Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Structural Basis of DEPTOR to Recognize Phosphatidic Acid Using its Tandem DEP Domains

DEP domain containing mTOR-interacting protein (DEPTOR) plays pivotal roles in regulating metabolism, growth, autophagy and apoptosis by functions as an endogenous inhibitor of mTOR signaling pathway. Activated by phosphatidic acid, a second messenger in mTOR signaling, DEPTOR dissociates from mTORC1 complex with unknown mechanism. Here, we present a 1.5 Å resolution crystal structure, which shows that the N-terminal two tandem DEP domains of hDEPTOR fold into a dumbbell-shaped structure, protruding the characteristic β-hairpin arms of DEP domains on each side. An 18 amino acids DDEX motif at the end of DEP2 interacts with DEP1 and stabilizes the structure. Biochemical studies showed that the tandem DEP domains directly interact with phosphatidic acid using two distinct positively charged patches. These results provide insights into mTOR activation upon phosphatidic acid stimulation.     

Extending the Horizon of Homology Detection with Coevolution-based Structure Prediction

Traditional sequence analysis algorithms fail to identify distant homologies when they lie beyond a detection horizon. In this review, we discuss how co-evolution-based contact and distance prediction methods are pushing back this homology detection horizon, thereby yielding new functional insights and experimentally testable hypotheses. Based on correlated substitutions, these methods divine three-dimensional constraints among amino acids in protein sequences that were previously devoid of all annotated domains and repeats. The new algorithms discern hidden structure in an otherwise featureless sequence landscape. Their revelatory impact promises to be as profound as the use, by archaeologists, of ground-penetrating radar to discern long-hidden, subterranean structures. As examples of this, we describe how triplicated structures reflecting longin domains in MON1A-like proteins, or UVR-like repeats in DISC1, emerge from their predicted contact and distance maps. These methods also help to resolve structures that do not conform to a “beads-on-a-string” model of protein domains. In one such example, we describe CFAP298 whose ubiquitin-like domain was previously challenging to perceive owing to a large sequence insertion within it. More generally, the new algorithms permit an easier appreciation of domain families and folds whose evolution involved structural insertion or rearrangement. As we exemplify with α1-antitrypsin, coevolution-based predicted contacts may also yield insights into protein dynamics and conformational change. This new combination of structure prediction (using innovative co-evolution based methods) and homology inference (using more traditional sequence analysis approaches) shows great promise for bringing into view a sea of evolutionary relationships that had hitherto lain far beyond the horizon of homology detection.     

The Structure and Ubiquitin Binding Properties of TRAF RING Heterodimers

Tumour necrosis factor (TNF) receptor associated factor (TRAF) family members share a common domain architecture, but play non-redundant physiological roles in cell signalling. At the N terminus, most TRAFs have a RING domain, followed by a series of Zinc finger (ZF) domains. The RING domain of TRAF6 dimerizes, and the RING homodimer together with the first ZF assembles ubiquitin chains that form a platform which facilitates activation of downstream kinases. The RING dimer interface is conserved amongst TRAF proteins, suggesting that functional heterodimers could be possible. Here we report the structure of the TRAF5-TRAF6 RING heterodimer, which accounts for the stability of the heterodimer as well as its ability to assemble ubiquitin chains. We also show that the RING domain of TRAF6 heterodimerizes with TRAF3 and TRAF2, and demonstrate that the linker helix and first ZF of TRAF2 can cooperate with TRAF6 to promote chain assembly. Collectively our results suggest that TRAF RING homo- and hetero-dimers have the potential to bridge interaction of nearby TRAF trimers and modulate TRAF-mediated signalling.     

Novel strategy for anchorage position control of GPI-attached proteins in the yeast cell wall using different GPI-anchoring domains

The yeast cell surface provides space to display functional proteins. Heterologous proteins can be covalently anchored to the yeast cell wall by fusing them with the anchoring domain of glycosylphosphatidylinositol (GPI)-anchored cell wall proteins (GPI-CWPs). In the yeast cell-surface display system, the anchorage position of the target protein in the cell wall is an important factor that maximizes the capabilities of engineered yeast cells because the yeast cell wall consists of a 100- to 200-nm-thick microfibrillar array of glucan chains. However, knowledge is limited regarding the anchorage position of GPI-attached proteins in the yeast cell wall. Here, we report a comparative study on the effect of GPI-anchoring domain–heterologous protein fusions on yeast cell wall localization. GPI-anchoring domains derived from well-characterized GPI-CWPs, namely Sed1p and Sag1p, were used for the cell-surface display of heterologous proteins in the yeast Saccharomyces cerevisiae. Immunoelectron-microscopic analysis of enhanced green fluorescent protein (eGFP)-displaying cells revealed that the anchorage position of the GPI-attached protein in the cell wall could be controlled by changing the fused anchoring domain. eGFP fused with the Sed1-anchoring domain predominantly localized to the external surface of the cell wall, whereas the anchorage position of eGFP fused with the Sag1-anchoring domain was mainly inside the cell wall. We also demonstrate the application of the anchorage position control technique to improve the cellulolytic ability of cellulase-displaying yeast. The ethanol titer during the simultaneous saccharification and fermentation of hydrothermally-processed rice straw was improved by 30% after repositioning the exo- and endo-cellulases using Sed1- and Sag1-anchor domains. This novel anchorage position control strategy will enable the efficient utilization of the cell wall space in various fields of yeast cell-surface display technology.     

Uncovering post-translational modification-associated protein–protein interactions

In living systems, the chemical space and functional repertoire of proteins are dramatically expanded through the post-translational modification (PTM) of various amino acid residues. These modifications frequently trigger unique protein–protein interactions (PPIs) – for example with reader proteins that directly bind the modified amino acid residue – which leads to downstream functional outcomes. The modification of a protein can also perturb its PPI network indirectly, for example, through altering its conformation or subcellular localization. Uncovering the network of unique PTM-triggered PPIs is essential to fully understand the roles of an ever-expanding list of PTMs in our biology. In this review, we discuss established strategies and current challenges associated with this endeavor.     

Mass Spectrometry–Based Proteomics Analysis of Human Substantia Nigra From Parkinson's Disease Patients Identifies Multiple Pathways Potentially Involved in the Disease

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) of the brain. Despite decades of studies, the precise pathogenic mechanism of PD is still elusive. An unbiased proteomic analysis of PD patient’s brain allows the identification of critical proteins and molecular pathways that lead to dopamine cell death and α-synuclein deposition and the resulting devastating clinical symptoms. In this study, we conducted an in-depth proteome analysis of human SN tissues from 15 PD patients and 15 healthy control individuals combining Orbitrap mass spectrometry with the isobaric tandem mass tag–based multiplexing technology. We identified 10,040 proteins with 1140 differentially expressed proteins in the SN of PD patients. Pathway analysis showed that the ribosome pathway was the most enriched one, followed by gamma-aminobutyric acidergic synapse, retrograde endocannabinoid signaling, cell adhesion molecules, morphine addiction, Prion disease, and PD pathways. Strikingly, the majority of the proteins enriched in the ribosome pathway were mitochondrial ribosomal proteins (mitoribosomes). The subsequent protein–protein interaction analysis and the weighted gene coexpression network analysis confirmed that the mitoribosome is the most enriched protein cluster. Furthermore, the mitoribosome was also identified in our analysis of a replication set of ten PD and nine healthy control SN tissues. This study provides potential disease pathways involved in PD and paves the way to study further the pathogenic mechanism of PD.     

The PHY Domain Dimer Interface of Bacteriophytochromes Mediates Cross-talk between Photosensory Modules and Output Domains

Protein dynamics play a major role for the catalytic function of enzymes, the interaction of protein complexes or signal integration in regulatory proteins. In the context of multi-domain proteins involved in light-regulation of enzymatic effectors, the central role of conformational dynamics is well established. Light activation of sensory modules is followed by long-range signal transduction to different effectors; rather than domino-style structural rearrangements, a complex interplay of functional elements is required to maintain functionality. One family of such sensor-effector systems are red-light-regulated phytochromes that control diguanylate cyclases involved in cyclic-dimeric-GMP formation. Based on structural and functional studies of one prototypic family member, the central role of the coiled-coil sensor-effector linker was established. Interestingly, subfamilies with different linker lengths feature strongly varying biochemical characteristics. The dynamic interplay of the domains involved, however, is presently not understood. Here we show that the PHY domain dimer interface plays an essential role in signal integration, and that a functional coupling with the coiled-coil linker element is crucial. Chimaeras of two biochemically different family members highlight the phytochrome-spanning helical spine as an essential structural element involved in light-dependent upregulation of enzymatic turnover. However, isolated structural elements can frequently not be assigned to individual characteristics, which further emphasises the importance of global conformational dynamics. Our results provide insights into the intricate processes at play during light signal integration and transduction in these photosensory systems and thus provide additional guidelines for a more directed design of novel sensor-effector combinations with potential applications as optogenetic tools.     

Loss of plasma membrane lipid asymmetry can induce ordered domain (raft) formation

In some cases, lipids in one leaflet of an asymmetric artificial lipid vesicle suppress the formation of ordered lipid domains (rafts) in the opposing leaflet. Whether this occurs in natural membranes is unknown. Here, we investigated this issue using plasma membrane vesicles (PMVs) from rat leukemia RBL-2H3 cells. Membrane domain formation and order was assessed by fluorescence resonance energy transfer and fluorescence anisotropy. We found that ordered domains in PMVs prepared from cells by N-ethyl maleimide (NEM) treatment formed up to ～37°C, whereas ordered domains in symmetric vesicles formed from the extracted PMV lipids were stable up to 55°C, indicating the stability of ordered domains was substantially decreased in intact PMVs. This behavior paralleled lesser ordered domain stability in artificial asymmetric lipid vesicles relative to the corresponding symmetric vesicles, suggesting intact PMVs exhibit some degree of lipid asymmetry. This was supported by phosphatidylserine mislocalization on PMV outer leaflets as judged by annexin binding, which indicated NEM-induced PMVs are much more asymmetric than PMVs formed by dithiothreitol/paraformaldehyde treatment. Destroying asymmetry by reconstitution of PMVs using detergent dilution also showed stabilization of domain formation, even though membrane proteins remained associated with reconstituted vesicles. Similar domain stabilization was observed in artificial asymmetric lipid vesicles after destroying asymmetry via detergent reconstitution. Proteinase K digestion of proteins had little effect on domain stability in NEM PMVs. We conclude that loss of PMV lipid asymmetry can induce ordered domain formation. The dynamic control of lipid asymmetry in cells may regulate domain formation in plasma membranes.     

Recent insight into intermediate filament structure

Intermediate filaments (IFs) are key players in multiple cellular processes throughout human tissues. Their biochemical and structural properties are important for understanding filament assembly mechanisms, for interactions between IFs and binding partners, and for developing pharmacological agents that target IFs. IF proteins share a conserved coiled-coil central-rod domain flanked by variable N-terminal ‘head’ and C-terminal ‘tail’ domains. There have been several recent advances in our understanding of IF structure from the study of keratins, glial fibrillary acidic protein, and lamin. These include discoveries of (i) a knob–pocket tetramer assembly mechanism in coil 1B; (ii) a lamin-specific coil 1B insert providing a one-half superhelix turn; (iii) helical, yet flexible, linkers within the rod domain; and (iv) the identification of coil 2B residues required for mature filament assembly. Furthermore, the head and tail domains of some IFs contain low-complexity aromatic-rich kinked segments, and structures of IFs with binding partners show electrostatic surfaces are a major contributor to complex formation. These new data advance the connection between IF structure, pathologic mutations, and clinical diseases in humans.     

SCFFBXO28-mediated self-ubiquitination of FBXO28 promotes its degradation

The F-box protein is the substrate recognition subunit of SCF (SKP1/CUL1/F-box) E3 ubiquitin ligase complex, a multicomponent RING-type E3 ligase involved in the regulation of numerous cellular processes by targeting critical regulatory proteins for ubiquitination. However, whether and how F-box proteins are regulated is largely unknown. Here we report that FBXO28, a poorly characterized F-box protein, is a novel substrate of SCF E3 ligase. Pharmaceutical or genetic inhibition of neddylation pathway that is required for the activation of SCF stabilizes FBXO28 and prolongs its half-life. Meanwhile, FBXO28 is subjected to ubiquitination and cullin1-based SCF complex promotes FBXO28 degradation. Moreover, deletion of F-box domain stabilizes FBXO28 and knockdown of endogenous FBXO28 strongly upregulates exogenous FBXO28 expression. Taken together, these data reveal that SCF^FBXO28 is the E3 ligase responsible for the self-ubiquitination and proteasomal degradation of FBXO28, providing a new clue for the upstream signaling regulation for F-box proteins.     

Effects of 1α,25-dihydroxyvitamin D3 and tacalcitol on cell signaling and anchorage-independent growth in T98G and U251 glioblastoma cells

The active hormonal form of vitamin D, 1α,25-dihydroxyvitamin D₃, is reported to have 1000s of biological targets. The growth-suppressive properties of 1α,25-dihydroxyvitamin D₃ and its synthetic analogs have attracted interest for the development of treatment and/or prevention of cancer. We examined effects of 1α,25-dihydroxyvitamin D₃ and the vitamin D analog tacalcitol on signaling pathways and anchorage-independent growth in T98G and U251 glioblastoma cells. Assay of signaling proteins important for cellular growth indicated suppression of p70-S6 kinase levels by 1α,25-dihydroxyvitamin D₃ and tacalcitol in T98G cells, whereas the levels of PLCγ, a target for phospholipid signaling, was slightly increased.Activation of STAT3, an important regulator of malignancy, was suppressed by 1α,25-dihydroxyvitamin D₃ and tacalcitol in T98G and U251 cells. However, despite the close structural similarity of these compounds, suppression was stronger by tacalcitol (1α,24-dihydroxyvitamin D₃), indicating that even minor modifications of a vitamin D analog can impact its effects on signaling. Experiments using soft agar colony formation assay in T98G and U251 cells revealed significant suppression by 1α,25-dihydroxyvitamin D₃ and tacalcitol on anchorage-independent growth, a property for cancer invasion and metastasis known to correlate with tumorigenicity. These findings indicate that vitamin D and its analogs may be able to counteract the oncogenic transformation, invasion and metastatic potential of glioblastoma and prompt further study of these compounds in the development of improved therapy for brain cancer.     

Structural Insight Into hnRNP A2/B1 Homodimerization and DNA Recognition

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) has been identified as a nuclear DNA sensor. Upon viral infection, hnRNP A2/B1 recognizes pathogen-derived DNA as a homodimer, which is a prerequisite for its translocation to the cytoplasm to activate the interferon response. However, the DNA binding mechanism inducing hnRNP A2/B1 homodimerization is unknown. Here, we show the crystal structure of the RNA recognition motif (RRM) of hnRNP A2/B1 in complex with a U-shaped ssDNA, which mediates the formation of a newly observed protein dimer. Our biochemical assays and mutagenesis studies confirm that the hnRNP A2/B1 homodimer forms in solution by binding to pre-generated ssDNA or dsDNA with a U-shaped bulge. These results depict a potential functional state of hnRNP A2/B1 in antiviral immunity and other cellular processes.     

Timing of TolA and TolQ Recruitment at the Septum Depends on the Functionality of the Tol-Pal System

Efficient cell division of Gram-negative bacteria requires the presence of the Tol-Pal system to coordinate outer membrane (OM) invagination with inner membrane invagination (IM) and peptidoglycan (PG) remodeling. The Tol-Pal system is a trans-envelope complex that connects the three layers of the cell envelope through an energy-dependent process. It is composed of the three IM proteins, TolA, TolQ and TolR, the periplasmic protein TolB and the OM lipoprotein Pal. The proteins of the Tol-Pal system are dynamically recruited to the cell septum during cell division. TolA, the central hub of the Tol-Pal system, has three domains: a transmembrane helix (TolA₁), a long second helical periplasmic domain (TolA₂) and a C-terminal globular domain (TolA₃). The TolQR complex uses the PMF to energize TolA, allowing its cyclic interaction via TolA₃ with the OM TolB-Pal complex. Here, we confirm that TolA₂ is sufficient to address TolA to the site of constriction, whereas TolA₁ is recruited by TolQ. Analysis of the protein localization as function of the bacterial cell age revealed that TolA and TolQ localize earlier at midcell in the absence of the other Tol-Pal proteins. These data suggest that TolA and TolQ are delayed from their septal recruitment by the multiple interactions of TolA with TolB-Pal in the cell envelope providing a new example of temporal regulation of proteins recruitment at the septum.     

Oligodendrocyte-derived transcellular signaling regulates axonal energy metabolism

The unique morphology and functionality of central nervous system (CNS) neurons necessitate specialized mechanisms to maintain energy metabolism throughout long axons and extensive terminals. Oligodendrocytes (OLs) enwrap CNS axons with myelin sheaths in a multilamellar fashion. Apart from their well-established function in action potential propagation, OLs also provide intercellular metabolic support to axons by transferring energy metabolites and delivering exosomes consisting of proteins, lipids, and RNAs. OL-derived metabolic support is crucial for the maintenance of axonal integrity; its dysfunction has emerged as an important player in neurological disorders that are associated with axonal energy deficits and degeneration. In this review, we discuss recent advances in how these transcellular signaling pathways maintain axonal energy metabolism in health and neurological disorders.     

Neutralizing Antibodies Against Allosteric Proteins: Insights From a Bacterial Adhesin

Allosteric proteins transition between ‘inactive’ and ‘active’ states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH – the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.