TRAPγ-CDG shows asymmetric glycosylation and an effect on processing of proteins required in higher organisms.

Newly synthesised glycoproteins enter the rough endoplasmic reticulum through a translocation pore. The translocon associated protein (TRAP) complex is located close to the pore. In a patient with a homozygous start codon variant in TRAPγ (SSR3), absence of TRAPγ causes disruption of the TRAP complex, impairs protein translocation into the endoplasmic reticulum and affects transport, for example, into the brush-border membrane. Furthermore, we observed an unbalanced non-occupancy of N-glycosylation sites. The major clinical features are intrauterine growth retardation, facial dysmorphism, congenital diarrhoea, failure to thrive, pulmonary disease and severe psychomotor disability.

The Human and Mouse Enteric Nervous System at Single-Cell Resolution.

The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease.

Novel mTORC1 Mechanism Suggests Therapeutic Targets for COMPopathies.

Cartilage oligomeric matrix protein (COMP) is a large, multifunctional extracellular protein that, when mutated, is retained in the rough endoplasmic reticulum (ER). This retention elicits ER stress, inflammation, and oxidative stress, resulting in dysfunction and death of growth plate chondrocytes. While identifying the cellular pathologic mechanisms underlying the murine mutant (MT)-COMP model of pseudoachondroplasia, increased midline-1 (MID1) expression and mammalian target of rapamycin complex 1 (mTORC1) signaling was found. This novel role for MID1/mTORC1 signaling was investigated since treatments shown to repress the pathology also reduced Mid1/mTORC1. Although ER stress-inducing drugs or tumor necrosis factor α (TNFα) in rat chondrosarcoma cells increased Mid1, oxidative stress did not, establishing that ER stress- or TNFα-driven inflammation alone is sufficient to elevate MID1 expression. Since MID1 ubiquitinates protein phosphatase 2A (PP2A), a negative regulator of mTORC1, PP2A was evaluated in MT-COMP growth plate chondrocytes. PP2A was decreased, indicating de-repression of mTORC1 signaling. Rapamycin treatment in MT-COMP mice reduced mTORC1 signaling and intracellular retention of COMP, and increased proliferation, but did not change inflammatory markers IL-16 and eosinophil peroxidase. Lastly, mRNA from tuberous sclerosis-1/2-null mice brain tissue exhibiting ER stress had increased Mid1 expression, confirming the relationship between ER stress and MID1/mTORC1 signaling. These findings suggest a mechanistic link between ER stress and MID1/mTORC1 signaling that has implications extending to other conditions involving ER stress.

Endometrial pinopode biomarkers: Molecules and microRNAs.

Ultrastructural changes on the apical surface of the luminal epithelium of the uterus are known as pinopodes. Their morphology in species and in special species is associated with different results about size, duration, and percentage of surface area covered by pinopodes. The content of pinopodes is different in rodents and humans. In mice and rats pinopodes have many vacuoles and no organelle that extends to the actin stalk above the microvilli. Human pinopodes do not have a large vacuole and contain the golgi complex, a rough endoplasmic reticulum, secretory vesicles, and mitochondria that extend from the entire cell surface. It has been suggested that pinopodes are good markers of endometrial receptivity and implantation window. There are several molecular markers related to the presence of pinopodes, including integrins, leukemia inhibiting factor (LIF), l-selectin, HOXA10, glutaredoxin, glycodelinA, heparin-binding epidermal growth factor, mucins, and microRNAs (miRNAs). Multiple lines of evidence have indicated that miRNAs could affect the expression of LIF and pinopodes in the endometrium and these molecules play key roles in implantation window processes. Here, we have summarized the morphology and function of pinopodes. Moreover, we have highlighted several molecules in relation to pinopodes that could be used as biomarkers.

Distinct mechanisms controlling rough and smooth endoplasmic reticulum contacts with mitochondria.

Gp78 (also known as AMFR), an endoplasmic-reticulum (ER)-associated protein degradation (ERAD) E3 ubiquitin ligase, localizes to mitochondria-associated ER and targets the mitofusin (Mfn1 and Mfn2) mitochondrial fusion proteins for degradation. Gp78 is also the cell surface receptor for autocrine motility factor (AMF), which prevents Gp78-dependent mitofusin degradation. Gp78 ubiquitin ligase activity promotes ER-mitochondria association and ER-mitochondria Ca(2+) coupling, processes that are reversed by AMF. Electron microscopy of HT-1080 fibrosarcoma cancer cells identified both smooth ER (SER; ∼8 nm) and wider (∼50-60 nm) rough ER (RER)-mitochondria contacts. Both short hairpin RNA (shRNA)-mediated knockdown of Gp78 (shGp78) and AMF treatment selectively reduced the extent of RER-mitochondria contacts without impacting on SER--mitochondria contacts. Concomitant small interfering RNA (siRNA)-mediated knockdown of Mfn1 increased SER-mitochondria contacts in both control and shGp78 cells, whereas knockdown of Mfn2 increased RER-mitochondria contacts selectively in shGp78 HT-1080 cells. The mitofusins therefore inhibit ER-mitochondria interaction. Regulation of close SER-mitochondria contacts by Mfn1 and of RER-mitochondria contacts by AMF-sensitive Gp78-mediated degradation of Mfn2 define new mechanisms that regulate ER-mitochondria interactions.

MicroRNAs in the intracellular space, regulation of organelle specific pathways in health and disease.

MicroRNAs (miRNA) are small (~22 nucleotide] non-coding RNA molecules originally characterised as nonsense or junk DNA. Emerging research suggests that these molecules have diverse regulatory roles in an array of molecular, cellular and physiological processes. MiRNAs are versatile and highly stable molecules, therefore, they are able to exist as intracellular or extracellular miRNAs. The purpose of this paper is to review the function and role of miRNAs in the intracellular space with specific focus on the interactions between miRNAs and organelles such as the mitochondria and the rough endoplasmic reticulum. Understanding the role of miRNAs in the intracellular space may be vital in understanding the mechanism of certain diseases.

An additional function of the rough endoplasmic reticulum protein complex prolyl 3-hydroxylase 1·cartilage-associated protein·cyclophilin B: the CXXXC motif reveals disulfide isomerase activity in vitro.

Collagen biosynthesis occurs in the rough endoplasmic reticulum, and many molecular chaperones and folding enzymes are involved in this process. The folding mechanism of type I procollagen has been well characterized, and protein disulfide isomerase (PDI) has been suggested as a key player in the formation of the correct disulfide bonds in the noncollagenous carboxyl-terminal and amino-terminal propeptides. Prolyl 3-hydroxylase 1 (P3H1) forms a hetero-trimeric complex with cartilage-associated protein and cyclophilin B (CypB). This complex is a multifunctional complex acting as a prolyl 3-hydroxylase, a peptidyl prolyl cis-trans isomerase, and a molecular chaperone. Two major domains are predicted from the primary sequence of P3H1: an amino-terminal domain and a carboxyl-terminal domain corresponding to the 2-oxoglutarate- and iron-dependent dioxygenase domains similar to the α-subunit of prolyl 4-hydroxylase and lysyl hydroxylases. The amino-terminal domain contains four CXXXC sequence repeats. The primary sequence of cartilage-associated protein is homologous to the amino-terminal domain of P3H1 and also contains four CXXXC sequence repeats. However, the function of the CXXXC sequence repeats is not known. Several publications have reported that short peptides containing a CXC or a CXXC sequence show oxido-reductase activity similar to PDI in vitro. We hypothesize that CXXXC motifs have oxido-reductase activity similar to the CXXC motif in PDI. We have tested the enzyme activities on model substrates in vitro using a GCRALCG peptide and the P3H1 complex. Our results suggest that this complex could function as a disulfide isomerase in the rough endoplasmic reticulum.

Multi-step down-regulation of the secretory pathway in mitosis: a fresh perspective on protein trafficking.

The secretory pathway delivers proteins synthesized at the rough endoplasmic reticulum (RER) to various subcellular locations via the Golgi apparatus. Currently, efforts are focused on understanding the molecular machineries driving individual processes at the RER and Golgi that package, modify and transport proteins. However, studies are routinely performed using non-dividing cells. This obscures the critical issue of how the secretory pathway is affected by cell division. Indeed, several studies have indicated that protein trafficking is down-regulated during mitosis. Moreover, the RER and Golgi apparatus exhibit gross reorganization in mitosis. Here I provide a relatively neglected perspective of how the mitotic cyclin-dependent kinase (CDK1) could regulate various stages of the secretory pathway. I highlight several aspects of the mitotic control of protein trafficking that remain unresolved and suggest that further studies on how the mitotic CDK1 influences the secretory pathway are necessary to obtain a deeper understanding of protein transport.

Understanding integration of α-helical membrane proteins: the next steps.

Integration of a protein into the endoplasmic reticulum (ER) membrane occurs through a series of multistep reactions that include targeting of ribosome-nascent polypeptide complexes to the ER, attachment of the ribosome to the protein translocation channel, lateral partitioning of α-helical transmembrane spans into the lipid bilayer, and folding of the lumenal, cytosolic and membrane-embedded domains of the protein. However, the molecular mechanisms and kinetics of these steps are still not entirely clear. To obtain a better understanding of the mechanism of membrane protein integration, we propose that it will be important to utilize in vivo experiments to examine the kinetics of membrane protein integration and in vitro experiments to characterize interactions between nascent membrane proteins, protein translocation factors and molecular chaperones.

The hydrophobic core of the Sec61 translocon defines the hydrophobicity threshold for membrane integration.

The Sec61 translocon mediates the translocation of proteins across the endoplasmic reticulum membrane and the lateral integration of transmembrane segments into the lipid bilayer. The structure of the idle translocon is closed by a lumenal plug domain and a hydrophobic constriction ring. To test the function of the apolar constriction, we have mutated all six ring residues of yeast Sec61p to more hydrophilic, bulky, or even charged amino acids (alanines, glycines, serines, tryptophans, lysines, or aspartates). The translocon was found to be surprisingly tolerant even to the charge mutations in the constriction ring, because growth and translocation efficiency were not drastically affected. Most interestingly, ring mutants were found to affect the integration of hydrophobic sequences into the lipid bilayer, indicating that the translocon does not simply catalyze the partitioning of potential transmembrane segments between an aqueous environment and the lipid bilayer but that it also plays an active role in setting the hydrophobicity threshold for membrane integration.

Loss of the Atp2c1 secretory pathway Ca(2+)-ATPase (SPCA1) in mice causes Golgi stress, apoptosis, and midgestational death in homozygous embryos and squamous cell tumors in adult heterozygotes.

Loss of one copy of the human ATP2C1 gene, encoding SPCA1 (secretory pathway Ca(2+)-ATPase isoform 1), causes Hailey-Hailey disease, a skin disorder. We performed targeted mutagenesis of the Atp2c1 gene in mice to analyze the functions of this Golgi membrane Ca(2+) pump. Breeding of heterozygous mutants yielded a normal Mendelian ratio among embryos on gestation day 9.5; however, null mutant (Spca1(-/-)) embryos exhibited growth retardation and did not survive beyond gestation day 10.5. Spca1(-/-) embryos had an open rostral neural tube, but hematopoiesis and cardiovascular development were ostensibly normal. Golgi membranes of Spca1(-/-) embryos were dilated, had fewer stacked leaflets, and were expanded in amount, consistent with increased Golgi biogenesis. The number of Golgi-associated vesicles was also increased, and rough endoplasmic reticulum had fewer ribosomes. Coated pits, junctional complexes, desmosomes, and basement membranes appeared normal in mutant embryos, indicating that processing and trafficking of proteins in the secretory pathway was not massively impaired. However, apoptosis was increased, possibly the result of secretory pathway stress, and a large increase in cytoplasmic lipid was observed in mutant embryos, consistent with impaired handling of lipid by the Golgi. Adult heterozygous mice appeared normal and exhibited no evidence of Hailey-Hailey disease; however, aged heterozygotes had an increased incidence of squamous cell tumors of keratinized epithelial cells of the skin and esophagus. These data show that loss of the Golgi Ca(2+) pump causes Golgi stress, expansion of the Golgi, increased apoptosis, and embryonic lethality and demonstrates that SPCA1 haploinsufficiency causes a genetic predisposition to cancer.