An interaction map of endoplasmic reticulum chaperones and foldases

Chaperones and foldases in the endoplasmic reticulum (ER) ensure correct protein folding. Extensive protein-protein interaction maps have defined the organization and function of many cellular complexes, but ER complexes are under-represented. Consequently, chaperone and foldase networks in the ER are largely uncharacterized. Using complementary ER-specific methods, we have mapped interactions between ER-lumenal chaperones and foldases and describe their organization in multiprotein complexes. We identify new functional chaperone modules, including interactions between protein-disulfide isomerases and peptidyl-prolyl cis-trans-isomerases. We have examined in detail a novel ERp72-cyclophilin B complex that enhances the rate of folding of immunoglobulin G. Deletion analysis and NMR reveal a conserved surface of cyclophilin B that interacts with polyacidic stretches of ERp72 and GRp94. Mutagenesis within this highly charged surface region abrogates interactions with its chaperone partners and reveals a new mechanism of ER protein-protein interaction. This ability of cyclophilin B to interact with different partners using the same molecular surface suggests that ER-chaperone/foldase partnerships may switch depending on the needs of different substrates, illustrating the flexibility of multichaperone complexes of the ER folding machinery.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Proteomic analysis during larval development and metamorphosis of the spionid polychaete Pseudopolydora vexillosa

Background  

While the larval-juvenile transition (metamorphosis) in the spionid polychaete Pseudopolydora vexillosa involves gradual morphological changes and does not require substantial development of juvenile organs, the opposite occurs in the barnacle Balanus amphitrite. We hypothesized that the proteome changes during metamorphosis in the spionids are less drastic than that in the barnacles. To test this, proteomes of pre-competent larvae, competent larvae (ready to metamorphose), and juveniles of P. vexillosa were compared using 2-dimensional gel electrophoresis (2-DE), and they were then compared to those of the barnacle.     

Quantitative proteomics reveals differentially expressed proteins in murine preneoplastic intestine in a model of intestinal tumorigenesis induced by low dietary folate and MTHFR deficiency

Colorectal cancer risk is increased when dietary folate intake is low, with or without a deficiency in methylenetetrahydrofolate reductase (MTHFR). We have observed that intestinal tumors are induced in mice fed low‐folate diets, and that tumor incidence is increased when these mice also have MTHFR deficiency. This study was undertaken to identify differentially expressed proteins in conditions favoring initial steps of murine carcinogenesis in normal preneoplastic intestine. We compared the proteome of BALB/c normal intestine in Mthfr^+/+ mice fed control diets for 1 year (low susceptibility to tumorigenesis) with the proteome of Mthfr^+/? animals fed low folate diets (higher tumor susceptibility). Our data suggest that the NuRD complex, KRAS‐related proteins, the protein synthetic machinery, and fatty acid‐related metabolic proteins are upregulated in the early stages of tumorigenesis. These proteins may serve as biomarkers or targets for colorectal cancer diagnosis or therapy.     

Two dimensional gel human protein databases offer a systematic approach to the study of cell proliferation and differentiation

Human cellular protein databases have been established using computer-analyzed 2D gel electrophoresis. These databases, which include information on various properties of proteins, offer a global approach to the study of regulation of cell proliferation and differentiation. Furthermore, thanks to the advent of microsequencing the databases make it possible to directly link protein and DNA information.     

NHS-A isoform of the NHS gene is a novel interactor of ZO-1

Mutations in the NHS (Nance-Horan Syndrome) gene lead to severe congenital cataracts, dental defects and sometimes mental retardation. NHS encodes two protein isoforms, NHS-A and -1A that display cell-type dependent differential expression and localization. Here we demonstrate that of these two isoforms, the NHS-A isoform associates with the cell membrane in the presence of intercellular contacts and it immunoprecipitates with the tight junction protein ZO-1 in MDCK (Madin Darby Canine Kidney) epithelial cells and in neonatal rat lens. The NHS-1A isoform however is a cytoplasmic protein. Both Nhs isoforms are expressed during mouse development. Immunolabelling of developing mouse with the anti-NHS antibody that detects both isoforms revealed the protein in the developing head including the eye and brain. It was primarily expressed in epithelium including neural epithelium and certain vascular endothelium but only weakly expressed in mesenchymal cells. In the epithelium and vascular endothelium the protein associated with the cell membrane and co-localized with ZO-1, which indirectly indicates expression of the Nhs-A isoform in these structures. Membrane localization of the protein in the lens vesicle similarly supports Nhs-A expression. In conclusion, the NHS-A isoform of NHS is a novel interactor of ZO-1 and may have a role at tight junctions. This isoform is important in mammalian development especially of the organs in the head.     

Genomic analysis of SARS-CoV-2 reveals local viral evolution in Ghana

The confirmed case fatality rate for the coronavirus disease 2019 (COVID-19) in Ghana has dropped from a peak of 2% in March to be consistently below 1% since May 2020. Globally, case fatality rates have been linked to the strains/clades of circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a specific country. Here we present 46 whole genomes of SARS-CoV-2 circulating in Ghana, from two separate sequencing batches: 15 isolates from the early epidemic (March 12–April 1 2020) and 31 from later time-points ( 25–27 May 2020). Sequencing was carried out on an Illumina MiSeq system following an amplicon-based enrichment for SARS-CoV-2 cDNA. After genome assembly and quality control processes, phylogenetic analysis showed that the first batch of 15 genomes clustered into five clades: 19A, 19B, 20A, 20B, and 20C, whereas the second batch of 31 genomes clustered to only three clades 19B, 20A, and 20B. The imported cases (6/46) mapped to circulating viruses in their countries of origin, namely, India, Hungary, Norway, the United Kingdom, and the United States of America. All genomes mapped to the original Wuhan strain with high similarity (99.5–99.8%). All imported strains mapped to the European superclade A, whereas 5/9 locally infected individuals harbored the B4 clade, from the East Asian superclade B. Ghana appears to have 19B and 20B as the two largest circulating clades based on our sequence analyses. In line with global reports, the D614G linked viruses seem to be predominating. Comparison of Ghanaian SARS-CoV-2 genomes with global genomes indicates that Ghanaian strains have not diverged significantly from circulating strains commonly imported into Africa. The low level of diversity in our genomes may indicate lower levels of transmission, even for D614G viruses, which is consistent with the relatively low levels of infection reported in Ghana.     

Cell Biology of the Endoplasmic Reticulum and the Golgi Apparatus through Proteomics

Enriched endoplasmic reticulum (ER) and Golgi membranes subjected to mass spectrometry have uncovered over a thousand different proteins assigned to the ER and Golgi apparatus of rat liver. This, in turn, led to the uncovering of several hundred proteins of poorly understood function and, through hierarchical clustering, showed that proteins distributed in patterns suggestive of microdomains in cognate organelles. This has led to new insights with respect to their intracellular localization and function. Another outcome has been the critical testing of the cisternal maturation hypothesis showing overwhelming support for a predominant role of COPI vesicles in the transport of resident proteins of the ER and Golgi apparatus (as opposed to biosynthetic cargo). Here we will discuss new insights gained and also highlight new avenues undertaken to further explore the cell biology of the ER and the Golgi apparatus through tandem mass spectrometry.Most biosynthetic proteins destined for the plasma membrane and secretion, as well as resident proteins of endosomes, lysosomes, the endoplasmic reticulum (ER), and the Golgi apparatus originate with cotranslational translocation into the ER. This is followed by carefully controlled folding and quality control. The ER is also a major site for sensing cellular stress and for cholesterol and phospholipid biosynthesis and constitutes a vast continuous endomembrane system that often pervades the entire cytoplasm. The ER commences as the nuclear envelope extends to rough (ribosome-studded) membranes, and ends with tripartite-like structures of tubular smooth (ribosome-free) ER.The ratio of rough and smooth ER is cell type specific. Stem cells have little rough ER and smooth ER (Murphy et al. 1971; Cheng and Leblond 1974), whereas cells highly specialized in protein secretion, such as in the exocrine pancreas, have extensive rough ER with little to no smooth ER (Palade and Siekevitz 1956; Jamieson and Palade 1967). By contrast, liver parenchymal hepatocytes have a near equal abundance of rough and smooth ER with the latter associated with glycogen storage and elimination of exogenous steroids through a multitude of P450-driven oxidation pathways (Bruni and Porter 1965; Loud 1968; Estabrook et al. 1971; Blouin et al. 1977; Reed and Backes 2012). At the other extreme, Leydig cells of the testis are specialized in cholesterol metabolism to form testosterone and are enriched with smooth ER (Mori and Christensen 1980). Emanating from the ER of all cell types in mammals are discrete export sites that are distributed throughout the cell, sometimes numbering in the hundreds. ER export is controlled by the COPII coat machinery, which has been characterized through proteomics analyses in yeast (Otte et al. 2001) and is coupled to microtubule- and dynein/dynactin-dependent transport directed toward the central juxta-nuclear Golgi apparatus (COPII vesicles are discussed in Lord et al. [2013]). This occurs in vesicular/tubular clusters (VTCs) that seemingly undergo a maturation/distillation process (Saraste and Kuismanen 1984) such that when arriving at the cis-face Golgi stacks, biosynthetic cargo appears more concentrated. This is achieved, at least in part, through a continuous removal of ER export machinery components (e.g., p58, p24, and SNARE proteins) that are then returned to the ER via vesicular transport intermediates controlled by the COPI coat machinery. This apparent “distillation” process continues throughout the secretory pathway from as far as the trans-part of the Golgi apparatus (Miesenbock and Rothman 1995). Whether or not such distillation occurs through intra-Golgi cisternal transport or through direct delivery to the ER is part of ongoing investigation, including the extent of recycling (see below).The Golgi apparatus is morphologically distinct from the ER because of its juxtanuclear position, and at the ultrastructural level, appears as a ribbon-like structure of laterally interconnected stacks of flattened cisternae, each having a network of extensive fenestrated membranes associated both at their cis- and trans-face. Biosynthetic cargo here, undergo extensive posttranslational modifications including the maturation of N-linked oligosaccharides, and the addition of O-linked oligosaccharides. At the trans-face, cargo is sorted and packaged for transport either to the plasma membrane or to the endosomal endomembrane system. Some specialized cargo is also packaged and concentrated into dedicated membrane structures for regulated secretion.The functional and morphological demarcation between the ER and the Golgi apparatus is usually assumed to be complete, giving rise to the notion of two independent organelles—each with their own distinct functions. Because of the extensive recycling that takes place, however, the two organelles appear functionally intertwined. Indeed, inhibition of the Golgi-located ARF1 guanine nucleotide exchange factor GBF1 results in a rapid collapse of the Golgi apparatus into the ER (Misumi et al. 1986; Oda et al. 1987; Fujiwara et al. 1988; Lippincott-Schwartz et al. 1989; Claude et al. 1999). As ARF1^GDP to ARF1^GTP conversion is obligatory to COPI recruitment and vesicle formation and subsequent recycling, the effect of BFA seems at first counter intuitive, but can be explained through the additional role of COPI as a protective coat on cisternal membranes, i.e., BFA-induced dissociation of COPI coat from Golgi membranes enables uncontrolled formation of Golgi-derived tubules that within minutes fuse with the ER, collapsing most of the Golgi into the ER. Strikingly, removal of BFA equally rapidly leads to the reformation of individual functional Golgi stacks positioned at the various ER exit sites and after microtubule-dependent transport, coalesce into the central and juxtanuclear Golgi apparatus. Thus, the two organelles are temporally as well as spatially linked to enable productive communication and trafficking for the process of protein secretion, as well as lipid biosynthesis. This link gives rise to some unexpected functions (see below).