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1.
J Biol Chem ; 299(12): 105450, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37949225

ABSTRACT

Protein folding, quality control, maturation, and trafficking are essential processes for proper cellular homeostasis. Around one-third of the human proteome is targeted to the endoplasmic reticulum (ER), the organelle that serves as entrance into the secretory pathway. Successful protein trafficking is paramount for proper cellular function and to that end there are many ER resident proteins that ensure efficient secretion. Here, biochemical and cell biological analysis was used to determine that TTC17 is a large, soluble, ER-localized protein that plays an important role in secretory trafficking. Transcriptional analysis identified the predominantly expressed protein isoform of TTC17 in various cell lines. Further, TTC17 localizes to the ER and interacts with a wide variety of chaperones and cochaperones normally associated with ER protein folding, quality control, and maturation processes. TTC17 was found to be significantly upregulated by ER stress and through the creation and use of TTC17-/- cell lines, quantitative mass spectrometry identified secretory pathway wide trafficking defects in the absence of TTC17. Notably, trafficking of insulin-like growth factor type 1 receptor, glycoprotein nonmetastatic melanoma protein B, clusterin, and UDP-glucose:glycoprotein glucosyltransferase 1 were significantly altered in H4 neuroglioma cells. This study defines a novel ER trafficking factor and provides insight into the protein-protein assisted trafficking in the early secretory pathway.


Subject(s)
Endoplasmic Reticulum Stress , Protein Folding , Humans , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Glycoproteins/metabolism , Cell Line
2.
Mol Biol Cell ; 31(3): 167-183, 2020 02 01.
Article in English | MEDLINE | ID: mdl-31851597

ABSTRACT

Protein glycosylation plays essential roles in protein structure, stability, and activity such as cell adhesion. The cadherin superfamily of adhesion molecules carry O-linked mannose glycans at conserved sites and it was recently demonstrated that the transmembrane and tetratricopeptide repeat-containing proteins 1-4 (TMTC1-4) gene products contribute to the addition of these O-linked mannoses. Here, biochemical, cell biological, and organismal analysis was used to determine that TMTC3 supports the O-mannosylation of E-cadherin, cellular adhesion, and embryonic gastrulation. Using genetically engineered cells lacking all four TMTC genes, overexpression of TMTC3 rescued O-linked glycosylation of E-cadherin and cell adherence. The knockdown of the Tmtcs in Xenopus laevis embryos caused a delay in gastrulation that was rescued by the addition of human TMTC3. Mutations in TMTC3 have been linked to neuronal cell migration diseases including Cobblestone lissencephaly. Analysis of TMTC3 mutations associated with Cobblestone lissencephaly found that three of the variants exhibit reduced stability and missence mutations were unable to complement TMTC3 rescue of gastrulation in Xenopus embryo development. Our study demonstrates that TMTC3 regulates O-linked glycosylation and cadherin-mediated adherence, providing insight into its effect on cellular adherence and migration, as well the basis of TMTC3-associated Cobblestone lissencephaly.


Subject(s)
Cadherins/metabolism , Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolism , Animals , COS Cells , Carrier Proteins/genetics , Cell Adhesion/physiology , Cell Adhesion Molecules/metabolism , Chlorocebus aethiops , Gastrulation/physiology , Glycosylation , HEK293 Cells , Humans , Mannose/metabolism , Membrane Proteins/genetics , Mutation , Neurons/cytology , Neurons/metabolism , Xenopus laevis
3.
Crit Rev Biochem Mol Biol ; 54(2): 103-118, 2019 04.
Article in English | MEDLINE | ID: mdl-31023093

ABSTRACT

The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into a number of functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein-protein interactions, supporting interactions with a wide range of ligands or substrates. Seven TPR-containing proteins have thus far been shown to localize to the ER and control protein organization and homeostasis within this multifunctional organelle. Here, we discuss the roles of these proteins in controlling ER processes and organization. The crucial roles that TPR-containing proteins play in the ER are highlighted by diseases or defects associated with their mutation or disruption.


Subject(s)
Endoplasmic Reticulum/metabolism , Proteins/metabolism , Proteostasis , Tetratricopeptide Repeat , Animals , Calcium/metabolism , Humans , Models, Molecular , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Protein Interaction Maps , Protein Transport , Proteins/chemistry
4.
Traffic ; 17(4): 308-26, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26676362

ABSTRACT

Helenius and colleagues proposed over 20-years ago a paradigm-shifting model for how chaperone binding in the endoplasmic reticulum was mediated and controlled for a new type of molecular chaperone- the carbohydrate-binding chaperones, calnexin and calreticulin. While the originally established basics for this lectin chaperone binding cycle holds true today, there has been a number of important advances that have expanded our understanding of its mechanisms of action, role in protein homeostasis, and its connection to disease states that are highlighted in this review.


Subject(s)
Calnexin/metabolism , Endoplasmic Reticulum/metabolism , Molecular Chaperones/metabolism , Polysaccharides/metabolism , Unfolded Protein Response , Animals , Humans , Protein Binding
5.
J Biol Chem ; 289(23): 16085-99, 2014 Jun 06.
Article in English | MEDLINE | ID: mdl-24764305

ABSTRACT

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.


Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , Calcium/metabolism , Carrier Proteins/physiology , Endoplasmic Reticulum/metabolism , Homeostasis , Membrane Proteins/physiology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Base Sequence , COS Cells , Carrier Proteins/genetics , Carrier Proteins/metabolism , Chlorocebus aethiops , Cytoplasm/metabolism , DNA Primers , DNA, Complementary , HEK293 Cells , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Real-Time Polymerase Chain Reaction
6.
Nature ; 473(7346): 226-9, 2011 May 12.
Article in English | MEDLINE | ID: mdl-21562563

ABSTRACT

Dietary restriction is a robust means of extending adult lifespan and postponing age-related disease in many species, including yeast, nematode worms, flies and rodents. Studies of the genetic requirements for lifespan extension by dietary restriction in the nematode Caenorhabditis elegans have implicated a number of key molecules in this process, including the nutrient-sensing target of rapamycin (TOR) pathway and the Foxa transcription factor PHA-4 (ref. 7). However, little is known about the metabolic signals that coordinate the organismal response to dietary restriction and maintain homeostasis when nutrients are limited. The endocannabinoid system is an excellent candidate for such a role given its involvement in regulating nutrient intake and energy balance. Despite this, a direct role for endocannabinoid signalling in dietary restriction or lifespan determination has yet to be demonstrated, in part due to the apparent absence of endocannabinoid signalling pathways in model organisms that are amenable to lifespan analysis. N-acylethanolamines (NAEs) are lipid-derived signalling molecules, which include the mammalian endocannabinoid arachidonoyl ethanolamide. Here we identify NAEs in C. elegans, show that NAE abundance is reduced under dietary restriction and that NAE deficiency is sufficient to extend lifespan through a dietary restriction mechanism requiring PHA-4. Conversely, dietary supplementation with the nematode NAE eicosapentaenoyl ethanolamide not only inhibits dietary-restriction-induced lifespan extension in wild-type worms, but also suppresses lifespan extension in a TOR pathway mutant. This demonstrates a role for NAE signalling in ageing and indicates that NAEs represent a signal that coordinates nutrient status with metabolic changes that ultimately determine lifespan.


Subject(s)
Caenorhabditis elegans/physiology , Diet , Ethanolamines/metabolism , Longevity/physiology , Signal Transduction , Amides/pharmacology , Amidohydrolases/metabolism , Animals , Caenorhabditis elegans/drug effects , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Caloric Restriction , Gene Expression Regulation, Developmental , Longevity/drug effects , Mutation , Trans-Activators/metabolism
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