I) and its splice variants Insulin-like growth factor-I isoform Ea (IGF-IEa) and mechano growth factor (MGF) may play an important role in muscular adaptations to resistance training (RT) that may be modulated by ageing. It has been suggested that IGF-I induces cellular responses via AKT8 virus oncogene cellular homolog (Akt) and Extracellular signal-regulated kinase (Erk) signalling pathways. Therefore, resistance exercise-induced changes in skeletal muscle IGF-IEa and MGF messenger ribonucleic acid (mRNA), and MGF, Erk1/2, Akt and p70S6K protein expression were investigated before and after 21 weeks of RT in younger (YM, 20–34 yrs., n = 7) and older men (OM, 51–71 yrs., n = 10). Experimental resistance exercises (RE) of 5 × 10 repetition maximum leg presses were performed pre- and post-RT. Muscle biopsies were obtained before and 48 h after REs, to study the late response to muscle loading. The muscle proteins or mRNAs of interest were not systematically influenced by the REs or RT, except for MGF mRNA expression which was increased (p

Proper functioning of the mitochondria is crucial for the survival of the cell. Viruses are able to interfere with mitochondrial functions as they infect the host cell. Parvoviruses are known to induce apoptosis in infected cells, but the role of the mitochondria in parvovirus induced cytopathy is only partially known. Here we demonstrate with confocal and electron microscopy that canine parvovirus (CPV) associated with the mitochondrial outer membrane from the onset of infection. During viral entry a transient depolarization of the mitochondrial transmembrane potential and increase in ROS level was detected. Subsequently, mitochondrial homeostasis was normalized shortly, as detected by repolarization of the mitochondrial membrane and decrease of ROS. Indeed, activation of cell survival signalling through ERK1/2 cascade was observed early in CPV infected cells. At 12 hours post infection, concurrent with the expression of viral non-structural protein 1, damage to the mitochondrial structure and depolarization of its membrane were apparent. Results of this study provide additional insight of parvovirus pathology and also more general information of virus-mitochondria association. peerReviewed

HIV- eli immuunikatovirustartuntaa kantaa maailmassa arviolta yli 30 miljoonaa ihmistä. Suomessa ilmoituksia HIV-positiivisista henkilöistä oli vuoden 2010 alkuun mennessä kirjattu runsas kolme tuhatta, ja määrä on viime vuosina lisääntynyt noin kahdellasadalla HIV-potilaalla vuosittain. Vaikka HIV:n lääkehoito on viime vuosina kehittynyt merkittävästi ja sairauden etenemistä pystytään tehokkaasti hidastamaan, ei virusta kuitenkaan pystytä nykyisillä lääkkeillä häätämään elimistöstä kokonaan. HIV infektoi pääasiassa ihmisen immuunijärjestelmän kannalta keskeisiä CD4(+) T-soluja sekä makrofageja. Lisääntyessään virus pystyy myös sekä suoraan että epäsuorasti tuhoamaan CD4(+) T-soluja eli auttajalymfosyyttejä. Tämän seurauksena on potilaan immuunipuolustuksen vähittäinen heikkeneminen ja HIV-infektion kehittyminen varsinaiseen AIDS-vaiheeseen, sekä useat siihen liittyvät sairaudet kuten kasvaimet ja muut tulehdukset. Nef on yksi HI-viruksen tuottamista virusproteiineista, ja keskeinen tekijä HIV-infektion kehittymiselle. Useat Nef-proteiinin toiminnot edistävät viruksen selviytymistä ja lisääntymistä potilaan elimistössä, ja infektion etenemisen onkin havaittu hidastuvan viallisen tai puuttuvan Nef-proteiinin seurauksena sekä luonnollisen HIV-infektion yhteydessä ihmisellä että kokeellisen SIV-infektion yhteydessä rhesusapinoilla. Nef ei itsessään ole entsymaattisesti aktiivinen proteiini, mutta sen kyky sitoutua solun omiin proteiineihin ja vaikuttaa niiden toimintaan selittää Nef:n vaikutusten laajan kirjon. Yksi tunnetuista Nef:iin sitoutuvista soluproteiineista on p21-aktivoituva kinaasi 2 (PAK2). Tämän työn tarkoituksena on ollut selvittää Nef/PAK2-kompleksin muodostumiseen vaikuttavia tekijöitä molekyylitasolla, sekä tämän kompleksin toimintaa solunsisäisessä viestinnässä. Tutkimuksessa kuvattiin uusi signalointimekanismi, joka mahdollistaa PAK2:n voimakkaan aktivoitumisen yhdistämällä kaksi viestintäreittiä. Molekyylibiologisia menetelmiä käyttäen tutkittiin myös PAK2:n aktivaatioon liittyvien proteiinin rakenteellisten muutosten, pienten GTPaasi-proteiinien sekä PAK2 kinaasin oman entsymaattisen aktiivisuuden merkitystä Nef/PAK2-kompleksin muodostumiselle. Proteiinikompleksin solunsisäistä sijaintia tutkittaessa sen havaittiin lokalisoituvan solussa tietyille, soluviestinnän kannalta keskeisille solukalvon ns. lipidilautta-alueille. GTPaaseja aktivoivan Vav1-proteiinin havaittiin osallistuvan Nef/PAK2-kompleksin muodostumiseen sekä kompleksin kulkeutumiseen solun kalvorakenteisiin. Lisäksi työssä tutkittiin Nef-molekyylin oletettua dimerisaatiopintaa, jonka vastakkaisesti varautuneilla aminohapoilla havaittiin olevan oleellinen merkitys Nef-Nef dimeerin muodostumiselle, jonka puolestaan todettiin olevan edellytys Nef/PAK2-kompleksin muodostumiselle sekä Nef:n välittämän HIV-partikkeleiden infektiivisyyden lisääntymiselle. Tulokset antavat uutta tietoa siitä, kuinka HIV käyttää solun omia signalointireittejä hyväkseen. HIV-1 Nef-proteiinin toiminnan kannalta tärkeiden vuorovaikutusten ymmärtäminen tarjoaa uusia hyödyntämismahdollisuuksia kohdennetulle lääkekehitykselle. Nef is an accessory protein encoded by the human and simian immunodeficiency viruses (HIV/SIV) and is a major factor in promoting viral pathogenesis. Several cellular functions of Nef, including downregulation of the surface molecules CD4 and MHC I, increase in viral infectivity, and Nef-mediated effects on intracellular signaling favor survival and propagation of the virus. Nef does not possess any intrinsic enzymatic activity; instead, it functions by interacting with a number of endogenous proteins within the host cell. One of the well-characterized Nef-associating protein partners and a central molecule in this study is p21-associating kinase 2 (PAK2). Previously, association of the HIV-1 Nef with PAK2 has been proposed to play a role in T cell activation, viral replication, apoptosis and progression of HIV infection to AIDS. Despite intense research, there is no consensus on the relevance of Nef-PAK2 association and its function in virus pathogenesis. Furthermore, selective association of Nef with a minute but highly active subpopulation of total cellular PAK2 has raised questions on the molecular requirements of Nef-PAK2 association. This work elucidated the interplay of Nef and PAK2 in cellular signaling by studying the mechanism of PAK2 activation and its role in Nef association. To clarify the role of additional proteins during Nef-PAK2 complex formation, the involvement of p21-GTPases and guanine nucleotide exchange factor (GEF) proteins was studied. Finally, the functional role of charge distribution within the putative Nef interaction surface was characterized. Here, we describe a new signaling pathway that potentiates PAK2 activation by converging signal transduction via Src tyrosine kinases with p21-GTPase-induced PAK activation. The p21-GTPase-induced conformational opening of PAK2 was associated with the ability of PAK2 to interact with Nef, and their association was stabilized by reduced kinase activity. The Nef-PAK2 (NAK) complex was specifically localized to detergent-resistant membranes (DRM), and Vav1 was identified as a GEF component of Nef-PAK2 complex, recruited to the DRMs by Nef. The reciprocal charges present at the putative dimer interface (PDI) of Nef were indispensable for Nef-dependent increase in viral infectivity. In addition, several other Nef activities, including NAK association, membrane localization, and activation of Hck were coordinated by charged residues at the Nef PDI. In conclusion, our results provide new insight on the cellular signaling upstream of Nef-PAK2 association, as well as on the molecular domains and binding partners that are important for the formation of the Nef-PAK2 complex. In addition, our data elucidate how Nef functions are related to charge distribution at the PDI surface. Detailed knowledge on the molecular mechanisms involved in Nef-PAK2-mediated signaling may provide clues for potential target sites that can be used to develop HIV inhibitory drugs.