Regulatory Binding Partners Research Articles

Three structures of PSI-LHCI from Chlamydomonas reinhardtii suggest a resting state re-activated by ferredoxin

Photosystem I (PSI) from the green alga Chlamydomonas reinhardtii, with various numbers of membrane bound antenna complexes (LHCI), has been described in great detail. In contrast, structural characterization of soluble binding partners is less advanced. Here, we used X-ray crystallography and single particle cryo-EM to investigate three structures of the PSI-LHCI supercomplex from Chlamydomonas reinhardtii. An X-ray structure demonstrates the absence of six chlorophylls from the luminal side of the LHCI belts, suggesting these pigments were either physically absent or less stably associated with the complex, potentially influencing excitation transfer significantly. CryoEM revealed extra densities on luminal and stromal sides of the supercomplex, situated in the vicinity of the electron transfer sites. These densities disappeared after the binding of oxidized ferredoxin to PSI-LHCI. Based on these structures, we propose the existence of a PSI-LHCI resting state with a reduced active chlorophyll content, electron donors docked in waiting positions and regulatory binding partners positioned at the electron acceptor site. The resting state PSI-LHCI supercomplex would be recruited to its active form by the availability of oxidized ferredoxin.

A comprehensive review of SHP2 and its role in cancer

Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase ubiquitously expressed mainly in the cytoplasm of several tissues. SHP2 modulates diverse cell signaling events that control metabolism, cell growth, differentiation, cell migration, transcription and oncogenic transformation. It interacts with diverse molecules in the cell, and regulates key signaling events including RAS/ERK, PI3K/AKT, JAK/STAT and PD-1 pathways downstream of several receptor tyrosine kinases (RTKs) upon stimulation by growth factors and cytokines. SHP2 acts as both a phosphatase and a scaffold, and plays prominently oncogenic functions but can be tumor suppressor in a context-dependent manner. It typically acts as a positive regulator of RTKs signaling with some inhibitory functions reported as well. SHP2 expression and activity is regulated by such factors as allosteric autoinhibition, microRNAs, ubiquitination and SUMOylation. Dysregulation of SHP2 expression or activity causes many developmental diseases, and hematological and solid tumors. Moreover, upregulated SHP2 expression or activity also decreases sensitivity of cancer cells to anticancer drugs. SHP2 is now considered as a compelling anticancer drug target and several classes of SHP2 inhibitors with different mode of action are developed with some already in clinical trial phases. Moreover, novel SHP2 substrates and functions are rapidly growing both in cell and cancer. In view of this, we comprehensively and thoroughly reviewed literatures about SHP2 regulatory mechanisms, substrates and binding partners, biological functions, roles in human cancers, and different classes of small molecule inhibitors target this oncoprotein in cancer.

Abstract 2306: BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models

Abstract Background: Cyclin-dependent kinases (CDK) are a class of enzymes that, along with their regulatory cyclin binding partners, drive cell cycle progression. Cell cycle dysregulation is a hallmark of cancer and targeting its genetic drivers can confer therapeutic benefits. Cyclin E1 (CCNE1) gene alterations are common in patients with ovarian cancer and can be found in ~20% of high-grade serous ovarian cancer cases, which tend to be platinum therapy resistant and therefore represent a high medical need. Cyclin E1 is the canonical binding partner of CDK2, which becomes constitutively active when CCNE1 is amplified and overexpressed. Selectively inhibiting CDK2 is an attractive therapeutic option for CCNE1-amplified tumors and may limit off-target CDK-driven toxicities. Here we report preclinical validation studies leading to the development of an orally available CDK2 inhibitor, BLU-222, for the treatment of ovarian cancer harboring a CCNE1 amplification. Methods: BLU-222 selectivity was measured by enzyme assays, cellular target engagement assays (NanoBRET), and proliferation assays in a panel of ovarian cancer cell lines. In vitro cellular potency was assessed by phospho-Rb levels. In vivo antitumor activity of BLU-222 as a single agent or in combination with carboplatin was measured in an OVCAR-3 cell line-derived xenograft (CDX) tumor model harboring a CCNE1 amplification. Results: BLU-222 demonstrated selectivity, both biochemically and in cells, with low nanomolar potency for CDK2 vs other CDK family members (CDK1, -4, -6, -7, and -9). In a panel of ovarian cancer cell lines, those with CCNE1 amplifications were highly sensitive to BLU-222. Consistent with the in vitro profiling, BLU-222 exhibited significant antitumor activity in the OVCAR-3 CDX model. While administration of single-agent carboplatin led to stasis in vivo, the combination of BLU-222 + carboplatin induced durable tumor regression even after treatment cessation. Conclusions: These data provide a strong rationale for advancing BLU-222 towards clinical development in patients with CCNE1-amplified ovarian cancer. Citation Format: Victoria Brown, Phil Ramsden, Nealia House, Richard Vargas, Jian Guo, Ruduan Wang, Riadh Lobbardi, Maxine Chen, Douglas Wilson, Joseph Kim, Neil Bifulco, Michelle Maynard, Emanuele Perola, Dean Zhang, Steve Wenglowsky, Yoon Jong Choi. BLU-222, an investigational, potent, and selective CDK2 inhibitor, demonstrated robust antitumor activity in CCNE1-amplified ovarian cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2306.

ONC212 is a Novel Mitocan Acting Synergistically with Glycolysis Inhibition in Pancreatic Cancer.

ONC212 is a fluorinated imipridone with preclinical efficacy against pancreatic and other malignancies. Although mitochondrial protease ClpP was identified as an ONC212-binding target, the mechanism leading to cancer cell death is incompletely understood. We investigated mitochondrial dysfunction and metabolic rewiring triggered by ONC212 in pancreatic cancer, a deadly malignancy with an urgent need for novel therapeutics. We found ClpP is expressed in pancreatic cancer cells and is required for ONC212 cytotoxicity. ClpX, the regulatory binding partner of ClpP, is suppressed upon ONC212 treatment. Immunoblotting and extracellular flux analysis showed ONC212 impairs oxidative phosphorylation (OXPHOS) with decrease in mitochondrial-derived ATP production. Although collapse of mitochondrial function is observed across ONC212-treated cell lines, only OXPHOS-dependent cells undergo apoptosis. Cells relying on glycolysis undergo growth arrest and upregulate glucose catabolism to prevent ERK1/2 inhibition and apoptosis. Glucose restriction or combination with glycolytic inhibitor 2-deoxy-D-glucose synergize with ONC212 and promote apoptosis in vitro and in vivo Thus, ONC212 is a novel mitocan targeting oxidative metabolism in pancreatic cancer, leading to different cellular outcomes based on divergent metabolic programs.

Selective Renal Silencing of the Calcineurin Homologous Protein‐1 Decreases Blood Pressure in Balb/cJ Mice

Hypertension has high incidence and health care cost. Its treatments are often ineffective; therefore, we are in need of new strategies to prevent, limit or reverse hypertension. Long-term regulation of blood pressure (BP) has fundamental renal components; in particular, the causes of impaired renal Na+ handling and possible strategies to overcome disturbances in renal Na+ balance in hypertension are not well defined. Recently, we have shown that a Ca2+-binding protein, the Calcineurin Homologous Protein-1 (CHP1), is a regulatory binding partner of a key player in the control of Na+ homeostasis, the epithelial brush-border Na+/H+ exchanger-3 (NHE3). Here, we proposed that CHP1 expression controls BP as a regulatory co-factor of NHE3 transport. Indeed, selective renal silencing of Chp1 via renal subcapsular infusion of Chp1 siRNA in Balb/cJ mice significantly reduced NHE3 protein expression (-50%). Importantly, Chp1 siRNA treated mice had a reduced systolic blood pressure compared to scrambled siRNA treated mice (-30%) measured in anesthetized mice from the aorta and femoral artery, and in conscious mice using noninvasive tail-cuff method. Chp1 siRNA treated mice also had markedly reduced serum Na+ and increased urinary Na+ excretion compared to scrambled siRNA treated mice. Serum K+ and Cl- did not change while urinary K+ and Cl- excretion were increased in Chp1 siRNA treated mice. Glomerular Filtration Rate estimated by creatinine clearance was reduced in Chp1 treated mice possibly as consequence of tubuloglomerular feedback. This is the first animal model in mammals where the effect of a reduction in CHP1 protein expression in the kidney was studied. Our findings provide new insights into the action of CHP1 on renal tubular Na+ transport and propose CHP1 protein expression as a novel tool to significantly control blood pressure.

Role of iRhoms 1 and 2 in Endochondral Ossification.

Growth of the axial and appendicular skeleton depends on endochondral ossification, which is controlled by tightly regulated cell–cell interactions in the developing growth plates. Previous studies have uncovered an important role of a disintegrin and metalloprotease 17 (ADAM17) in the normal development of the mineralized zone of hypertrophic chondrocytes during endochondral ossification. ADAM17 regulates EGF-receptor signaling by cleaving EGFR-ligands such as TGFα from their membrane-anchored precursor. The activity of ADAM17 is controlled by two regulatory binding partners, the inactive Rhomboids 1 and 2 (iRhom1, 2), raising questions about their role in endochondral ossification. To address this question, we generated mice lacking iRhom2 (iR2−/−) with floxed alleles of iRhom1 that were specifically deleted in chondrocytes by Col2a1-Cre (iR1∆Ch). The resulting iR2−/−iR1∆Ch mice had retarded bone growth compared to iR2−/− mice, caused by a significantly expanded zone of hypertrophic mineralizing chondrocytes in the growth plate. Primary iR2−/−iR1∆Ch chondrocytes had strongly reduced shedding of TGFα and other ADAM17-dependent EGFR-ligands. The enlarged zone of mineralized hypertrophic chondrocytes in iR2−/−iR1∆Ch mice closely resembled the abnormal growth plate in A17∆Ch mice and was similar to growth plates in Tgfα−/− mice or mice with EGFR mutations. These data support a model in which iRhom1 and 2 regulate bone growth by controlling the ADAM17/TGFα/EGFR signaling axis during endochondral ossification.

Simultaneous Fluorescence Imaging Reveals N-Methyl-d-aspartic Acid Receptor Dependent Zn2+/H+ Flux in the Brains of Mice with Depression

Depression is immensely attributed to the overactivation of N-methyl-d-aspartic acid (NMDA) receptor in the brains. As regulatory binding partners of NMDA receptor, both Zn2+ and H+ are intimately interrelated to NMDA receptor's activity. Therefore, exploring synergistic changes on the levels of Zn2+ and H+ in brains will promote the knowledge and treatment of depression. However, the lack of efficient, appropriate imaging tools limits simultaneously tracking Zn2+ and H+ in living mouse brains. Thus, a well-designed dual-color fluorescent probe (DNP) was fabricated for the simultaneous monitoring of Zn2+ and H+ in the brains of mice with depression. Encountering Zn2+, the probe evoked bright blue fluorescence at 460 nm. Meanwhile, the red fluorescence at 680 nm was decreased with H+ addition. With blue/red dual fluorescence signal of DNP, we observed the synchronous increased Zn2+ and H+ in PC12 cells under oxidative stress. Notably, in vivo imaging for the first time revealed the simultaneous reduction of Zn2+ and pH in brains of mice with depression-like behaviors. Further results implied that the NMDA receptor might be responsible for the coinstantaneous fluctuation of Zn2+ and H+ during depression. Altogether, this work is conducive to the knowledge of neural signal transduction mechanisms, advancing our understanding of the pathogenesis in depression.

Class I phosphoinositide 3-kinase (PI3K) regulatory subunits and their roles in signaling and disease.

The Class I phosphoinositide 3-kinases (PI3Ks) are a group of heterodimeric lipid kinases that regulate crucial cellular processes including proliferation, survival, growth, and metabolism. The diversity in functions controlled by the various catalytic isoforms (p110α, p110β, p110δ, and p110γ) depends on their abilities to be activated by distinct stimuli such as receptor tyrosine kinases (RTKs), G-protein coupled receptors (GPCRs), and the Ras family of small G-proteins. A major factor determining the ability of each p110 enzyme to be activated is the presence of regulatory binding partners. Given the overwhelming evidence for the involvement of PI3Ks in diseases such as cancer, inflammation, immunodeficiency and diabetes, an understanding of how these regulatory proteins influence PI3K function is essential. This article highlights research deciphering the role of regulatory subunits in PI3K signaling and their involvement in human disease.

Collagen-binding proteins: insights from the Collagen Toolkits.

The Collagen Toolkits are libraries of 56 and 57 triple-helical synthetic peptides spanning the length of the collagen II and collagen III helices. These have been used in solid-phase binding assays to locate sites where collagen receptors and extracellular matrix components bind to collagens. Truncation and substitution allowed exact binding sites to be identified, and corresponding minimal peptides to be synthesised for use in structural and functional studies. 170 sites where over 30 proteins bind to collagen II have been mapped, providing firm conclusions about the amino acid distribution within such binding sites. Protein binding to collagen II is not random, but displays a periodicity of approximately 28 nm, with several prominent nodes where multiple proteins bind. Notably, the vicinity of the collagenase-cleavage site in Toolkit peptide II-44 is highly promiscuous, binding over 20 different proteins. This may reflect either the diverse chemistry of that locus or its diverse function, together with the interplay between regulatory binding partners. Peptides derived from Toolkit studies have been used to determine atomic level resolution of interactions between collagen and several of its binding partners and are finding practical application in tissue engineering.

Neuregulin-1β Plays a Neuroprotective Role by Inhibiting the Cdk5 Signaling Pathway after Cerebral Ischemia-Reperfusion Injury in Rats

This study investigated the effects of neuregulin-1β (NRG1β) after middle cerebral artery occlusion/reperfusion (MCAO/R) in rats to evaluate whether they occur via the cyclin-dependent kinase (Cdk)5 signaling pathway. One hundred adult male Wistar rats were randomly divided into sham, MCAO/R, treatment (NRG1β), inhibitor (roscovitine; Ros), and inhibitor + treatment (Ros + NRG1β) groups. The MCAO/R model was established using the intraluminal thread method. The neurobehavioral function was evaluated by the modified neurological severity score (mNSS). The cerebral infarction volume (CIV) was measured by triphenyl tetrazolium chloride (TTC) staining. Morphological changes were observed by hematoxylin-eosin (HE) staining. The apoptotic cell index (ACI) was detected by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Immunohistochemistry and Western blotting were performed to detect the expression of calpain 1, p35/p25 (regulatory binding partners of Cdk5), Cdk5, and p-Tau in neurons. The neuronal morphology in the MCAO/R, NRG1β, Ros + NRG1β, and Ros groups differed compared to the sham group; the mNSS, CIV, ACI, and the expression of calpain 1, p35/p25, Cdk5, and p-Tau were significantly increased in all four groups (P < 0.05). In the NRG1β, Ros and Ros + NRG1β groups, the neuronal morphology was significantly improved compared to the MCAO/R group, as were the mNSS, CIV, and ACI. The levels of calpain 1, p35/p25, and p-Tau were decreased compared with the MCAO/R group (P < 0.05), while the Cdk5 expression was not significantly different (P > 0.05). NRG1β may exert neuroprotective effects by inhibiting the expression of calpain 1, p35/p25, and p-Tau after cerebral ischemia-reperfusion injury.

A class of their own: exploring the nondeacetylase roles of class IIa HDACs in cardiovascular disease.

Histone deacetylases (HDACs) play integral roles in many cardiovascular biological processes ranging from transcriptional and translational regulation to protein stabilization and localization. There are 18 known HDACs categorized into 4 classes that can differ on the basis of substrate targets, subcellular localization, and regulatory binding partners. HDACs are classically known for their ability to remove acetyl groups from histone and nonhistone proteins that have lysine residues. However, despite their nomenclature and classical functions, discoveries from many research groups over the past decade have suggested that nondeacetylase roles exist for class IIa HDACs. This is not surprising given that class IIa HDACs have, for example, relatively poor deacetylase capabilities and are often shuttled in and out of nuclei upon specific pathological and nonpathological cardiac events. This review aims to consolidate and elucidate putative nondeacetylase roles for class IIa HDACs and, where possible, highlight studies that provide evidence for their noncanonical roles, especially in the context of cardiovascular maladies. There has been great interest recently in exploring the pharmacological regulators of HDACs for use in therapeutic interventions for treating cardiovascular diseases and inflammation. Thus it is of interest to earnestly consider nonenzymatic and or nondeacetylase roles of HDACs that might be key in potentiating or abrogating pathologies. These noncanonical HDAC functions may possibly yield new mechanisms and targets for drug discovery.

Invited review: Activation of G proteins by GTP and the mechanism of Gα-catalyzed GTP hydrolysis.

This review addresses the regulatory consequences of the binding of GTP to the alpha subunits (Gα) of heterotrimeric G proteins, the reaction mechanism of GTP hydrolysis catalyzed by Gα and the means by which GTPase activating proteins (GAPs) stimulate the GTPase activity of Gα. The high energy of GTP binding is used to restrain and stabilize the conformation of the Gα switch segments, particularly switch II, to afford stable complementary to the surfaces of Gα effectors, while excluding interaction with Gβγ, the regulatory binding partner of GDP-bound Gα. Upon GTP hydrolysis, the energy of these conformational restraints is dissipated and the two switch segments, particularly switch II, become flexible and are able to adopt a conformation suitable for tight binding to Gβγ. Catalytic site pre-organization presents a significant activation energy barrier to Gα GTPase activity. The glutamine residue near the N-terminus of switch II (Glncat ) must adopt a conformation in which it orients and stabilizes the γ phosphate and the water nucleophile for an in-line attack. The transition state is probably loose with dissociative character; phosphoryl transfer may be concerted. The catalytic arginine in switch I (Argcat ), together with amide hydrogen bonds from the phosphate binding loop, stabilize charge at the β-γ bridge oxygen of the leaving group. GAPs that harbor "regulator of protein signaling" (RGS) domains, or structurally unrelated domains within G protein effectors that function as GAPs, accelerate catalysis by stabilizing the pre-transition state for Gα-catalyzed GTP hydrolysis, primarily by restraining Argcat and Glncat to their catalytic conformations. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 449-462, 2016.

Regulation of Blood Pressure by the Calcineurin Homologous Protein‐1

Hypertension has high incidence and health care cost. Its treatments are often ineffective; therefore, we are in need of new strategies to prevent, limit or reverse hypertension. Long‐term regulation of blood pressure (BP) has fundamental renal components; in particular, the causes of impaired renal Na+ handling and possible strategies to overcome disturbances in renal Na+ balance in hypertension are not well defined. Recently, we have shown that a Ca2+‐binding protein, the Calcineurin Homologous Protein‐1 (CHP1), is a regulatory binding partner of a key player in the control of Na+ homeostasis, the epithelial brush‐border Na+/H+ exchanger‐3 (NHE3). Here, we proposed that CHP1 expression controls BP as a regulatory co‐factor of NHE3 transport. Indeed, selective renal silencing of Chp1 via renal subcapsular infusion of Chp1 siRNA in Balb/cJ mice significantly reduced NHE3 protein expression (‐50%). Importantly, Chp1 siRNA treated mice had a reduced systolic blood pressure compared to scrambled siRNA treated mice (‐30%). Chp1 siRNA treated mice also had markedly reduced serum Na+ and increased urinary Na+ excretion compared to scrambled siRNA treated mice. Serum K+ and Cl‐ did not change while urinary K+ and Cl‐ excretion were increased in Chp1 siRNA treated mice. Glomerular Filtration Rate estimated by creatinine clearance was reduced in Chp1 treated mice possibly as consequence of tubuloglomerular feedback. This is the first animal model in mammals where the effect of a reduction in CHP1 protein expression in the kidney was studied. Our findings provide new insights into the action of CHP1 on renal tubular Na+ transport and propose CHP1 protein expression as a novel tool to significantly control blood pressure.

Abstract 16905: Fluorescence Resonance Energy Transfer Reveals that Cardiac Calcium ATPase Dimerizes and Forms a Complex with Phospholamban in a 2:1 Stoichiometry

The sarco/endoplasmic reticulum calcium ATPase (SERCA) has been proposed to form functional dimers in vitro. In order to investigate whether SERCA forms homo-dimers in live cells, we fused canine SERCA2a to cerulean (Cer) or yellow fluorescent protein (YFP), and quantified SERCA-SERCA interactions by fluorescence resonance energy transfer (FRET). SERCA-SERCA FRET efficiency was dependent on the labeling position of the fluorescent protein tags, with the highest FRET efficiency achieved when the respective fluorescent proteins were fused to SERCA N-termini. FRET was reduced by competition with unlabeled SERCA, suggesting that the observed FRET was due to specific protein-protein interactions. Progressive photobleaching of YFP showed that Cer intensity increased linearly with decreasing YFP intensity, suggesting that the stoichiometry of the SERCA complex is a dimer. In contrast, a control experiment with phospholamban (PLB) oligomer showed a non-linear YFP/Cer relationship, consistent with its well-known pentameric stoichiometry. We also investigated whether SERCA dimers could interact with PLB, the regulatory binding partner of SERCA. Interestingly, while average maximal FRET was 28% between SERCA and PLB, fluorescence lifetime measurements revealed two different lifetimes, consistent with two different populations of FRET donors. One population showed very low FRET, while the other population exhibited high FRET- approximately double the measured average maximal FRET efficiency. The data are consistent with a single PLB bound to each SERCA homo-dimer; in this regulatory complex one SERCA protomer is in close proximity to PLB (50 Å), while the other is too far away to participate in FRET with PLB.

Abstract 5386: Disrupting p53:Mdm2 and p53:Mdm4 - Comparative cell-based assays for drug screening

Abstract The important interconnected protein-protein interactions between the tumor suppressor p53 and its regulatory binding partners Mdm2 and Mdm4 (= MdmX/ HdmX) are implicated in pathogenesis of various cancers. However, until now there is a lack of reversible cell-based assays for concurrent analysis for both PPIs: p53/Mdm2 and p53/Mdm4. Our aim was to develop two comparative assays based on the Fluorescent 2-Hybrid (F2H) assay principle, which enable side-by-side analysis of antagonistic compounds. The F2H assay is a fully reversible microscopy-assisted assay for the direct intracellular analysis of PPIs. It offers a fast and straight-forward readout: an interaction-dependent co-localization of two fluorescent signals at a defined spot in the nucleus of mammalian cells. With these assays we analyzed a set of newly developed stapled peptides as potent p53:Mdm2 and/ or p53:Mdm4 inhibitors. Live cell data generated by the F2H assays enabled us to discriminate and describe the peptides according to their ability to penetrate into the cells, their efficacy on the PPIs and their cytotoxic side effects in one single assay. In parallel we performed a pilot F2H screen with a sub-set or 20 small molecule compounds (including Nutlin-3) that exhibited activity against one or both interactions at various potencies in ELISAs before. We could identify 5 potent compounds, which were dramatically reducing the number of p53:Mdm2 interactions. However, none of the small molecules, but only the stapled peptides exhibited an intracellular activity on p53:Mdm4. Furthermore, we were able to expand these assays and to identify mutants of Mdm2 resistant to Nutlin inhibition. In summary, we show that the p53:Mdm2 and p53:Mdm4 F2H assays described here, - enable side-by-side analysis of substances' dual Mdm2-Mdm4 activity - are suitable for screening and testing various types of compounds, such as peptidic inhibitors or small molecules - concurrently provide initial data on compound cell-permeability and cytotoxicity - allow real-time visualization of PPI dynamics in living cells. Citation Format: Larisa Yurlova, Christopher J. Brown, Maarten Derks, Farid John Ghadessy, Ian Hickson, David P. Lane, Eberhard Krausz, Kourosh Zolghadr. Disrupting p53:Mdm2 and p53:Mdm4 - Comparative cell-based assays for drug screening. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2014-5386

Regulatory Proteins Enable the Kinesin Kip2 to Overpower Cytoplasmic Dynein

Cytoplasmic dynein and kinesin are opposite-polarity, microtubule-based motors that create movement and spatial organization within eukaryotic cells. As a minus-end-directed motor that typically moves cargo toward the cell interior, dynein faces a directionality problem: how is dynein initially targeted to the cell periphery? Previous studies have shown that in S. cerevisiae, this problem is solved in part by the action of Kip2, a plus-end-directed motor that is posited to co-transport dynein and another protein called Bik1 toward the microtubule plus-end. Here, we investigate the interplay between dynein and Kip2 by coupling them to a three-dimensional DNA origami scaffold, or chassis, and using fluorescence microscopy to visualize the emergent motile behavior of these assemblies. In the absence of regulators, dynein-chassis-Kip2 structures move predominantly in the minus-end (dynein) direction. However, the frequency of plus-end-directed movements is markedly enhanced by the addition of Bik1 (a homolog of the cytoplasmic linker protein Clip170) and Bim1 (a member of the EB [end-binding] protein family). Moreover, Bik1, Bim1 and Kip2 co-elute as a ternary complex by size-exclusion chromatography. Thus, the addition of two regulatory binding partners can enable Kip2 to overcome dynein's intrinsic minus-end-directed motility and transport dynein toward the microtubule plus-end.

Phosphorylation of the Rab exchange factor Sec2p directs a switch in regulatory binding partners

Sec2p is a guanine nucleotide exchange factor that promotes exocytosis by activating the Rab GTPase Sec4p. Sec2p is highly phosphorylated, and we have explored the role of phosphorylation in the regulation of its function. We have identified three phosphosites and demonstrate that phosphorylation regulates the interaction of Sec2p with its binding partners Ypt32p, Sec15p, and phosphatidyl-inositol-4-phosphate. In its nonphosphorylated form, Sec2p binds preferentially to the upstream Rab, Ypt32p-GTP, thus forming a Rab guanine nucleotide exchange factor cascade that leads to the activation of the downstream Rab, Sec4p. The nonphosphorylated form of Sec2p also binds to the Golgi-associated phosphatidyl-inositol-4-phosphate, which works in concert with Ypt32p-GTP to recruit Sec2p to Golgi-derived secretory vesicles. In contrast, the phosphorylated form of Sec2p binds preferentially to Sec15p, a downstream effector of Sec4p and a component of the exocyst tethering complex, thus forming a positive-feedback loop that prepares the secretory vesicle for fusion with the plasma membrane. Our results suggest that the phosphorylation state of Sec2p can direct a switch in its regulatory binding partners that facilitates maturation of the secretory vesicle and helps to promote the directionality of vesicular transport.

Pathological hypertrophy amelioration by PRAS40-mediated inhibition of mTORC1

Mechanistic target of rapamycin complex 1 (mTORC1), necessary for cellular growth, is regulated by intracellular signaling mediating inhibition of mTORC1 activation. Among mTORC1 regulatory binding partners, the role of Proline Rich AKT Substrate of 40 kDa (PRAS40) in controlling mTORC1 activity and cellular growth in response to pathological and physiological stress in the heart has never been addressed. This report shows PRAS40 is regulated by AKT in cardiomyocytes and that AKT-driven phosphorylation relieves the inhibitory function of PRAS40. PRAS40 overexpression in vitro blocks mTORC1 in cardiomyocytes and decreases pathological growth. Cardiomyocyte-specific overexpression in vivo blunts pathological remodeling after pressure overload and preserves cardiac function. Inhibition of mTORC1 by PRAS40 preferentially promotes protective mTORC2 signaling in chronic diseased myocardium. In contrast, strong PRAS40 phosphorylation by AKT allows for physiological hypertrophy both in vitro and in vivo, whereas cardiomyocyte-specific overexpression of a PRAS40 mutant lacking capacity for AKT-phosphorylation inhibits physiological growth in vivo, demonstrating that AKT-mediated PRAS40 phosphorylation is necessary for induction of physiological hypertrophy. Therefore, PRAS40 phosphorylation acts as a molecular switch allowing mTORC1 activation during physiological growth, opening up unique possibilities for therapeutic regulation of the mTORC1 complex to mitigate pathologic myocardial hypertrophy by PRAS40.

Glucose-Specific Enzyme IIA Has Unique Binding Partners in The Vibrio cholerae Biofilm

ABSTRACTGlucose-specific enzyme IIA (EIIAGlc) is a central regulator of bacterial metabolism and an intermediate in the phosphoenolpyruvate phosphotransferase system (PTS), a conserved phosphotransfer cascade that controls carbohydrate transport. We previously reported that EIIAGlc activates transcription of the genes required for Vibrio cholerae biofilm formation. While EIIAGlc modulates the function of many proteins through a direct interaction, none of the known regulatory binding partners of EIIAGlc activates biofilm formation. Therefore, we used tandem affinity purification (TAP) to compare binding partners of EIIAGlc in both planktonic and biofilm cells. A surprising number of novel EIIAGlc binding partners were identified predominantly under one condition or the other. Studies of planktonic cells revealed established partners of EIIAGlc, such as adenylate cyclase and glycerol kinase. In biofilms, MshH, a homolog of Escherichia coli CsrD, was found to be a dominant binding partner of EIIAGlc. Further studies revealed that MshH inhibits biofilm formation. This function was independent of the Carbon storage regulator (Csr) pathway and dependent on EIIAGlc. To explore the existence of multiprotein complexes centered on EIIAGlc, we also affinity purified the binding partners of adenylate cyclase from biofilm cells. In addition to EIIAGlc, this analysis yielded many of the same proteins that copurified with EIIAGlc. We hypothesize that EIIAGlc serves as a hub for multiprotein complexes and furthermore that these complexes may provide a mechanism for competitive and cooperative interactions between binding partners.

Protein and nucleic acid methylating enzymes: mechanisms and regulation

Protein and nucleic acid methylating enzymes are implicated in myriad cellular processes. These enzymes utilize diverse chemical mechanisms ranging from nucleophilic substitution-displacement to a novel radical-based reaction found in bacterial iron-sulfur cluster proteins. Within the cell, methylation activity is governed by interactions with endogenous molecular machinery. Of particular interest are the observations that methylating enzyme activity can be allosterically controlled by regulatory binding partners. Recent advances and emerging trends in the study of methylating enzyme mechanisms and regulation highlight the importance of protein and nucleic acid methylation in cellular physiology and disease.