scholarly journals Secreted HHIP1 interacts with heparan sulfate and regulates Hedgehog ligand localization and function

2015 ◽  
Vol 209 (5) ◽  
pp. 739-758 ◽  
Author(s):  
Alexander M. Holtz ◽  
Samuel C. Griffiths ◽  
Samantha J. Davis ◽  
Benjamin Bishop ◽  
Christian Siebold ◽  
...  
Keyword(s):  
Heparan Sulfate ◽  
Molecular Mechanisms ◽  
Binding Motif ◽  
Interacting Protein ◽  
Rich Domain ◽  
Pathway Function ◽  
Cell Type Specific ◽  
Hh Signaling ◽  
Patched 1 ◽  
And Function

Vertebrate Hedgehog (HH) signaling is controlled by several ligand-binding antagonists including Patched-1 (PTCH1), PTCH2, and HH-interacting protein 1 (HHIP1), whose collective action is essential for proper HH pathway activity. However, the molecular mechanisms used by these inhibitors remain poorly understood. In this paper, we investigated the mechanisms underlying HHIP1 antagonism of HH signaling. Strikingly, we found evidence that HHIP1 non–cell-autonomously inhibits HH-dependent neural progenitor patterning and proliferation. Furthermore, this non–cell-autonomous antagonism of HH signaling results from the secretion of HHIP1 that is modulated by cell type–specific interactions with heparan sulfate (HS). These interactions are mediated by an HS-binding motif in the cysteine-rich domain of HHIP1 that is required for its localization to the neuroepithelial basement membrane (BM) to effectively antagonize HH pathway function. Our data also suggest that endogenous, secreted HHIP1 localization to HS-containing BMs regulates HH ligand distribution. Overall, the secreted activity of HHIP1 represents a novel mechanism to regulate HH ligand localization and function during embryogenesis.

scholarly journals CNPY4 inhibits the Hedgehog pathway by modulating membrane sterol lipids

2021 ◽  
Author(s):  
Megan Lo ◽  
Amnon Sharir ◽  
Michael D Paul ◽  
Hayarpi Torosyan ◽  
Christopher Agnew ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Primary Cilia ◽  
Molecular Mechanisms ◽  
Negative Regulator ◽  
Membrane Composition ◽  
Hh Signaling ◽  
Pathway Regulation ◽  
Patched 1 ◽  
Cancer Signal Transduction

The Hedgehog (HH) pathway is critical for development and adult tissue homeostasis. Aberrant HH signaling can cause congenital malformations, such as digit anomalies and holoprosencephaly, and other diseases, including cancer. Signal transduction is initiated by HH ligand binding to the Patched 1 (PTCH1) receptor on primary cilia, thereby releasing inhibition of Smoothened (SMO), a HH pathway activator. Although cholesterol and several oxysterol lipids, which are enriched in the ciliary membrane, play a crucial role in HH activation, the molecular mechanisms governing the regulation of these lipid molecules remain unresolved. Here, we identify Canopy 4 (CNPY4), a Saposin-like protein, as a regulator of the HH pathway that controls membrane sterol lipid levels. Cnpy4—/— embryos exhibit multiple defects consistent with HH signaling perturbations, most notably changes in digit number. Knockdown of Cnpy4 hyperactivates the HH pathway at the level of SMO in vitro, and elevates membrane levels of accessible sterol lipids such as cholesterol, an endogenous ligand involved in SMO activation. Thus, our data demonstrate that CNPY4 is a negative regulator that fine-tunes the initial steps of HH signal transduction, revealing a previously undescribed facet of HH pathway regulation that operates through control of membrane composition.

scholarly journals Cx50 requires an intact PDZ-binding motif and ZO-1 for the formation of functional intercellular channels

2011 ◽  
Vol 22 (23) ◽  
pp. 4503-4512 ◽  
Author(s):  
Zhifang Chai ◽  
Daniel A. Goodenough ◽  
David L. Paul
Keyword(s):  
Gap Junction ◽  
Hela Cells ◽  
Pdz Domain ◽  
Cell Communication ◽  
Normal Function ◽  
Scaffolding Protein ◽  
Binding Motif ◽  
Interacting Protein ◽  
And Function

The three connexins expressed in the ocular lens each contain PDZ domain–binding motifs directing a physical association with the scaffolding protein ZO-1, but the significance of the interaction is unknown. We found that Cx50 with PDZ-binding motif mutations did not form gap junction plaques or induce cell–cell communication in HeLa cells, whereas the addition of a seven–amino acid PDZ-binding motif restored normal function to Cx50 lacking its entire C-terminal cytoplasmic domain. C-Terminal deletion had a similar although weaker effect on Cx46 but little if any effect on targeting and function of Cx43. Furthermore, small interfering RNA knockdown of ZO-1 completely inhibited the formation of gap junctions by wild-type Cx50 in HeLa cells. Thus both a PDZ-binding motif and ZO-1 are necessary for Cx50 intercellular channel formation in HeLa cells. Knock-in mice expressing Cx50 with a PDZ-binding motif mutation phenocopied Cx50 knockouts. Furthermore, differentiating lens fibers in the knock-in displayed extensive intracellular Cx50, whereas plaques in mature fibers contained only Cx46. Thus normal Cx50 function in vivo also requires an intact PDZ domain–binding motif. This is the first demonstration of a connexin-specific requirement for a connexin-interacting protein in gap junction assembly.

scholarly journals Glypican-5 stimulates rhabdomyosarcoma cell proliferation by activating Hedgehog signaling

2011 ◽  
Vol 192 (4) ◽  
pp. 691-704 ◽  
Author(s):  
Fuchuan Li ◽  
Wen Shi ◽  
Mariana Capurro ◽  
Jorge Filmus
Keyword(s):  
Cell Proliferation ◽  
Heparan Sulfate ◽  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Core Protein ◽  
Stimulatory Activity ◽  
Rhabdomyosarcoma Cell ◽  
Hh Signaling ◽  
Patched 1

Glypican-5 (GPC5) is one of the six members of the glypican family. It has been previously reported that GPC5 stimulates the proliferation of rhabdomyosarcoma cells. In this study, we show that this stimulatory activity of GPC5 is a result of its ability to promote Hedgehog (Hh) signaling. We have previously shown that GPC3, another member of the glypican family, inhibits Hh signaling by competing with Patched 1 (Ptc1) for Hh binding. Furthermore, we showed that GPC3 binds to Hh through its core protein but not to Ptc1. In this paper, we demonstrate that GPC5 increases the binding of Sonic Hh to Ptc1. We also show that GPC5 binds to both Hh and Ptc1 through its glycosaminoglycan chains and that, unlike GPC3, GPC5 localizes to the primary cilia. Interestingly, we found that the heparan sulfate chains of GPC5 display a significantly higher degree of sulfation than those of GPC3. Based on these results, we propose that GPC5 stimulates Hh signaling by facilitating/stabilizing the interaction between Hh and Ptc1.

Interactome Analysis of the Differentially Expressed Proteins in Uterine Leiomyoma

2019 ◽  
Vol 19 (10) ◽  
pp. 1293-1312 ◽  
Author(s):  
Tahreem Sahar ◽  
Aruna Nigam ◽  
Shadab Anjum ◽  
Farheen Waziri ◽  
Shipie Biswas ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Uterine Leiomyoma ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Differentially Expressed ◽  
Binding Motif ◽  
Differentially Expressed Proteins ◽  
Protein Patterns ◽  
Interacting Protein ◽  
Fatty Acid Binding

Background: Recent advances in proteomics present enormous opportunities to discover proteome related disparities and thus understanding the molecular mechanisms related to a disease. Uterine leiomyoma is a benign monoclonal tumor, located in the pelvic region, and affecting 40% of reproductive aged female. Objective: Identification and characterization of the differentially expressed proteins associated with leiomyogenesis by comparing uterine leiomyoma and normal myometrium. Methods: Paired samples of uterine leiomyoma and adjacent myometrium retrieved from twenty-five females suffering from uterine leiomyoma (n=50) were submitted to two-dimensional electrophoresis (2-DE), matrixassisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and to reverse transcription polymerase chain reaction (RT-PCR). Results: Comparison of protein patterns revealed seven proteins with concordantly increased spot intensities in leiomyoma samples. E3 ubiquitin-protein ligase MIB2 (MIB2), Mediator of RNA polymerase II transcription subunit 10 (MED10), HIRA-interacting protein (HIRP3) and Fatty acid binding protein brain (FABP7) were found to be upregulated. While, Biogenesis of lysosome-related organelles complex 1 subunit 2 (BL1S2), Shadow of prion protein (SPRN) and RNA binding motif protein X linked like 2 (RMXL2) were found to be exclusively present in leiomyoma sample. The expression modulations of the corresponding genes were further validated which corroborated with the 2-DE result showing significant upregulation in leiomyoma. We have generated a master network showing the interactions of the experimentally identified proteins with their close neighbors and further scrutinized the network to prioritize the routes leading to cell proliferation and tumorigenesis. Conclusion: This study highlights the importance of identified proteins as potential targets for therapeutic purpose. This work provides an insight into the mechanism underlying the overexpression of the proteins but warrants further investigations.

scholarly journals A Novel Deoxynivalenol-Activated Wheat Arl6ip4 Gene Encodes an Antifungal Peptide with Deoxynivalenol Affinity and Protects Plants against Fusarium Pathogens and Mycotoxins

2021 ◽  
Vol 7 (11) ◽  
pp. 941
Author(s):  
Gang Liu ◽  
Dong-Yun Zuo ◽  
Peng Yang ◽  
Wei-Jie He ◽  
Zheng Yang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fusarium Graminearum ◽  
Molecular Mechanisms ◽  
Subtractive Hybridization ◽  
Vital Role ◽  
Binding Motif ◽  
Suspension Cells ◽  
Multiple Sequence ◽  
Interacting Protein ◽  
Cereal Products

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium–wheat interactions are not yet well understood. In this study, a novel wheat ADP-ribosylation factor-like protein 6-interacting protein 4 gene, TaArl6ip4, was identified from DON-treated wheat suspension cells by suppression subtractive hybridization (SSH). The qRT-PCR result suggested that TaArl6ip4 expression is specifically activated by DON in both the Fusarium intermediate susceptible wheat cultivar Zhengmai9023 and the Fusarium resistant cultivar Sumai3. The transient expression results of the TaARL6IP4::GFP fusion protein indicate that TaArl6ip4 encodes a plasma membrane and nucleus-localized protein. Multiple sequence alignment using microscale thermophoresis showed that TaARL6IP4 comprises a conserved DON binding motif, 67HXXXG71, and exhibits DON affinity with a dissociation constant (KD) of 91 ± 2.6 µM. Moreover, TaARL6IP4 exhibited antifungal activity with IC50 values of 22 ± 1.5 µM and 25 ± 2.6 µM against Fusarium graminearum and Alternaria alternata, respectively. Furthermore, TaArl6ip4 interacted with the plasma membrane of Fusarium graminearum spores, resulting in membrane disruption and the leakage of cytoplasmic materials. The heterologous over-expression of TaArl6ip4 conferred greater DON tolerance and Fusarium resistance in Arabidopsis. Finally, we describe a novel DON-induced wheat gene, TaArl6ip4, exhibiting antifungal function and DON affinity that may play a key role in Fusarium–wheat interactions.

scholarly journals Identification of Elongation Factor-2 as a Novel Regulator of Mitochondrial Fission

2021 ◽  
Author(s):  
Jinhwan Kim ◽  
Yan Cheng ◽  
Yanfeng Li ◽  
Yi Zhang ◽  
Ji Cheng ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Critical Role ◽  
Elongation Factor ◽  
Binding Motif ◽  
Gtpase Activity ◽  
Elongation Factor 2 ◽  
Gtp Binding ◽  
And Function

Abstract Mitochondria continuously undergo morphologically dynamic processes of fusion and fission to maintain their size, shape, amount, and function; yet the precise molecular mechanisms by which mitochondrial dynamics is regulated remain to be fully elucidated. Here, we report a previous unappreciated but critical role of eukaryotic elongation factor 2 (eEF2) in regulating mitochondrial fission. eEF2, a G-protein superfamily member encoded by EEF2 gene in human, has long been appreciated as a promoter of the GTP-dependent translocation of the ribosome during protein synthesis. We found unexpectedly in several types of cells that eEF2 was not only present in the cytosol but also in the mitochondria. Furthermore, we showed that mitochondrial length was significantly increased when the cells were subjected to silencing of eEF2 expression, suggesting a promotive role for eEF2 in the mitochondrial fission. Inversely, overexpression of eEF2 decreased mitochondrial length, suggesting an increase of mitochondrial fission. Inhibition of mitochondrial fission caused by eEF2 depletion was accompanied by alterations of cellular metabolism, as evidenced by a reduction of oxygen consumption and an increase of oxidative stress in the mitochondria. We further demonstrated that eEF2 and Drp1, a key driver of mitochondrial fission, co-localized at the mitochondria, as evidenced by microscopic observation, co-immunoprecipitation, and GST pulldown assay. Deletion of the GTP binding motif of eEF2 decreased its association with Drp1 and abrogated its effect on mitochondria fission. Moreover, we showed that wild-type eEF2 stimulated GTPase activity of Drp1, whereas deletion of the GTP binding site of eEF2 diminished its stimulatory effect on GTPase activity. This work not only reveals a previously unrecognized function of eEF2 (i.e., promoting mitochondrial fission), but also uncovers the interaction of eEF2 with Drp1 as a novel regulatory mechanism of the mitochondrial dynamics. Therefore, eEF2 warrants further exploration for its potential as a therapeutic target for the mitochondria-related diseases.

scholarly journals Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Morgane Rosendale ◽  
Thi Nhu Ngoc Van ◽  
Dolors Grillo-Bosch ◽  
Silvia Sposini ◽  
Léa Claverie ◽  
...  
Keyword(s):  
Binding Motif ◽  
Concerted Action ◽  
Sh3 Domains ◽  
Knock Out ◽  
Rich Domain ◽  
Single Motif ◽  
Src Homology ◽  
Domain 3 ◽  
And Function

Abstract During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin’s efficient recruitment and function. First, we show that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, partially rescue CME in dynamin triple knock-out cells. However, mutating two motifs largely prevents that ability. Furthermore, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME much more effectively than with monovalent ones. We conclude that dynamin drives vesicle scission via multivalent interactions in cells.

scholarly journals The Enigmatic HOX Genes: Can We Crack Their Code?

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 323 ◽  
Author(s):  
Zhifei Luo ◽  
Suhn Rhie ◽  
Peggy Farnham
Keyword(s):  
Transcription Factors ◽  
Embryonic Development ◽  
Molecular Mechanisms ◽  
Hox Genes ◽  
Homeobox Genes ◽  
Large Family ◽  
Cell Types ◽  
Interacting Protein ◽  
Developmental Problems ◽  
And Function

Homeobox genes (HOX) are a large family of transcription factors that direct the formation of many body structures during early embryonic development. There are 39 genes in the subgroup of homeobox genes that constitute the human HOX gene family. Correct embryonic development of flies and vertebrates is, in part, mediated by the unique and highly regulated expression pattern of the HOX genes. Disruptions in these fine-tuned regulatory mechanisms can lead to developmental problems and to human diseases such as cancer. Unfortunately, the molecular mechanisms of action of the HOX family of transcription factors are severely under-studied, likely due to idiosyncratic details of their structure, expression, and function. We suggest that a concerted and collaborative effort to identify interacting protein partners, produce genome-wide binding profiles, and develop HOX network inhibitors in a variety of human cell types will lead to a deeper understanding of human development and disease. Within, we review the technological challenges and possible approaches needed to achieve this goal.

scholarly journals Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

2018 ◽  
Author(s):  
Morgane Rosendale ◽  
Thi Nhu Ngoc Van ◽  
Dolors Grillo-Bosch ◽  
Silvia Sposini ◽  
Léa Claverie ◽  
...  
Keyword(s):  
Binding Motif ◽  
Knock Out ◽  
Rich Domain ◽  
Single Motif ◽  
Src Homology ◽  
Domain 3 ◽  
And Function ◽  
Kinetics Of

AbstractDuring clathrin mediated endocytosis (CME), membrane scission is achieved by the concerted action of dynamin and its interacting partners. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin’s efficient recruitment and function. First, we show in dynamin triple knock-out cells that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, are partially capable of rescuing CME. However, mutating two motifs largely prevents that ability. To support this observation, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent onesin vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME 2 to 3 times more effectively than with monovalent ones. Finally, the frequency of endocytic events decreases with competing peptides or hypomorphic rescue mutants but the kinetics of dynamin recruitment is unaffected. This suggests that PRD-SH3 interactions act upstream of dynamin accumulation at the neck of nascent vesicles. We conclude from these data that dynamin drives vesicle scissionviamultivalent interactionsin vivo.

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