Evolutionarily conserved structural and functional roles of the FYVE domain

2007 ◽  
Vol 74 ◽  
pp. 95-105 ◽  
Author(s):  
Akira Hayakawa ◽  
Susan Hayes ◽  
Deborah Leonard ◽  
David Lambright ◽  
Silvia Corvera
Keyword(s):  
Mammalian Cells ◽  
Electrostatic Interactions ◽  
Head Group ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Intact Cells ◽  
C Elegans ◽  
Fyve Domain ◽  
Early Endosomes

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12–13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4′. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

scholarly journals The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

2021 ◽  
Author(s):  
Krishna Chintaluri
Keyword(s):  
Toxoplasma Gondii ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Effector Proteins ◽  
Ph Domain ◽  
Fyve Domain ◽  
Px Domain ◽  
Early Endosomes ◽  
Phosphatidylinositol 3

Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

scholarly journals The PH domain from the Toxoplasma gondii PH-containing protein-1 (TgPH1) serves as an ectopic reporter of phosphatidylinositol 3-phosphate in mammalian cells

2021 ◽  
Author(s):  
Krishna Chintaluri
Keyword(s):  
Toxoplasma Gondii ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Effector Proteins ◽  
Ph Domain ◽  
Fyve Domain ◽  
Px Domain ◽  
Early Endosomes ◽  
Phosphatidylinositol 3

Phosphoinositides (PtdInsPs) lipids recruit effector proteins to membranes to mediate a variety of functions including signal transduction and membrane trafficking. Each PtdInsP binds to a specific set of effectors through characteristic protein domains such as the PH, FYVE and PX domains. Domains with high affinity for a single PtdInsP species are useful as probes to visualize the distribution and dynamics of that PtdInsP. The endolysosomal system is governed by two primary PtdInsPs: phosphatidylinositol-3-phosphate [PtdIns(3)P] and phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2], which are thought to localize and control early endosomes and lysosomes, respectively. While PtdIns(3)P has been analysed with mammalian-derived PX and FYVE domains, PtdIns(3,5)P2 indicators remain controversial. Thus, complementary probes against these PtdInsPs are needed, including those originating from non-mammalian proteins. Here, we characterized in mammalian cells the dynamics of the PH domain from PH-containing protein-1 from the parasite Toxoplasma gondii (TgPH1), which was previously shown to bind PtdIns(3,5)P2 in vitro. However, we show that TgPH1 retains membrane-binding in PIKfyve-inhibited cells, suggesting that TgPH1 is not a viable PtdIns(3,5)P2 marker in mammalian cells. Instead, PtdIns(3)P depletion using pharmacological treatments dissociated TgPH1 from membranes. Indeed, TgPH1 co-localized to EEA1-positive endosomes. In addition, TgPH1 co-localized and behaved similarly to the PX domain of p40phox and tandem FYVE domain of EEA1, which are commonly used as PtdIns(3)P indicators. Collectively, TgPH1 offers a complementary reporter for PtdIns(3)P derived from a non-mammalian protein and that is distinct from commonly employed PX and FYVE domain-based probes.

Identification and characterisation of novel Mss4-binding Rab GTPases

2011 ◽  
Vol 392 (3) ◽  
Author(s):  
Viktor Wixler ◽  
Ludmilla Wixler ◽  
Anika Altenfeld ◽  
Stephan Ludwig ◽  
Roger S. Goody ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mutational Analysis ◽  
Binding Capacity ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Nucleotide Exchange ◽  
Binding Studies ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor

Abstract The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.

scholarly journals Cathepsin S Cleaves BAX as a Novel and Therapeutically Important Regulatory Mechanism for Apoptosis

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 339
Author(s):  
Surinder M. Soond ◽  
Lyudmila V. Savvateeva ◽  
Vladimir A. Makarov ◽  
Neonila V. Gorokhovets ◽  
Paul A. Townsend ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Mechanism ◽  
Renal Clear Cell Carcinoma ◽  
Cathepsin S ◽  
Apoptotic Pathway ◽  
Intact Cells ◽  
Unique Role ◽  
Bax Protein ◽  
Anti Cancer

Certain lysosomal cathepsin proteins have come into focus as being good candidates for therapeutic targeting, based on them being over-expressed in a variety of cancers and based on their regulation of the apoptotic pathway. Here, we report novel findings that highlight the ability of cathepsin S expression to be up-regulated under Paclitaxel-stimulatory conditions in kidney cell lines and it being able to cleave the apoptotic p21 BAX protein in intact cells and in vitro. Consistent with this, we demonstrate that this effect can be abrogated in vitro and in mammalian cells under conditions that utilize dominant-inhibitory cathepsin S expression, cathepsin S expression-knockdown and through the activity of a novel peptide inhibitor, CS-PEP1. Moreover, we report a unique role for cathepsin S in that it can cleave a polyubiquitinated-BAX protein intermediate and is a step that may contribute to down-regulating post-translationally-modified levels of BAX protein. Finally, CS-PEP1 may possess promising activity as a potential anti-cancer therapeutic against chemotherapeutic-resistant Renal Clear Cell Carcinoma kidney cancer cells and for combined uses with therapeutics such as Paclitaxel.

scholarly journals Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4

2005 ◽  
Vol 16 (10) ◽  
pp. 4648-4659 ◽  
Author(s):  
Brandon E. Kremer ◽  
Timothy Haystead ◽  
Ian G. Macara
Keyword(s):  
Mammalian Cells ◽  
Spectroscopic Analysis ◽  
Molecular Function ◽  
Rich Region ◽  
Intact Cells ◽  
Pronounced Increase ◽  
Binding Partner ◽  
Microtubule Stability ◽  
Proline Rich Region

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.

scholarly journals Molecular Basis for Rab Prenylation

2000 ◽  
Vol 150 (1) ◽  
pp. 89-104 ◽  
Author(s):  
Christelle Alory ◽  
William E. Balch
Keyword(s):  
Mammalian Cells ◽  
Normal Growth ◽  
Kinetic Properties ◽  
Temperature Sensitive ◽  
Rab Gtpases ◽  
Inherited Disease ◽  
Vesicular Traffic ◽  
Null Strain

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Δ null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Δ null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6ts) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

scholarly journals A role for Rab5 in structuring the endoplasmic reticulum

2007 ◽  
Vol 178 (1) ◽  
pp. 43-56 ◽  
Author(s):  
Anjon Audhya ◽  
Arshad Desai ◽  
Karen Oegema
Keyword(s):  
Endoplasmic Reticulum ◽  
Caenorhabditis Elegans ◽  
Nuclear Envelope ◽  
Rab Gtpases ◽  
C Elegans ◽  
Rab Family ◽  
Kinetics Of

The endoplasmic reticulum (ER) is a contiguous network of interconnected membrane sheets and tubules. The ER is differentiated into distinct domains, including the peripheral ER and nuclear envelope. Inhibition of two ER proteins, Rtn4a and DP1/NogoA, was previously shown to inhibit the formation of ER tubules in vitro. We show that the formation of ER tubules in vitro also requires a Rab family GTPase. Characterization of the 29 Caenorhabditis elegans Rab GTPases reveals that depletion of RAB-5 phenocopies the defects in peripheral ER structure that result from depletion of RET-1 and YOP-1, the C. elegans homologues of Rtn4a and DP1/NogoA. Perturbation of endocytosis by other means did not affect ER structure; the role of RAB-5 in ER morphology is thus independent of its well-studied requirement for endocytosis. RAB-5 and YOP-1/RET-1 also control the kinetics of nuclear envelope disassembly, which suggests an important role for the morphology of the peripheral ER in this process.

scholarly journals Proteasome pathway operates for the degradation of ornithine decarboxylase in intact cells

1996 ◽  
Vol 317 (1) ◽  
pp. 77-80 ◽  
Author(s):  
Yasuko MURAKAMI ◽  
Nobuyuki TANAHASHI ◽  
Keiji TANAKA ◽  
Satoshi ŌMURA ◽  
Shin-ichi HAYASHI
Keyword(s):  
Ornithine Decarboxylase ◽  
Proteasome Inhibitor ◽  
Mammalian Cells ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Intact Cells ◽  
Htc Cells ◽  
Proteasome Pathway ◽  
Hypertonic Shock

Ornithine decarboxylase (ODC) is degraded in an ATP-dependent manner in vitro by the 26 S proteasome in the presence of antizyme, an ODC destabilizing protein induced by polyamines. In the present study we examined whether the proteasome catalyses ODC degradation in living mammalian cells. Lactacystin, the most selective proteasome inhibitor, strongly inhibited the degradation of ODC that had been induced in hepatoma tissue-culture (HTC) cells by refeeding with fresh medium. Furthermore the inhibitor inhibited the rapid degradation of ODC that had been induced by hypotonic shock. Interestingly, hypertonic shock was found to increase the proportion of ODC present as a complex with antizyme (the ratio of ODC–antizyme complex to total ODC). Cycloheximide, which partly inhibits rapid ODC degradation caused by hypertonic shock, also partly inhibited the increase in the ratio of ODC–antizyme complex to total ODC. These results suggest that a common ODC degradation pathway, namely the antizyme-dependent and 26 S proteasome-catalysed ODC degradation pathway, is also operating in intact cells for osmoregulated ODC degradation.

scholarly journals Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

2013 ◽  
Vol 3 (1) ◽  
Author(s):  
Divyanshu Mahajan ◽  
Boon Kim Boh ◽  
Yan Zhou ◽  
Li Chen ◽  
Tobias Carl Cornvik ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Active Form ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Vitro Assay ◽  
Guanine Nucleotide Exchange ◽  
And Function ◽  
Golgi Network

Abstract Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.

scholarly journals Mimovirus: a Novel Form of Vaccine That Induces Hepatitis B Virus-Specific Cytotoxic T-Lymphocyte Responses In Vivo

2002 ◽  
Vol 76 (20) ◽  
pp. 10264-10269 ◽  
Author(s):  
Yu-Zhang Wu ◽  
Jian-Ping Zhao ◽  
Ying Wan ◽  
Zheng-Cai Jia ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Electrostatic Interactions ◽  
Cytotoxic T Lymphocyte ◽  
Interleukin 12 ◽  
Antigen Delivery ◽  
Intracellular Pathogens ◽  
Virus Size

ABSTRACT CD8+ cytotoxic T lymphocytes (CTLs) are now recognized as important mediators of immunity against intracellular pathogens, including human immunodeficiency virus and tumors. How to efficiently evoke antigen-specific CTL responses in vivo has become a crucial problem in the development of modern vaccines. Here, we developed a completely novel CTL vaccine—mimovirus, which is a kind of virus-size particulate antigen delivery system. It was formed by the self-assembly of a cationic peptide containing 18 lysines and a CTL-epitope peptide of HBsAg28-39, with a plasmid encoding mouse interleukin-12 (IL-12) through electrostatic interactions. We examined the formation of mimovirus by DNA retardation assay, DNase I protection assay, and transmission electron microscopy and demonstrated that mimovirus could efficiently transfer the plasmid encoding IL-12 into mammalian cells such as P815 cells in vitro. Furthermore, it was proved that mimovirus could induce an HBsAg28-39-specific CTL response in vivo. Considering its effectiveness, flexibility, and defined composition, mimovirus is potentially a novel system for vaccination against intracellular pathogens and tumors.

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