scholarly journals Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

2016 ◽  
Vol 82 (17) ◽  
pp. 5060-5067 ◽  
Author(s):  
Mariela P. Mezzina ◽  
M. Julia Pettinari
Keyword(s):  
Binding Capacity ◽  
Coiled Coil ◽  
Active Role ◽  
Structural Features ◽  
Structural Function ◽  
Associated Proteins ◽  
Α Helix ◽  
Pha Granule

ABSTRACTPhasins are the major polyhydroxyalkanoate (PHA) granule-associated proteins. They promote bacterial growth and PHA synthesis and affect the number, size, and distribution of the granules. These proteins can be classified in 4 families with distinctive characteristics. Low-resolution structural studies andin silicopredictions were performed in order to elucidate the structure of different phasins. Most of these proteins share some common structural features, such as a preponderant α-helix composition, the presence of disordered regions that provide flexibility to the protein, and coiled-coil interacting regions that form oligomerization domains. Due to their amphiphilic nature, these proteins play an important structural function, forming an interphase between the hydrophobic content of PHA granules and the hydrophilic cytoplasm content. Phasins have been observed to affect both PHA accumulation and utilization. Apart from their role as granule structural proteins, phasins have a remarkable variety of additional functions. Different phasins have been determined to (i) activate PHA depolymerization, (ii) increase the expression and activity of PHA synthases, (iii) participate in PHA granule segregation, and (iv) have bothin vivoandin vitrochaperone activities. These properties suggest that phasins might play an active role in PHA-related stress protection and fitness enhancement. Due to their granule binding capacity and structural flexibility, several biotechnological applications have been developed using different phasins, increasing the interest in the study of these remarkable proteins.

scholarly journals Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A

2009 ◽  
Vol 186 (4) ◽  
pp. 481-490 ◽  
Author(s):  
Vito Mennella ◽  
Dong-Yan Tan ◽  
Daniel W. Buster ◽  
Ana B. Asenjo ◽  
Uttama Rath ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Molecular Dynamics ◽  
Drosophila Melanogaster ◽  
Structural Features ◽  
Motor Domain ◽  
Α Helix ◽  
Dynamics Simulations

Microtubule (MT)-destabilizing kinesin 13s perform fundamental roles throughout the cell cycle. In this study, we show that the Drosophila melanogaster kinesin 13, KLP10A, is phosphorylated in vivo at a conserved serine (S573) positioned within the α-helix 5 of the motor domain. In vitro, a phosphomimic KLP10A S573E mutant displays a reduced capacity to depolymerize MTs but normal affinity for the MT lattice. In cells, replacement of endogenous KLP10A with KLP10A S573E dampens MT plus end dynamics throughout the cell cycle, whereas a nonphosphorylatable S573A mutant apparently enhances activity during mitosis. Electron microscopy suggests that KLP10A S573 phosphorylation alters its association with the MT lattice, whereas molecular dynamics simulations reveal how KLP10A phosphorylation can alter the kinesin–MT interface without changing important structural features within the motor’s core. Finally, we identify casein kinase 1α as a possible candidate for KLP10A phosphorylation. We propose a model in which phosphorylation of the KLP10A motor domain provides a regulatory switch controlling the time and place of MT depolymerization.

scholarly journals The SUMO-specific isopeptidase SENP2 is targeted to intracellular membranes via a predicted N-terminal amphipathic α-helix

2018 ◽  
Vol 29 (15) ◽  
pp. 1878-1890 ◽  
Author(s):  
Hana M. Odeh ◽  
Etienne Coyaud ◽  
Brian Raught ◽  
Michael J. Matunis
Keyword(s):  
Binding Assays ◽  
Cellular Functions ◽  
Intracellular Membranes ◽  
Vitro Binding ◽  
Wide Range ◽  
Associated Functions ◽  
Associated Proteins ◽  
Α Helix

Sumoylation regulates a wide range of essential cellular functions, many of which are associated with activities in the nucleus. Although there is also emerging evidence for the involvement of the small ubiquitin-related modifier (SUMO) at intracellular membranes, the mechanisms by which sumoylation is regulated at membranes is largely unexplored. In this study, we report that the SUMO-specific isopeptidase, SENP2, uniquely associates with intracellular membranes. Using in vivo analyses and in vitro binding assays, we show that SENP2 is targeted to intracellular membranes via a predicted N-terminal amphipathic α-helix that promotes direct membrane binding. Furthermore, we demonstrate that SENP2 binding to intracellular membranes is regulated by interactions with the nuclear import receptor karyopherin-α. Consistent with membrane association, biotin identification (BioID) revealed interactions between SENP2 and endoplasmic reticulum, Golgi, and inner nuclear membrane-associated proteins. Collectively, our findings indicate that SENP2 binds to intracellular membranes where it interacts with membrane-associated proteins and has the potential to regulate their sumoylation and membrane-associated functions.

scholarly journals p150Glued, the largest subunit of the dynactin complex, is nonessential in Neurospora but required for nuclear distribution.

1996 ◽  
Vol 7 (5) ◽  
pp. 731-742 ◽  
Author(s):  
J H Tinsley ◽  
P F Minke ◽  
K S Bruno ◽  
M Plamann
Keyword(s):  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Structural Features ◽  
Gene Encoding ◽  
Nuclear Distribution ◽  
Transport Vesicles ◽  
Dynactin Complex ◽  
Intermediate Chains

Dynactin is a multisubunit complex that is required for cytoplasmic dynein, a minus-end-directed, microtubule-associated motor, to efficiently transport vesicles along microtubules in vitro. p150Glued, the largest subunit of dynactin, has been identified in vertebrates and Drosophila and recently has been shown to interact with cytoplasmic dynein intermediate chains in vitro. The mechanism by which dynactin facilitates cytoplasmic dynein-dependent vesicle transport is unknown. We have devised a genetic screen for cytoplasmic dynein/dynactin mutants in the filamentous fungus Neurospora crassa. In this paper, we report that one of these mutants, ro-3, defines a gene encoding an apparent homologue of p150Glued, and we provide genetic evidence that cytoplasmic dynein and dynactin interact in vivo. The major structural features of vertebrate and Drosophila p150Glued, a microtubule-binding site at the N-terminus and two large alpha-helical coiled-coil regions contained within the distal two-thirds of the polypeptide, are conserved in Ro3. Drosophila p150Glued is essential for viability; however, ro-3 null mutants are viable, indicating that dynactin is not an essential complex in N. crassa. We show that N. crassa cytoplasmic dynein and dynactin mutants have abnormal nuclear distribution but retain the ability to organize cytoplasmic microtubules and actin in anucleate hyphae.

scholarly journals Testing the 3Q:1R “Rule”: Mutational Analysis of the Ionic “Zero” Layer in the Yeast Exocytic SNARE Complex Reveals No Requirement for Arginine

2000 ◽  
Vol 11 (11) ◽  
pp. 3849-3858 ◽  
Author(s):  
Luba Katz ◽  
Patrick Brennwald
Keyword(s):  
Mutational Analysis ◽  
Coiled Coil ◽  
Snare Complex ◽  
Hydrophobic Core ◽  
Arginine Residues ◽  
Strict Requirement ◽  
Α Helix ◽  
Ionic Layer

The crystal structure of the synaptic SNARE complex reveals a parallel four-helix coiled-coil arrangement; buried in the hydrophobic core of the complex is an unusual ionic layer composed of three glutamines and one arginine, each provided by a separate α-helix. The presence of glutamine or arginine residues in this position is highly conserved across the t- and v-SNARE families, and it was recently suggested that a 3Q:1R ratio is likely to be a general feature common to all SNARE complexes. In this study, we have used genetic and biochemical assays to test this prediction with the yeast exocytic SNARE complex. We have determined that the relative position of Qs and Rs within the layer is not critical for biological activity and that Q-to-R substitutions in the layer reduce complex stability and result in lethal or conditional lethal growth defects. Surprisingly, SNARE complexes composed of four glutamines are fully functional for assembly in vitro and exocytic function in vivo. We conclude that the 3Q:1R layer composition is not required within the yeast exocytic SNARE complex because complexes containing four Q residues in the ionic layer appear by all criteria to be functionally equivalent. The unexpected flexibility of this layer suggests that there is no strict requirement for the 3Q:1R combination and that the SNARE complexes at other stages of transport may be composed entirely of Q-SNAREs or other noncanonical combinations.

scholarly journals Active role of free RNA during the early phase of proteostasis stress

2019 ◽  
Author(s):  
Marion Alriquet ◽  
Adrían Martínez-Limón ◽  
Gerd Hanspach ◽  
Martin Hengesbach ◽  
Gian G. Tartaglia ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Active Form ◽  
Active Role ◽  
Structural Features ◽  
Cellular Functions ◽  
Granule Formation

ABSTRACTTransient sequestration of proteins and RNA is an essential principle of cellular reaction to stress. Compared to polypeptides, less is known about the role of RNA released from polysomes during acute proteostasis stress. Using quantitative mass spectrometry, we identified a set of proteins assembled by free RNA in the heat-shocked mammalian cytosol. RNA-associated proteins displayed higher disorder and larger size, which supports the role of multivalent interactions during the initial phase of the RNA granule formation. Structural features of the free RNA interactors defined them as a subset of RNA-binding proteins. The interactome contained preferentially the active form of eIF2α. The interaction between assembled proteins in vivo required RNA. The reconstitution of the association process in vitro indicated to the multimolecular basis for the increased binding to RNA upon heat shock in the cytosol. Our results reveal how free RNA can participate in reorganization of cellular functions during proteostasis stress.

scholarly journals Engineered Single-Chain, Antiparallel, Coiled CoilMimics the MerR Metal BindingSite

2004 ◽  
Vol 186 (6) ◽  
pp. 1861-1868 ◽  
Author(s):  
Lingyun Song ◽  
Jonathan Caguiat ◽  
Zhongrui Li ◽  
Jacob Shokes ◽  
Robert A. Scott ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Metal Binding ◽  
Coiled Coil ◽  
Mercury Resistance ◽  
Binding Motif ◽  
Single Chain ◽  
Metal Binding Domain ◽  
Α Helix

ABSTRACT The repressor-activator MerR that controls transcription of the mercury resistance (mer) operon is unusual for its high sensitivity and specificity for Hg(II) in in vivo and in vitro transcriptional assays. The metal-recognition domain of MerR resides at the homodimer interface in a novel antiparallel arrangement of α-helix 5 that forms a coiled-coil motif. To facilitate the study of this novel metal binding motif, we assembled this antiparallel coiled coil into a single chain by directly fusing two copies of the 48-residue α-helix 5 of MerR. The resulting 107-residue polypeptide, called the metal binding domain (MBD), and wild-type MerR were overproduced and purified, and their metal-binding properties were determined in vivo and in vitro. In vitro MBD bound ca. 1.0 equivalent of Hg(II) per pair of binding sites, just as MerR does, and it showed only a slightly lower affinity for Hg(II) than did MerR. Extended X-ray absorption fine structure data showed that MBD has essentially the same Hg(II) coordination environment as MerR. In vivo, cells overexpressing MBD accumulated 70 to 100% more 203Hg(II) than cells bearing the vector alone, without deleterious effects on cell growth. Both MerR and MBD variously bound other thiophilic metal ions, including Cd(II), Zn(II), Pb(II), and As(III), in vitro and in vivo. We conclude that (i) it is possible to simulate in a single polypeptide chain the in vitro and in vivo metal-binding ability of dimeric, full-length MerR and (ii) MerR's specificity in transcriptional activation does not reside solely in the metal-binding step.

scholarly journals The Golgi-Associated Hook3 Protein Is a Member of a Novel Family of Microtubule-Binding Proteins

2001 ◽  
Vol 152 (5) ◽  
pp. 923-934 ◽  
Author(s):  
Jason H. Walenta ◽  
Aaron J. Didier ◽  
Xinran Liu ◽  
Helmut Krämer
Keyword(s):  
Golgi Complex ◽  
Membrane Trafficking ◽  
Spatial Organization ◽  
Large Fraction ◽  
Coiled Coil ◽  
Brefeldin A ◽  
Associated Proteins ◽  
Terminal Domains

Microtubules are central to the spatial organization of diverse membrane-trafficking systems. Here, we report that Hook proteins constitute a novel family of cytosolic coiled coil proteins that bind to organelles and to microtubules. The conserved NH2-terminal domains of Hook proteins mediate attachment to microtubules, whereas the more divergent COOH-terminal domains mediate the binding to organelles. Human Hook3 bound to Golgi membranes in vitro and was enriched in the cis-Golgi in vivo. Unlike other cis-Golgi–associated proteins, however, a large fraction of Hook3 maintained its juxtanuclear localization after Brefeldin A treatment, indicating a Golgi-independent mechanism for Hook3 localization. Because overexpression of Hook3 caused fragmentation of the Golgi complex, we propose that Hook3 participates in defining the architecture and localization of the mammalian Golgi complex.

In Vitro and in Vivo Comparison of Binding of 99m-Tc-Iabeled Anti-CEA MAb F33-104 with 99m-Tc-labeled Anti-CEA MAb BW431/26

1999 ◽  
Vol 38 (04) ◽  
pp. 115-119
Author(s):  
N. Oriuchi ◽  
S. Sugiyama ◽  
M. Kuroki ◽  
Y. Matsuoka ◽  
S. Tanada ◽  
...  
Keyword(s):  
Nude Mice ◽  
Binding Capacity ◽  
Colon Adenocarcinoma ◽  
Human Colon ◽  
Cell Binding ◽  
Vitro Binding ◽  
Labeling Method ◽  
Human Colon Adenocarcinoma

Summary Aim: The purpose of this study was to assess the potential for radioimmunodetection (RAID) of murine anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb) F33-104 labeled with technetium-99m (99m-Tc) by a reduction-mediated labeling method. Methods: The binding capacity of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA by means of in vitro procedures such as immunoradiometric assay and cell binding assay and the biodistribution of 99m-Tc-labeled anti-CEA MAb F33-104 in normal nude mice and nude mice bearing human colon adenocarcinoma LS180 tumor were investigated and compared with 99m-Tc-labeled anti-CEA MAb BW431/26. Results: The in vitro binding rate of 99m-Tc-labeled anti-CEA MAb F33-104 with CEA in solution and attached to the cell membrane was significantly higher than 99m-Tclabeled anti-CEA MAb BW431/261 (31.4 ± 0.95% vs. 11.9 ± 0.55% at 100 ng/mL of soluble CEA, 83.5 ± 2.84% vs. 54.0 ± 2.54% at 107 of LS 180 cells). In vivo, accumulation of 99m-Tc-labeled anti-CEA MAb F33-104 was higher at 18 h postinjection than 99m-Tc-labeled anti-CEA MAb BW431/26 (20.1 ± 3.50% ID/g vs. 14.4 ± 3.30% ID/g). 99m-Tcactivity in the kidneys of nude mice bearing tumor was higher at 18 h postinjection than at 3 h (12.8 ± 2.10% ID/g vs. 8.01 ± 2.40% ID/g of 99m-Tc-labeled anti-CEA MAb F33-104, 10.7 ± 1.70% ID/g vs. 8.10 ± 1.75% ID/g of 99m-Tc-labeled anti-CEA MAb BW431/26). Conclusion: 99m-Tc-labeled anti-CEA MAb F33-104 is a potential novel agent for RAID of recurrent colorectal cancer.

Traceable Peptidic Ligands that Target Ghrelin Receptors

2020 ◽  
Vol 21 (10) ◽  
pp. 955-964 ◽  
Author(s):  
Mengjie Liu ◽  
John Wade ◽  
Mohammed Akhter Hossain
Keyword(s):  
In Vivo Imaging ◽  
Peptide Hormone ◽  
Structural Features ◽  
Pharmacological Properties ◽  
Imaging Studies ◽  
Cognate Receptor ◽  
Ghrelin Receptors ◽  
Biochemical Research

: Ghrelin is a 28-amino acid octanoylated peptide hormone that is implicated in many physiological and pathophysiological processes. Specific visualization of ghrelin and its cognate receptor using traceable ligands is crucial in elucidating the localization, functions, and expression pattern of the peptide’s signaling pathway. Here 12 representative radio- and fluorescently-labeled peptide-based ligands are reviewed for in vitro and in vivo imaging studies. In particular, the focus is on their structural features, pharmacological properties, and applications in further biochemical research.

Polyamines and Related Nitrogen Compounds in the Chemotherapy of Neglected Diseases Caused by Kinetoplastids

2018 ◽  
Vol 18 (5) ◽  
pp. 321-368 ◽  
Author(s):  
Juan A. Bisceglia ◽  
Maria C. Mollo ◽  
Nadia Gruber ◽  
Liliana R. Orelli
Keyword(s):  
Drug Targets ◽  
Redox Homeostasis ◽  
Cost Effective ◽  
Structural Features ◽  
Mammalian Host ◽  
Neglected Diseases ◽  
Nitrogen Compounds ◽  
Chemical Structures

Neglected diseases due to the parasitic protozoa Leishmania and Trypanosoma (kinetoplastids) affect millions of people worldwide, and the lack of suitable treatments has promoted an ongoing drug discovery effort to identify novel nontoxic and cost-effective chemotherapies. Polyamines are ubiquitous small organic molecules that play key roles in kinetoplastid parasites metabolism, redox homeostasis and in the normal progression of cell cycles, which differ from those found in the mammalian host. These features make polyamines attractive in terms of antiparasitic drug development. The present work provides a comprehensive insight on the use of polyamine derivatives and related nitrogen compounds in the chemotherapy of kinetoplastid diseases. The amount of literature on this subject is considerable, and a classification considering drug targets and chemical structures were made. Polyamines, aminoalcohols and basic heterocycles designed to target the relevant parasitic enzyme trypanothione reductase are discussed in the first section, followed by compounds directed to less common targets, like parasite SOD and the aminopurine P2 transporter. Finally, the third section comprises nitrogen compounds structurally derived from antimalaric agents. References on the chemical synthesis of the selected compounds are reported together with their in vivo and/or in vitro IC50 values, and structureactivity relationships within each group are analyzed. Some favourable structural features were identified from the SAR analyses comprising protonable sites, hydrophobic groups and optimum distances between them. The importance of certain pharmacophoric groups or amino acid residues in the bioactivity of polyamine derived compounds is also discussed.

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