scholarly journals Seipin traps triacylglycerols to facilitate their nanoscale clustering in the ER membrane

2020 ◽  
Author(s):  
Xavier Prasanna ◽  
Veijo T. Salo ◽  
Shiqian Li ◽  
Katharina Ven ◽  
Helena Vihinen ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
In Silico ◽  
Lipid Membrane ◽  
Wild Type ◽  
Binding Interface ◽  
Biomolecular Simulations ◽  
Hydrophobic Helices ◽  
Tag Clustering ◽  
Er Membrane ◽  
In Cells

AbstractSeipin is a disk-like oligomeric ER protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While seipin-S166D mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

scholarly journals Seipin traps triacylglycerols to facilitate their nanoscale clustering in the endoplasmic reticulum membrane

PLoS Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
pp. e3000998
Author(s):  
Xavier Prasanna ◽  
Veijo T. Salo ◽  
Shiqian Li ◽  
Katharina Ven ◽  
Helena Vihinen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplet ◽  
In Silico ◽  
Lipid Membrane ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
Binding Interface ◽  
Biomolecular Simulations ◽  
Hydrophobic Helices ◽  
Tag Clustering

Seipin is a disk-like oligomeric endoplasmic reticulum (ER) protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While the S166D-seipin mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

scholarly journals Insulin activates p21Ras and guanine nucleotide releasing factor in cells expressing wild type and mutant insulin receptors.

1993 ◽  
Vol 268 (27) ◽  
pp. 19998-20001
Author(s):  
B Draznin ◽  
L Chang ◽  
J.W. Leitner ◽  
Y Takata ◽  
J.M. Olefsky
Keyword(s):  
Insulin Receptors ◽  
Guanine Nucleotide ◽  
Wild Type ◽  
Mutant Insulin ◽  
In Cells

Overexpression of cysteine dioxygenase reduces intracellular cysteine and glutathione pools in HepG2/C3A cells

2007 ◽  
Vol 293 (1) ◽  
pp. E62-E69 ◽  
Author(s):  
John E. Dominy ◽  
Jesse Hwang ◽  
Martha H. Stipanuk
Keyword(s):  
Steady State ◽  
Experimental Evidence ◽  
Cysteine Dioxygenase ◽  
Wild Type ◽  
Hepatic Expression ◽  
Direct Experimental Evidence ◽  
Homeostatic Response ◽  
Catabolic Enzyme ◽  
In Cells

Cysteine levels are carefully regulated in mammals to balance metabolic needs against the potential for cytotoxicity. It has been postulated that one of the major regulators of intracellular cysteine levels in mammals is cysteine dioxygenase (CDO). Hepatic expression of this catabolic enzyme increases dramatically in response to increased cysteine availability and may therefore be part of a homeostatic response to shunt excess toxic cysteine to more benign metabolites such as sulfate or taurine. Direct experimental evidence, however, is lacking to support the hypothesis that CDO is capable of altering steady-state intracellular cysteine levels. In this study, we expressed either the wild-type (WT) or a catalytically inactivated mutant (H86A) isoform of CDO in HepG2/C3A cells (which do not express endogenous CDO protein) and cultured them in different concentrations of extracellular cysteine. WT CDO, but not H86A CDO, was capable of reducing intracellular cysteine levels in cells incubated in physiologically relevant concentrations of cysteine. WT CDO also decreased the glutathione pool and potentiated the toxicity of CdCl2. These results demonstrate that CDO is capable of altering intracellular cysteine levels as well as glutathione levels.

scholarly journals DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

1994 ◽  
Vol 125 (3) ◽  
pp. 625-638 ◽  
Author(s):  
J Lukas ◽  
H Müller ◽  
J Bartkova ◽  
D Spitkovsky ◽  
A A Kjerulff ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Complex Formation ◽  
Cyclin D1 ◽  
Cell Lines ◽  
T Antigen ◽  
Regulatory Function ◽  
Retinoblastoma Gene ◽  
Checkpoint Control ◽  
Wild Type ◽  
In Cells

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

scholarly journals Stage- and ribosome-specific alterations in nascent chain-Sec61p interactions accompany translocation across the ER membrane.

1995 ◽  
Vol 129 (4) ◽  
pp. 957-970 ◽  
Author(s):  
C V Nicchitta ◽  
E C Murphy ◽  
R Haynes ◽  
G S Shelness
Keyword(s):  
Protein Translocation ◽  
Signal Sequence ◽  
Near Neighbor ◽  
Cross Linking ◽  
Dramatic Reduction ◽  
Wild Type ◽  
Early Stages ◽  
Nascent Chain ◽  
Chemical Cross Linking ◽  
Er Membrane

Near-neighbor interactions between translocating nascent chains and Sec61p were investigated by chemical cross-linking. At stages of translocation before signal sequence cleavage, nascent chains could be cross-linked to Sec61p at high (60-80%) efficiencies. Cross-linking occurred through the signal sequence and the mature portion of wild-type and signal cleavage mutant nascent chains. At later stages of translocation, as represented through truncated translocation intermediates, cross-linking to Sec61p was markedly reduced. Dissociation of the ribosome into its large and small subunits after assembly of the precursor into the translocon, but before cross-linking, resulted in a dramatic reduction in subsequent cross-linking yield, indicating that at early stages of translocation, nascent chain-Sec61p interactions are in part mediated through interactions of the ribosome with components of the ER membrane, such as Sec61p. Dissociation of the ribosome was, however, without effect on subsequent translocation. These results are discussed with respect to a model in which Sec61p performs a function essential for the initiation of protein translocation.

scholarly journals Synthetic Derivatives against Wild-Type and Non-Wild-Type Sporothrix brasiliensis: In Vitro and In Silico Analyses

2022 ◽  
Vol 15 (1) ◽  
pp. 55
Author(s):  
Lais Cavalcanti dos Santos Velasco de Souza ◽  
Lucas Martins Alcântara ◽  
Pãmella Antunes de Macêdo-Sales ◽  
Nathália Faria Reis ◽  
Débora Sena de Oliveira ◽  
...  
Keyword(s):  
In Silico ◽  
Wide Distribution ◽  
Wild Type ◽  
Sporothrix Brasiliensis ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Therapeutic Alternatives ◽  
Synthetic Derivatives ◽  
Prevalent Species

Recently, the well-known geographically wide distribution of sporotrichosis in Brazil, combined with the difficulties of effective domestic feline treatment, has emphasized the pressing need for new therapeutic alternatives. This work considers a range of synthetic derivatives as potential antifungals against Sporothrix brasiliensis isolated from cats from the hyperendemic Brazilian region. Six S. brasiliensis isolates from the sporotrichotic lesions of itraconazole responsive or non-responsive domestic cats were studied. The minimum inhibitory concentrations (MICs) of three novel hydrazone derivatives and eleven novel quinone derivatives were determined using the broth microdilution method (M38-A2). In silico tests were also used to predict the pharmacological profile and toxicity parameters of these synthetic derivatives. MICs and MFCs ranged from 1 to >128 µg/mL. The ADMET computational analysis failed to detect toxicity while a good pharmacological predictive profile, with parameters similar to itraconazole, was obtained. Three hydrazone derivatives were particularly promising candidates as antifungal agents against itraconazole-resistant S. brasiliensis from the Brazilian hyperendemic region. Since sporotrichosis is a neglected zoonosis currently spreading in Latin America, particularly in Brazil, the present data can contribute to its future control by alternative antifungal drug design against S. brasiliensis, the most virulent and prevalent species of the hyperendemic context.

scholarly journals Deciphering the evolution of microbial interactions: in silico studies of two-member microbial communities

2022 ◽  
Author(s):  
Gayathri Sambamoorthy ◽  
Karthik Raman
Keyword(s):  
Microbial Communities ◽  
In Silico ◽  
Microbial Interactions ◽  
Interaction Patterns ◽  
Wild Type ◽  
Community Function ◽  
Evolutionary Forces ◽  
In Silico Studies ◽  
Wide Range ◽  
In The Wild

Microbes thrive in communities, embedded in a complex web of interactions. These interactions, particularly metabolic interactions, play a crucial role in maintaining the community structure and function. As the organisms thrive and evolve, a variety of evolutionary processes alter the interactions among the organisms in the community, although the community function remains intact. In this work, we simulate the evolution of two-member microbial communities in silico to study how evolutionary forces can shape the interactions between organisms. We employ genomescale metabolic models of organisms from the human gut, which exhibit a range of interaction patterns, from mutualism to parasitism. We observe that the evolution of microbial interactions varies depending upon the starting interaction and also on the metabolic capabilities of the organisms in the community. We find that evolutionary constraints play a significant role in shaping the dependencies of organisms in the community. Evolution of microbial communities yields fitness benefits in only a small fraction of the communities, and is also dependent on the interaction type of the wild-type communities. The metabolites cross-fed in the wild-type communities appear in only less than 50% of the evolved communities. A wide range of new metabolites are cross-fed as the communities evolve. Further, the dynamics of microbial interactions are not specific to the interaction of the wild-type community but vary depending on the organisms present in the community. Our approach of evolving microbial communities in silico provides an exciting glimpse of the dynamics of microbial interactions and offers several avenues for future investigations.

scholarly journals The Potential for SARS-CoV-2 to Evade Both Natural and Vaccine-induced Immunity

2020 ◽  
Author(s):  
Emily Shang ◽  
Paul Axelsen
Keyword(s):  
Human Infection ◽  
Antibody Binding ◽  
Spike Protein ◽  
Wild Type ◽  
Binding Interface ◽  
Naturally Occurring ◽  
Binding Interfaces ◽  
Induced Immunity ◽  
Wild Type Form

SARS-CoV-2 attaches to the surface of susceptible cells through extensive interactions between the receptor binding domain (RBD) of its spike protein and angiotensin converting enzyme type 2 (ACE2) anchored in cell membranes. To investigate whether naturally occurring mutations in the spike protein are able to prevent antibody binding, yet while maintaining the ability to bind ACE2 and viral infectivity, mutations in the spike protein identified in cases of human infection were mapped to the crystallographically-determined interfaces between the spike protein and ACE2 (PDB entry 6M0J), antibody CC12.1 (PDB entry 6XC2), and antibody P2B-2F6 (PDB entry 7BWJ). Both antibody binding interfaces partially overlap with the ACE2 binding interface. Among 16 mutations that map to the RBD:CC12.1 interface, 11 are likely to disrupt CC12.1 binding but not ACE2 binding. Among 12 mutations that map to the RBD:P2B-2F6 interface, 8 are likely to disrupt P2B-2F6 binding but not ACE2 binding. As expected, none of the mutations observed to date appear likely to disrupt the RBD:ACE2 interface. We conclude that SARS-CoV-2 with mutated forms of the spike protein may retain the ability to bind ACE2 while evading recognition by antibodies that arise in response to the original wild-type form of the spike protein. It seems likely that immune evasion will be possible regardless of whether the spike protein was encountered in the form of infectious virus, or as the immunogen in a vaccine. Therefore, it also seems likely that reinfection with a variant strain of SARS-CoV-2 may occur among people who recover from Covid-19, and that vaccines with the ability to generate antibodies against multiple variant forms of the spike protein will be necessary to protect against variant forms of SARS-CoV-2 that are already circulating in the human population.

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