Search

The extracellular matrix (ECM) is central to the physiology of animal tissues, through its multifaceted roles in tissue structure, mechanical properties, and cell interactions, and by its cell-signaling activities that regulate cell phenotype and behavior. The secretion of ECM proteins typically involves multiple transport and processing steps within the endoplasmic reticulum and the subsequent compartments of the secretory pathway. Many ECM proteins are substituted with various posttranslational modifications (PTMs) and there is increasing evidence of how PTM additions are required for ECM protein secretion or functionality within the extracellular milieu. The targeting of PTM-addition steps may thus offer opportunities to manipulate ECM quality or quantity, in vitro or in vivo. This review discusses selected examples of PTMs of ECM proteins for which the PTM has known importance for anterograde trafficking and secretion of the core protein, and/or loss-of-function of the respectively modifying enzyme leads to alterations of ECM structure or function with pathophysiological consequences in humans. Members of the protein disulfide isomerase (PDI) family have central roles in disulfide bond formation and isomerization within the endoplasmic reticulum, and are discussed in relation to emerging knowledge of the roles of certain PDIs in ECM production in the pathophysiological context of breast cancer. Cumulative data suggest the possible applicability of inhibition of PDIA3 activity to modulate ECM composition and functionality within the tumor microenvironment.

Metastasis and recurrence of breast cancer remain major causes of patient mortality, and there is an ongoing need to identify new therapeutic targets relevant to tumor invasion. Protein disulfide isomerase A3 (PDIA3) is a disulfide oxidoreductase and isomerase of the endoplasmic reticulum that has known extracellular substrates and has been correlated with aggressive breast cancers. We show that either prior PDIA3 inhibition by the disulfide isomerase inhibitor 16F16 or depletion of heparin-binding proteins strongly reduces the activity of conditioned medium (CM) of MDA-MB-231 human breast cancer cells to support promigratory cell spreading and F-actin organization by newly adherent MDA-MB-231 cells. Quantitative proteomics to investigate effects of 16F16 inhibition on heparin-binding proteins in the CM of MDA-MB-231 cells identified 80 proteins reproducibly decreased at least twofold (at q ≤ 0.05) after 16F16 treatment. By Gene Ontology analysis, many of these have roles in extracellular matrix (ECM) structure and function and cell adhesion; ribosomal proteins that also correlate with extracellular vesicles were also identified. Protein-protein interaction analysis showed that many of the extracellular proteins have known network interactions with each other. The predominant types of disulfide-bonded domains in the extracellular proteins contained β-hairpin folds, with the knottin fold the most common. From human breast cancer data sets, the extracellular proteins were found to correlate specifically with the basal subtype of breast cancer and their high expression in tumors correlated with reduced distant metastasis-free survival. These data provide new evidence that PDIA3 may be a relevant therapeutic target to alter properties of the ECM-associated microenvironment in basal breast cancer.

The matricellular glycoprotein thrombospondin-1 (TSP1) has complex roles in the extracellular matrix (ECM) and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulfide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalize in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3−/− mouse embryo fibroblasts ( Pdia3−/− MEFs), led to alterations in the composition of cell-derived extracellular matrix, involving changed abundance of fibronectin and TSP1, was correlated with reduced cell spreading, altered organization of F-actin, and reduced focal adhesions. These cellular phenotypes of Pdia3−/− MEFs were normalized by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEFs. Rescue depended on PDIA3 activity in WT-MEFs and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEFs and Pdia3−/− MEFs identified multiple ECM and growth factor proteins to be downregulated in the CM of Pdia3−/− MEFs. Of these, cell communication network 2 (CCN2) was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3−/− MEFs and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, proadhesive microenvironment, with implications for PDIA3 as a translational target.

Thrombospondins (TSPs) are multidomain, secreted proteins that associate with cell surfaces and extracellular matrix. In mammals, there is a large body of data on functional roles of various TSP family members in cardiovascular disease (CVD), including stroke, cardiac remodeling and fibrosis, atherosclerosis, and aortic aneurysms. Coding single nucleotide polymorphisms (SNPs) of TSP1 or TSP4 are also associated with increased risk of several forms of CVD. Whereas interactions and functional effects of TSPs on a variety of cell types have been studied extensively, the molecular and cellular basis for the differential effects of the SNPs remains under investigation. Here, we provide an integrative review on TSPs, their roles in CVD and cardiovascular cell physiology, and known properties and mechanisms of TSP SNPs relevant to CVD. In considering recent expansions to knowledge of the fundamental cellular roles and mechanisms of TSPs, as well as the effects of wild-type and variant TSPs on cells of the cardiovascular system, we aim to highlight knowledge gaps and areas for future research or of translational potential.

Animals (metazoans) include some of the most complex living organisms on Earth, with regard to their multicellularity, numbers of differentiated cell types, and lifecycles. The metazoan extracellular matrix (ECM) is well-known to have major roles in the development of tissues during embryogenesis and in maintaining homoeostasis throughout life, yet insight into the ECM proteins which may have contributed to the transition from unicellular eukaryotes to multicellular animals remains sparse. Recent phylogenetic studies place either ctenophores or poriferans as the closest modern relatives of the earliest emerging metazoans. Here, we review the literature and representative genomic and transcriptomic databases for evidence of ECM and ECM-affiliated components known to be conserved in bilaterians, that are also present in ctenophores and/or poriferans. Whereas an extensive set of related proteins are identifiable in poriferans, there is a strikingly lack of conservation in ctenophores. From this perspective, much remains to be learnt about the composition of ctenophore mesoglea. The principal ECM-related proteins conserved between ctenophores, poriferans, and bilaterians include collagen IV, laminin-like proteins, thrombospondin superfamily members, integrins, membrane-associated proteoglycans, and tissue transglutaminase. These are candidates for a putative ancestral ECM that may have contributed to the emergence of the metazoans.

Since the 1970s, laboratory methods have been developed in molecular biology that allow for the manipulation of the DNA sequences of genes or the genome of an organism. Expression of proteins in bacteria, genetic modifications of mice and other animals or plants, and the CRISPR/cas9 gene editing technology are described. Finally the possibility of, and ethical issues around, gene editing in humans are discussed.

Early ideas about the origin of the cosmos and of life on Earth can be found in the myths of different nations and in religions. A number of these reflections are reviewed. The myths are based on imagination and form impressive pieces of literature.

A major human-made and general risk to life arises from the large number of weapons designed for mass destruction. The history and present potential of nuclear and chemical weapons are briefly reviewed. Hydrogen bombs are now owned by many countries and have an overkill potential. Extremely dangerous new missiles have been designed. Nuclear weapons pollute large areas with radioactivity thus killing most organisms and preventing further use of the land. We discuss current international treaties to limit the stock-piling of nuclear weapons and the Organisation for Prohibition of Chemical Weapons.

We discuss how environmental conditions of the early Earth about 4.5 billion years ago have been estimated from astronomy, geology and chemistry. Of major interest are the prebiotic chemical constituents from which life began. We discuss models for precursors of the first cells, including more detailed models: the hypercycle model, the metabolism first model, the protocell model and a model based on phylogenetic methods of a last universal common ancestor of cells.

This chapter discusses the development of computers capable of machine-learning and artificial intelligence. We discuss examples of beneficial applications in bioscience research and potential utility in clinical diagnosis along with the huge potential for great risks. We introduce the idea of trans-humanism, that aims to generate partially inorganic beings that could transcend ageing and mortality and facilitate long-distance space travel. We discuss the major legal issues raised by the development of intelligent machines.

In this chapter, we discuss the evidence for climate change through global warming and the evidence that this global heating has an anthropogenic origin. We discuss the inter-linked issue of the increasing human population and then turn to the ongoing efforts to mitigate or arrest climate change. Finally, we discuss why massive efforts to decrease greenhouse gas emissions are needed so urgently. This area has also become a major political issue.

We discuss some prominent major natural risks to life including the epidemics and pandemics caused by micro-organisms such as bacteria or viruses, with detail on the ongoing covid-19 pandemic. We also consider localised severe climatic or geological events and extra-terrestrial risks from hits by asteroids. We discuss current knowledge of the likely causes of historic mass extinctions, such as the Cretaceous-Paleogene extinction which abolished the majority of the dinosaurs.

It has long been discussed if life exists outside the Earth. We discuss the environmental conditions thought to be necessary to support life; calculations of the number of possible habitable planets; ongoing research on the environment of Mars, and efforts to search for life outside the Solar system. The idea of sending humans to Mars poses enormous challenges but appears possible to be achieved during the twenty-first century.

[No abstract]

Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER)-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix (ECM) proteins. PDIA3 is up-regulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified ‘scratch wound’ assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wildtype (WT) or Pdia3−/− mouse embryonic fibroblasts (MEFs). The conditioned medium (CM) of Pdia3−/− MEFs was less effective in promoting cell spreading and F-actin organisation or supporting ‘scratch wound’ closure. Similarly, ECM prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.

[No abstract]

Extracellular matrix (ECM) is considered central to the evolution of metazoan multicellularity, however the repertoire of ECM proteins in non-bilaterians remains unclear. Thrombospondins (TSPs) are known to be well-conserved from cnidarians to vertebrates, yet to date have been considered a unique family, principally studied for matricellular functions in vertebrates. Through searches utilizing the highlyconserved C-terminal region of TSPs we identify undisclosed new families of thrombospondin-related proteins in metazoans, designated mega-thrombospondin, sushi-thrombospondin and poriferan-thrombospondin, each with a distinctive phylogenetic distribution. These proteins share the TSP C-terminal region domain architecture, as determined by domain composition and analysis of molecular models against known structures. Mega-thrombospondins, the only form identified in ctenophores, are typically >2700aa and are also characterized by N-terminal leucine-rich repeats and central cadherin/immunoglobulin domains. In cnidarians, which have a well-defined ECM, Mega-TSP was expressed throughout embryogenesis in Nematostella vectensis, with dynamic endodermal expression in larvae and primary polyps and widespread ectodermal expression in adult N. vectensis and Hydra magnipapillata polyps. Hydra Mega-TSP was also expressed during regeneration and siRNA-silencing of Mega-TSP in Hydra caused specific blockade of head regeneration. Molecular phylogenetic analyses based on the conserved TSP C-terminal region identified each of the TSP-related groups to form clades distinct from the canonical TSPs. We discuss models for the evolution of the newly-defined TSP superfamily by gene duplications, radiation and gene losses from a debut in the last metazoan common ancestor. Together, the data provide new insight into the evolution of ECM and tissue organization in metazoans.

Dystroglycan (DG) is an adhesion complex that links the cytoskeleton to the surrounding extracellular matrix in skeletal muscle and a wide variety of other tissues. It is composed of a highly glycosylated extracellular alpha-DG associated noncovalently with a transmembrane beta-DG whose cytodomain interacts with dystrophin and its isoforms. Alpha-dystroglycan (alpha-DG) binds tightly and in a calcium-dependent fashion to multiple extracellular proteins and proteoglycans, each of which harbors at least one, or, more frequently, tandem arrays of laminin-globular (LG) domains. Considerable biochemical and structural work has accumulated on the alpha-DG-binding LG domains, highlighting a significant heterogeneity in ligand-binding properties of domains from different proteins as well as between single and multiple LG domains within the same protein. Here we review biochemical, structural, and functional information on the LG domains reported to bind alpha-dystroglycan. In addition, we have incorporated bioinformatics and modeling to explore whether specific motifs responsible for alpha-dystroglycan recognition can be identified within isolated LG domains. In particular, we analyzed the LG domains of slits and agrin as well as those of paradigmatic alpha-DG non-binders such as laminin-alpha 3. While some stretches of basic residues may be important, no universally conserved motifs could be identified. However, the data confirm that the coordinated calcium atom within the LG domain is needed to establish an interaction with the sugars of alpha-DG, although it appears that this alone is insufficient to mediate significant alpha-DG binding. We develop a scenario involving different binding modes of a single LG domain unit, or tandemly repeated units, with alpha-DG. A variability of binding modes might be important to generate a range of affinities to allow physiological regulation of this interaction, reflecting its crucial biological importance.

The extracellular matrix (ECM) has central roles in tissue integrity and remodeling throughout the life span of animals. While collagens are the most abundant structural components of ECM in most tissues, tissue-specific molecular complexity is contributed by ECM glycoproteins. The matricellular glycoproteins are categorized primarily according to functional criteria and represented predominantly by the thrombospondin, tenascin, SPARC/osteonectin, and CCN families. These proteins do not self-assemble into ECM fibrils; nevertheless, they shape ECM properties through interactions with structural ECM proteins, growth factors, and cells. Matricellular proteins also promote cell migration or morphological changes through adhesion-modulating or counter-adhesive actions on cell-ECM adhesions, intracellular signaling, and the actin cytoskeleton. Typically, matricellular proteins are most highly expressed during embryonic development. In adult tissues, expression is more limited unless activated by cues for dynamic tissue remodeling and cell motility, such as occur during inflammatory response and wound repair. Many insights in the complex roles of matricellular proteins have been obtained from studies of gene knockout mice. However, with the exception of chordate-specific tenascins, these are highly conserved proteins that are encoded in many animal phyla. This review will consider the increasing body of research on matricellular proteins in nonmammalian animal models. These models provide better access to the very earliest stages of embryonic development and opportunities to study biological processes such as limb and organ regeneration. In aggregate, this research is expanding concepts of the functions and mechanisms of action of matricellular proteins.

Fibrillar collagens of the extracellular matrix are critical for tissue structure and physiology; however, excessive or abnormal deposition of collagens is a defining feature of fibrosis. Regulatory mechanisms that act on collagen fibril assembly potentially offer new targets for antifibrotic treatments. Tissue weakening, altered collagen fibril morphologies, or both, are shared phenotypes of mice lacking matricellular thrombospondins. Thrombospondin-1 (TSP1) plays an indirect role in collagen homeostasis through interactions with matrix metalloproteinases and transforming growth factor–1 (TGF-1). We found that TSP1 also affects collagen fibril formation directly. Compared to skin from wild-type mice, skin from Thbs1-/- mice had reduced collagen cross-linking and reduced prolysyl oxidase (proLOX) abundance with increased conversion to catalytically active LOX. In vitro, TSP1 bound to both the C-propeptide domain of collagen I and the highly conserved KGHR sequences of the collagen triple-helical domain that participate in cross-linking. TSP1 also bound to proLOX and inhibited proLOX processing by bone morphogenetic protein-1. In human dermal fibroblasts (HDFs), TSP1 and collagen I colocalized in intracellular vesicles and on extracellular collagen fibrils, whereas TSP1 and proLOX colocalized only in intracellular vesicles. Inhibition of LOX-mediated collagen cross-linking did not prevent the extracellular association between collagen and TSP1; however, treatment of HDFs with KGHR-containing, TSP1-binding, triple-helical peptides disrupted the collagen-TSP1 association, perturbed the collagen extracellular matrix, and increased myofibroblastic differentiation in a manner that depended on TGF- receptor 1. Thus, the extracellular KGHR-dependent interaction of TSP1 with fibrillar collagens contributes to fibroblast homeostasis.

The extracellular matrix (ECM) has central roles in tissue integrity and remodeling throughout the life span of animals. While collagens are the most abundant structural components of ECM in most tissues, tissue-specific molecular complexity is contributed by ECM glycoproteins. The matricellular glycoproteins are categorized primarily according to functional criteria and represented predominantly by the thrombospondin, tenascin, SPARC/osteonectin, and CCN families. These proteins do not self-assemble into ECM fibrils; nevertheless, they shape ECM properties through interactions with structural ECM proteins, growth factors, and cells. Matricellular proteins also promote cell migration or morphological changes through adhesion-modulating or counter-adhesive actions on cell-ECM adhesions, intracellular signaling, and the actin cytoskeleton. Typically, matricellular proteins are most highly expressed during embryonic development. In adult tissues, expression is more limited unless activated by cues for dynamic tissue remodeling and cell motility, such as occur during inflammatory response and wound repair. Many insights in the complex roles of matricellular proteins have been obtained from studies of gene knockout mice. However, with the exception of chordate-specific tenascins, these are highly conserved proteins that are encoded in many animal phyla. This review will consider the increasing body of research on matricellular proteins in nonmammalian animal models. These models provide better access to the very earliest stages of embryonic development and opportunities to study biological processes such as limb and organ regeneration. In aggregate, this research is expanding concepts of the functions and mechanisms of action of matricellular proteins.

[no abstract]

Two prescient 1953 publications set the stage for the elucidation of a novel endocrine system: Schatzmann’s report that cardiotonic steroids (CTSs) are all Na+ pump inhibitors, and Szent-Gyorgi’s suggestion that there is an endogenous “missing screw” in heart failure that CTSs like digoxin may replace. In 1977 I postulated that an endogenous Na+ pump inhibitor acts as a natriuretic hormone and simultaneously elevates blood pressure (BP) in salt-dependent hypertension. This hypothesis was based on the idea that excess renal salt retention promoted the secretion of a CTS-like hormone that inhibits renal Na+ pumps and salt reabsorption. The hormone also inhibits arterial Na+ pumps, elevates myocyte Na+ and promotes Na/Ca exchanger-mediated Ca2+ gain. This enhances vasoconstriction and arterial tone—the hallmark of hypertension. Here I describe how those ideas led to the discovery that the CTS-like hormone is endogenous ouabain (EO), a key factor in the pathogenesis of hypertension and heart failure. Seminal observations that underlie the still-emerging picture of the EO-Na+ pump endocrine system in the physiology and pathophysiology of multiple organ systems are summarized. Milestones include: 1) cloning the Na+ pump isoforms and physiological studies of mutated pumps in mice; 2) discovery that Na+ pumps are also EO-triggered signaling molecules; 3) demonstration that ouabain, but not digoxin, is hypertensinogenic; 4) elucidation of EO’s roles in kidney development and cardiovascular and renal physiology and pathophysiology; 5) discovery of “brain ouabain”, a component of a novel hypothalamic neuromodulatory pathway; and 6) finding that EO and its brain receptors modulate behavior and learning.

Fascin-1 is a filamentous actin-binding protein that crosslinks actin microfilaments into tight, parallel bundles. These bundles are important for the extension of microspikes, filopodia and invadopodia from cell surfaces and for the functionality of these protrusions in cell migration and/or dynamic sensing of the local microenvironment. Fascin-1 is absent in normal colonic epithelium, but its upregulation in colorectal adenomas and adenocarcinomas and its correlation with an aggressive clinical course has piqued the interest of many laboratories with research interests in cancer metastasis. This report summarizes current knowledge of the molecular interactions of fascin-1 in relation to its activities and mechanisms of upregulation in colorectal carcinoma cells. The status and key questions surrounding investigations of fascin-1 as a novel, early prognostic biomarker in colorectal cancer are discussed. Ongoing pre-clinical research into new migration inhibitory and anti-metastatic compounds that alter the actin cytoskeleton, and the goal of targeting fascin-1, is also discussed.

Thrombospondins (TSPs) are multidomain glycoproteins with complex matricellular functions in tissue homeostasis and remodeling. We describe a novel role of TSP as a Wnt signaling target in the basal eumetazoan Hydra. Proteome analysis identified Hydra magnipapillata TSP (HmTSP) as a major component of the cnidarian mesoglea. In general, the domain organization of cnidarian TSPs is related to the pentameric TSPs of bilaterians, and in phylogenetic analyses cnidarian TSPs formed a separate clade of high sequence diversity. HmTSP expression in polyps was restricted to the hypostomal tip and tentacle bases that harbor Wnt-regulated organizer tissues. In the hypostome, HmTSP- and Wnt3-expressing cells were identical or in close vicinity to each other, and regions of ectopic tentacle formation induced by pharmacological β-Catenin activation (Alsterpaullone) corresponded to foci of HmTSP expression. Chromatin immunoprecipitation (ChIP) confirmed binding of Hydra TCF to conserved elements in the HmTSP promotor region. Accordingly, β-Catenin knockdown by siRNAs reduced normal HmTSP expression at the head organizer. In contrast, knockdown of HmTSP expression led to increased numbers of ectopic organizers in Alsterpaullone-treated animals, indicating a negative regulatory function. Our data suggest an unexpected role for HmTSP as a feedback inhibitor of Wnt signaling during Hydra body axis patterning and maintenance.

In a recent publication in Bioscience Reports “Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies”, Brown et al. identified by mass spectrometry and immunoblotting that certain commercial preparations of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan, in fact, contained a mix of several proteoglycans that also included fibromodulin and aggrecan. The preparations were thus not suitable to study specific activities of decorin or biglycan. Decorin and biglycan are widely studied SLRPs that are considered to have highly multi-functional effects on cells. Decorin is of interest as a transforming growth factor-β antagonist and is also finding use in tissue engineering materials. This Commentary discusses Brown et al.’s findings and general issues raised for researchers who work with commercially sourced purified proteoglycans.

in 2015, aps council and the APS Publications Committee, the latter led by Dr. Curt D. Sigmund, began an initiative on the transparency of research reporting in the APS Journals. At this time, concerns over biosciences research reproducibility, especially in areas such as preclinical cancer biology

Although prostate-specific antigen (PSA) testing can identify early-stage prostate cancers, additional biomarkers are needed for risk stratification. In one study, high levels of the actin-bundling protein, fascin-1, were correlated with lethal-phase, hormone-refractory prostate cancer. Analyses of independent samples are needed to establish the value of fascin-1 as a possible biomarker. We examined fascin-1 by immunohistochemistry in tumour specimens from the Wales Cancer Bank in comparison with nuclear-located ETS-related gene (ERG), an emerging marker for aggressive prostate cancer. Fascin-1 was elevated in focal areas of a minority of tumours, yet fascin-1-positivity did not differentiate tumours of low-, intermediate-, or high-risk Gleason scores and did not correlate with PSA status or biochemical relapse after surgery. Stromal fascin-1 correlated with high Gleason score. Nuclear ERG was upregulated in tumours but not in stroma. The complexities of fascin-1 status indicate that fascin-1 is unlikely to provide a suitable biomarker for prediction of aggressive prostate cancers.

The mammalian muskelin/RanBP9/C-terminal to LisH (CTLH) complex and the Saccharomyces cerevisiae glucose-induced degradation (GID) complex are large, multi-protein complexes that each contain a RING E3 ubiquitin ligase. The yeast GID complex acts to degrade a key enzyme of gluconeogenesis, fructose 1,6-bisphosphatase, under conditions of abundant fermentable carbon sources. However, the assembly and functions of the mammalian complex remain poorly understood. A striking feature of these complexes is the presence of multiple proteins that contain contiguous lissencephaly-1 homology (LisH), CTLH and C-terminal CT11-RanBP9 (CRA) domains. TWA1/Gid8, the smallest constituent protein of these complexes, consists only of LisH, CTLH and CRA domains and is highly conserved in eukaryotes. Towards better knowledge of the role of TWA1 in these multi-protein complexes, we established a method for bacterial expression and purification of mouse TWA1 that yields tag-free, recombinant TWA1 in quantities suitable for biophysical and biochemical studies. CD spectroscopy of recombinant TWA1 indicated a predominantly α-helical protein. Gel filtration chromatography, size-exclusion chromatography (SEC) with multi-angle light scattering (SEC-MALS) and native PAGE demonstrated a propensity of untagged TWA1 to form stable dimers and, to a lesser extent, higher order oligomers. TWA1 has a single cysteine residue, Cys139, yet the dimeric form was preserved when TWA1 was purified in the presence of the reducing agent tris(2-carboxyethyl)phosphine (TCEP). These findings have implications for understanding the molecular role of TWA1 in the yeast GID complex and related multi-protein E3 ubiquitin ligases identified in other eukaryotes.

central to the homeostatic life of cells is the need to coordinate responses to external and internal changes. These processes become even more important in the context of the sustained cell-cell interactions that take place in multicellular organisms. As amply studied in metazoans, intricate