Supramolecular Protein Complexes Research Articles

Hereditary periodic fevers in children

Fevers in children is a pressing challenge in paediatrics, as they are one of the most common symptoms people seek medical help. Autoinflammatory diseases (AIDs) constitute a group of diseases, where fever is one of the main symptoms, and fever attacks have a certain frequency and duration. Autoinflammatory diseases are rooted in the systemic aseptic inflammation associated with activation of the innate immune system, without elevated levels of autoantibodies. The most common autoinflammatory diseases include hereditary periodic fevers, among which are familial Mediterranean fever (FMF), HIDS/MKD, CAPS-, TRAPS-syndromes and rarer diseases (deficiency of natural interleukin receptor antagonists). The pathogenesis of this group of diseases is based on the process of accelerated formation of a supramolecular protein complex (inflammasome), which subsequently leads to the transition of the inactive form of IL1β into its active form. The clinical presentations of these diseases may be similar and include episodes of fever, abdominal pain, arthralgia, various rashes, etc., as well as an increase in acute phase parameters. These diseases are characterized by symptoms at certain intervals; they usually have intervals without showing any symptoms. Familial Mediterranean fever is characterized by a certain ethnic background (Armenians, Turks, Arabs, Jews). Today, molecular genetic testing is the most accurate method for diagnosing familial Mediterranean fever. Current therapy aims to prevent attacks of the disease, but it is also important to use symptomatic therapy to relieve the child’s condition when an attack has already developed. Modern treatment strategies include both drugs that have long been used in paediatric practice (colchicine, non-steroidal anti-inflammatory drugs (ibuprofen), glucocorticosteroids) and novel genetically engineered biological drugs (interleukin antagonists and tumour necrosis factor inhibitors, etc.).

Stochastic chain termination in bacterial pilus assembly

Adhesive type 1 pili from uropathogenic Escherichia coli strains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reaction in vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembled in vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction. In addition, we demonstrate that FimI contributes to anchoring the pilus to the outer membrane and report the crystal structures of (i) FimI in complex with the assembly chaperone FimC, (ii) the FimI-FimC complex bound to the N-terminal domain of FimD, and (iii) a ternary complex between FimI, FimA and FimC that provides structural insights on pilus assembly termination and pilus anchoring by FimI.

SNARE Modulators and SNARE Mimetic Peptides.

The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins play a central role in most forms of intracellular membrane trafficking, a key process that allows for membrane and biocargo shuffling between multiple compartments within the cell and extracellular environment. The structural organization of SNARE proteins is relatively simple, with several intrinsically disordered and folded elements (e.g., SNARE motif, N-terminal domain, transmembrane region) that interact with other SNAREs, SNARE-regulating proteins and biological membranes. In this review, we discuss recent advances in the development of functional peptides that can modify SNARE-binding interfaces and modulate SNARE function. The ability of the relatively short SNARE motif to assemble spontaneously into stable coiled coil tetrahelical bundles has inspired the development of reduced SNARE-mimetic systems that use peptides for biological membrane fusion and for making large supramolecular protein complexes. We evaluate two such systems, based on peptide-nucleic acids (PNAs) and coiled coil peptides. We also review how the self-assembly of SNARE motifs can be exploited to drive on-demand assembly of complex re-engineered polypeptides.

Electronic Structure of de Novo Peptide ACC-Hex from First Principles.

Proteins are promising components for bioelectronic devices due in part to their biocompatibility, flexibility, and chemical diversity, which enable tuning of material properties. Indeed, an increasingly broad range of conductive protein supramolecular materials have been reported. However, due to their structural and environmental complexity, the electronic structure, and hence conductivity, of protein assemblies is not well-understood. Here we perform an all-atom simulation of the physical and electronic structure of a recently synthesized self-assembled peptide antiparallel coiled-coil hexamer, ACC-Hex. Using classical molecular dynamics and first-principles density functional theory, we examine the interactions of each peptide, containing phenylalanine residues along a hydrophobic core, to form a hexamer structure. We find that while frontier electronic orbitals are composed of phenylalanine, the peptide backbone and remaining residues, including those influenced by solvent, also contribute to the electronic density. Additionally, by studying dimers extracted from the hexamer, we show that structural distortions due to atomic fluctuations significantly impact the electronic structure of the peptide bundle. These results indicate that it is necessary to consider the full atomistic picture when using the electronic structure of supramolecular protein complexes to predict electronic properties.

Rational Design of Self-Assembling Supramolecular Protein Nanostructures Utilizing the Cucurbit[8]Uril Macrocyclic Host.

Self-assembly is a phenomenon that governs molecular structural organization in nature, therefore raising research interest for the fabrication of novel nanomaterials with diverse applications in biocatalysis, biomedicine, material templating, and biosensor development. In this chapter we provide protocols for the development of supramolecular protein complexes based on host-guest interactions in the presence of the macrocyclic host, cucurbit[8]uril (CB[8]). CB[8] is reported to exhibit high binding affinity towards the tripeptide Phe-Gly-Gly (FGG), therefore it can be utilized as a selective adhesive of protein molecules, after fusion of FGG to an accessible protein surface.

The kinetochore protein NNF1 has a moonlighting role in the vegetative development of Arabidopsis thaliana

The kinetochore is a supramolecular protein complex assembled on the chromosomes, essential for faithful segregation of the genome during cell divisions. More than 100 proteins are known to constitute the eukaryotic kinetochore architecture, primarily identified using non-plant organisms. A majority of them are fast evolving and are under positive selection. Thus, functional characterization of the plant kinetochore proteins is limited as only a few conserved orthologs sharing sequence similarity with their animal counterparts have been examined. Here, we report the functional characterization of the Arabidopsis thaliana homolog of the yeast NNF1/human PMF1 outer kinetochore protein and show that it has both kinetochore and non-kinetochore functions in plant growth and development. Knockout of NNF1 causes embryo lethality implying its essential role in cell division. AtNNF1 interacts with MIS12 in Y2H and co-immunoprecipitation assays, confirming it is one of the constituents of the plant MIS12 complex. GFP-NNF1 localizes to the kinetochore, rescuing the embryo lethal nnf1-1-/- phenotype, but the rescued plants (GFP-NNF1nnf1-/- ) are dwarf, displaying hypomorphic phenotypes with no evidence of mitotic or meiotic segregation defects. GFP-NNF1nnf1-/- dwarf plants have reduced levels of endogenous polyamines, which are partially rescued to wild-type levels upon exogenous application of polyamines. Mutations in the putative leucine zipper-like binding motif of NNF1 gave rise to a dominant-negative tall plant phenotype reminiscent of constitutive gibberellic acid (GA) action. These contrasting hypomorphic dwarf and antimorphic tall phenotypes facilitated us to attribute a moonlighting role to Arabidopsis NNF1 affecting polyamine and GA metabolism apart from its primary role in kinetochores.

Visualizing transiently associated catalytic domains in assembly-line biosynthesis using cryo-electron microscopy

While cryo-electron microscopy (cryo-EM) has revolutionized the structure determination of supramolecular protein complexes that are refractory to structure determination by X-ray crystallography, structure determination by cryo-EM can nonetheless be complicated by excessive conformational flexibility or structural heterogeneity resulting from weak or transient protein–protein association. Since such transient complexes are often critical for function, specialized approaches must be employed for the determination of meaningful structure–function relationships. Here, we outline examples in which transient protein–protein interactions have been visualized successfully by cryo-EM in the biosynthesis of fatty acids, polyketides, and terpenes. These studies demonstrate the utility of chemical crosslinking to stabilize transient protein–protein complexes for cryo-EM structural analysis, as well as the use of partial signal subtraction and localized reconstruction to extract useful structural information out of cryo-EM data collected from inherently dynamic systems. While these approaches do not always yield atomic resolution insights on protein–protein interactions, they nonetheless enable direct experimental observation of complexes in assembly-line biosynthesis that would otherwise be too fleeting for structural analysis.

Exploring proteins in living cells

<p indent="0mm">Proteins have been studied for over <sc>200 years,</sc> but much of that work has been performed <italic>in vitro</italic>. This article briefly summarized the current status of protein exploration in living cells, and then presented some of my own representative findings in this field. First, I summarized the major methodologies hitherto developed for studying proteins <italic>in vivo</italic>, including visualizing a target protein by fusing it with the green fluorescent protein; investigating the tertiary structure of the target protein via in-cell nuclear magnetic resonance spectroscopy; and exploring the behaviors of a target protein by genetically labeling it with different types of unnatural amino acids. Next, I described how my laboratory applied and modified an <italic>in vivo</italic> methodology in which the genetic introduction of a photoactivatable, unnatural amino acid at a selected residue position in a target protein then mediates crosslinking of that protein with other proteins when the cells are irradiated by UV light. By improving that method, my laboratory contributed the following: (1) The creation of a bacterial strain with a genome that has a gene that encodes the orthogonal aminoacyl-tRNA synthetase and one that encodes the tRNA that are both needed to incorporate an unnatural amino acid (i.e., Bpa) into any target protein. (2) The development of a system in which the unnatural amino acid is randomly introduced at each of the target protein’s amino acid residue positions and, after detecting a meaningful interaction, DNA sequencing subsequently identifies that unnatural amino acid’s exact position. (3) The incorporation of the unnatural amino acid into a target protein by directly modifying the endogenous target gene in the genomic DNA so that the variant protein is produced spatiotemporally identical to that of the wild-type protein. (4) The development of a high-throughput polyacrylamide gel electrophoresis system in which 384 protein samples can be analyzed in a single gel. Finally, I described a few representative results from my lab by using <italic>in vivo</italic> protein photocrosslinking analysis: (1) The serendipitous discovery of a reversible subcellular structure (regrowth-delay body) that forms when a highly selected group of important proteins assembles only in non-dividing bacterial cells, and then dissolves when the cells resume growth. Their presence thus marks dormant persister bacterial cells that are known to tolerate antibiotics. (2) A revelation of a “gear shifting” mechanism that modulates bacterial ATP synthase activity via adaptation of its ε-subunit to the “inserted” or “non-inserted” conformation as the cell responds to a variety of favorable to unfavorable cellular conditions. (3) The identification of a supramolecular protein complex that is composed of proteins present in the cytoplasm, inner membrane, periplasm and outer membrane of Gram-negative bacteria, and that mediates the biogenesis of a group of outer membrane proteins. (4) The unveiling of a unique way that the acid stress chaperone HdeA acts. It exhibits biological activity while its tertiary structure becomes largely disordered, and then it protects other chaperones under acidic conditions. (5) A clarification that the long-designated bifunctional protein DegP acts primarily as a proteinase with little chaperone activity. (6) The discovery that small heat shock proteins are activated in a temperature-responsive, cascade manner that allows them to significantly increase their biological activity at high temperatures. I then conclude with a list of unresolved issues regarding protein exploration in living cells.

Exploiting Complex Fluorophore Interactions to Monitor Virus Capsid Disassembly.

Supramolecular protein complexes are the corner stone of biological processes; they are essential for many biological functions. Unraveling the interactions responsible for the (dis)assembly of these complexes is required to understand nature and to exploit such systems in future applications. Virus capsids are well-defined assemblies of hundreds of proteins and form the outer shell of non-enveloped viruses. Due to their potential as a drug carriers or nano-reactors and the need for virus inactivation strategies, assessing the intactness of virus capsids is of great interest. Current methods to evaluate the (dis)assembly of these protein assemblies are experimentally demanding in terms of instrumentation, expertise and time. Here we investigate a new strategy to monitor the disassembly of fluorescently labeled virus capsids. To monitor surfactant-induced capsid disassembly, we exploit the complex photophysical interplay between multiple fluorophores conjugated to capsid proteins. The disassembly of the capsid changes the photophysical interactions between the fluorophores, and this can be spectrally monitored. The presented data show that this low complexity method can be used to study and monitor the disassembly of supramolecular protein complexes like virus capsids. However, the range of labeling densities that is suitable for this assay is surprisingly narrow.

Inflammasomes in Alveolar Bone Loss.

Bone remodeling is tightly controlled by osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Fine tuning of the osteoclast–osteoblast balance results in strict synchronization of bone resorption and formation, which maintains structural integrity and bone tissue homeostasis; in contrast, dysregulated bone remodeling may cause pathological osteolysis, in which inflammation plays a vital role in promoting bone destruction. The alveolar bone presents high turnover rate, complex associations with the tooth and periodontium, and susceptibility to oral pathogenic insults and mechanical stress, which enhance its complexity in host defense and bone remodeling. Alveolar bone loss is also involved in systemic bone destruction and is affected by medication or systemic pathological factors. Therefore, it is essential to investigate the osteoimmunological mechanisms involved in the dysregulation of alveolar bone remodeling. The inflammasome is a supramolecular protein complex assembled in response to pattern recognition receptors and damage-associated molecular patterns, leading to the maturation and secretion of pro-inflammatory cytokines and activation of inflammatory responses. Pyroptosis downstream of inflammasome activation also facilitates the clearance of intracellular pathogens and irritants. However, inadequate or excessive activity of the inflammasome may allow for persistent infection and infection spreading or uncontrolled destruction of the alveolar bone, as commonly observed in periodontitis, periapical periodontitis, peri-implantitis, orthodontic tooth movement, medication-related osteonecrosis of the jaw, nonsterile or sterile osteomyelitis of the jaw, and osteoporosis. In this review, we present a framework for understanding the role and mechanism of canonical and noncanonical inflammasomes in the pathogenesis and development of etiologically diverse diseases associated with alveolar bone loss. Inappropriate inflammasome activation may drive alveolar osteolysis by regulating cellular players, including osteoclasts, osteoblasts, osteocytes, periodontal ligament cells, macrophages, monocytes, neutrophils, and adaptive immune cells, such as T helper 17 cells, causing increased osteoclast activity, decreased osteoblast activity, and enhanced periodontium inflammation by creating a pro-inflammatory milieu in a context- and cell type-dependent manner. We also discuss promising therapeutic strategies targeting inappropriate inflammasome activity in the treatment of alveolar bone loss. Novel strategies for inhibiting inflammasome signaling may facilitate the development of versatile drugs that carefully balance the beneficial contributions of inflammasomes to host defense.

Rational thermostabilisation of four-helix bundle dimeric de novo proteins

The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

Poly(styrene-co-maleic acid)-mediated isolation of supramolecular membrane protein complexes from plant thylakoids

Derivatives of poly(styrene-co-maleic acid) (pSMA), have recently emerged as effective reagents for extracting membrane protein complexes from biological membranes. Despite recent progress in using SMAs to study artificial and bacterial membranes, very few reports have addressed their use in studying the highly abundant and well characterized thylakoid membranes. Recently, we tested the ability of twelve commercially available SMA copolymers with different physicochemical properties to extract membrane protein complexes (MPCs) from spinach thylakoid membrane. Based on the efficacy of both protein and chlorophyll extraction, we have found five highly efficient SMA copolymers: SMA® 1440, XIRAN® 25010, XIRAN® 30010, SMA® 17352, and SMA® PRO 10235, that show promise in extracting MPCs from chloroplast thylakoids. To further advance the application of these polymers for studying biomembrane organization, we have examined the composition of thylakoid supramolecular protein complexes extracted by the five SMA polymers mentioned above. Two commonly studied plants, spinach (Spinacia oleracea) and pea (Pisum sativum), were used for extraction as model biomembranes. We found that the pSMAs differentially extract protein complexes from spinach and pea thylakoids. Based on their differential activity, which correlates with the polymer chemical structure, pSMAs can be divided into two groups: unfunctionalized polymers and ester derivatives.

Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging

The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.

A Biological Nanomachine at Work: Watching the Cellulosome Degrade Crystalline Cellulose.

The cellulosome is a supramolecular multienzymatic protein complex that functions as a biological nanomachine of cellulosic biomass degradation. How the megadalton-size cellulosome adapts to a solid substrate is central to its mechanism of action and is also key for its efficient use in bioconversion applications. We report time-lapse visualization of crystalline cellulose degradation by individual cellulosomes from Clostridium thermocellum by atomic force microscopy. Upon binding to cellulose, the cellulosomes switch to elongated, even filamentous shapes and morph these dynamically at below 1 min time scale according to requirements of the substrate surface under attack. Compared with noncomplexed cellulases that peel off material while sliding along crystalline cellulose surfaces, the cellulosomes remain bound locally for minutes and remove the material lying underneath. The consequent roughening up of the surface leads to an efficient deconstruction of cellulose nanocrystals both from the ends and through fissions within. Distinct modes of cellulose nanocrystal deconstruction by nature’s major cellulase systems are thus revealed.

The NLRP3 inflammasome: a new player in neurological diseases.

Inflammasomes are supramolecular protein complexes implicated in the detection of pathogens or danger-associated molecules and are responsible for mounting the first line of innate immune response to counteract these signals and restore tissue homeostasis. Among different inflammasomes identified so far, NLRP3 is of main interest since mutations in Nlrp3 gene are associated with autoinflammatory diseases such as Muckle–Wells syndrome, neonatal onset multisystem inflammatory disease, and familial cold urticaria/autoinflammatory syndrome. On the other hand, whereas other inflammasomes are mainly detectors of specific molecular motifs, NLRP3 is acting as a general sensor of cellular perturbations including potassium efflux, lysosomal damage, and ROS production. Besides this central role of NLRP3 in inflammation, recent publications show that the NLRP3 inflammasome is also involved in the physiopathology of several neurological disorders including Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. This review gives an overview of the established functions of the NLRP3 inflammasome in mediating inflammation in macrophages and describes its recently discovered roles in neurological disorders in promoting neuroinflammation, as well as modulating key proteins mediating the disorders. Finally, we discuss the targeting of NLRP3 in neurological diseases and present some examples of NLRP3 inhibitors that could be used in neurological disorder treatments.

Recent developments in isotope-aided NMR methods for supramolecular protein complexes –SAIL aromatic TROSY

BackgroundThe structure-function relationships for large protein complexes at the atomic level would be comprehensively understood, if hitherto unexplored aromatic ring NMR signals became accessible in addition to the currently used backbone amide and side-chain methyl signals. MethodsThe 82 kDa malate synthase G (MSG) proteins, selectively labeled with Trp and Phe bearing relaxation optimized isotope-labeled rings, were prepared to investigate the optimal conditions for obtaining the aromatic TROSY spectra. ResultsThe MSG proteins, selectively labeled with either [δ1,ε1,ε3,η2]-SAIL Trp or ζ-SAIL Phe, provided well-separated, narrow TROSY signals for the 12 Trp and 19 Phe residues in MSG. The signals were assigned sequence-specifically, using the set of single amino acid substitution mutants. The site-specific substitution of each Phe with Tyr or Leu induced substantial chemical shifts for the other aromatic ring signals, allowing us to identify the aromatic clusters in MSG, which were comparable to the structural domains proposed previously. ConclusionsWe demonstrated that the aromatic ring 13CH pairs without directly bonded 13C and adjacent 1H spins provide surprisingly narrow TROSY signals, if the rings are surrounded by fully deuterated amino acids. The observed signals can be readily assigned by either the single amino acid substitution or the NOEs between the aromatic and methyl protons, if the methyl assignments are available. General significanceThe method described here should be generally applicable for difficult targets, such as proteins in lipid bilayers or possibly in living cells, thus providing unprecedented opportunities to use these new probes in structural biology.

Native supramolecular protein complexes in pulmonary surfactant: Evidences for SP-A/SP-B interactions

Pulmonary surfactant is a lipid-protein complex which coats lung alveoli. It displays the essential function of reducing surface tension at the air-liquid interface, avoiding alveolar collapse during expiration. The optimized biophysical properties of surfactant rely on its defined composition, constituted mainly by phospholipids and tiny amounts of lipid-associated specific proteins. Due to the highly hydrophobic nature of surfactant, organic solvents have been traditionally employed to obtain and characterize surfactant lipids and proteins, very likely leading to disruption of native interactions among its components. In the present work we have addressed the search of native protein complexes in pulmonary surfactant, which could have an essential role in the optimal function of the system. By solubilizing native lipid-protein membranes of surfactant with non-denaturing detergents, and with the use of a two-dimensional electrophoresis strategy, we have been able to detect the presence of supramolecular complexes composed of surfactant proteins SP-A, SP-B and SP-C. Furthermore, by co-immunoprecipitation assays, we have confirmed for the first time the existence of a direct interaction between SP-A and SP-B, an important feature which could explain the known functional cooperation of both proteins in several aspects of surfactant biology. SignificanceThis paper deepens for the first time in the existence of complex interaction networks of surfactant proteins in native surfactant membranes. By the use of non-denaturing detergents, two-dimensional electrophoresis and immunoprecipitation, we have been able to make progress in the elucidation of native protein complexes in this essential system, that had been previously hindered by the classical purification protocols employing organic solvents. In this work, we have described the presence of interactions between SP-B and SP-A, two important proteins whose functional cooperation has been broadly reported in the literature. Pioneer determination of such native complexes could have potential implications for understanding the wide variety of roles of pulmonary surfactant system.

Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions.

The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho-GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a "mix-and-match" approach containing the specific Rho-inhibiting clostridial C3 enzyme and three PMN-binding peptides using an avidin platform. Selective delivery of the C3 Rho-inhibitor with these complexes into the cytosol of human neutrophil-like NB-4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type-selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post-traumatic injury.

Ubiquitin-Independent Degradation of Proteins in Proteasomes

Proteasomes are large supramolecular protein complexes present in all prokaryotic and eukaryotic cells, where they perform targeted degradation of intracellular proteins. Until recently, it was generally accepted that prior to proteolytic degradation in proteasomes the proteins had to be targeted by ubiquitination: ATP-dependent attachment of (typically four sequential) residues of the low-molecular protein, ubiquitin, which involves the ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin ligase. Cytoplasmic and nucleoplasmic proteins labeled in this way are then digested in 26S proteasomes. However, it becomes increasingly clear that using this route the cell eliminates only a part of unwanted proteins. Many proteins can be cleaved by the 20S proteasome in an ATP-independent manner and without previous ubiquitination. Ubiquitin-independent degradation of proteins in proteasomes is a relatively new area of studies of the role of the ubiquitin-proteasome system. However, recent data obtained in this direction already correct existing concepts about proteasomal degradation of proteins and its regulation. Ubiquitin-independent proteasome degradation needs the main structural precondition in proteins: the presence of unstructured regions in the amino acid sequences that provide interaction with the proteasome. Taking into consideration that in humans almost half of all genes encode proteins that contain a certain proportion of intrinsically disordered regions, it appears that the list of proteins undergoing ubiquitin-independent degradation will demonstrate a further increase. Since 26S proteasomes account for only 30% of the total proteasome content in mammalian cells, most of the proteasomes exist in the form of 20S complexes. The latter suggests that ubiquitin-independent proteolysis performed by the 20S proteasome is a natural process of removing damaged proteins from the cell and maintaining a constant level of intrinsically disordered proteins. In this case, the functional overload of proteasomes in aging and/or other types of pathological processes, if it is not accompanied by triggering more radical mechanisms for the elimination of damaged proteins, organelles, and whole cells, has the most serious consequences for the whole organism.

TRAPPC11 and GOSR2 mutations associate with hypoglycosylation of \u03b1-dystroglycan and muscular dystrophy

BackgroundTransport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation.Case presentationSubject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and liver abnormalities. Her serum aminotransferases and creatine kinase were abnormally high. Subjects 2 and 3 are siblings from a family unrelated to subject 1. Both siblings displayed hypotonia, muscle weakness, low muscle bulk, and elevated creatine kinase levels. Subject 3 also developed a seizure disorder. Muscle biopsies from subjects 1 and 3 were severely dystrophic with abnormal immunofluorescence and western blotting indicative of α-dystroglycan hypoglycosylation. Compound heterozygous mutations in TRAPPC11 were identified in subject 1: c.851A>C and c.965+5G>T. Cellular biological analyses on fibroblasts confirmed abnormal membrane trafficking. Subject 3 was found to have compound heterozygous mutations in GOSR2: c.430G>T and c.2T>G. Cellular biological analyses on fibroblasts from subject 3 using two different model cargo proteins did not reveal defects in protein transport. No mutations were found in any of the genes currently known to cause dystroglycanopathy in either individual.ConclusionRecessive mutations in TRAPPC11 and GOSR2 are associated with congenital muscular dystrophy and hypoglycosylation of α-dystroglycan. This is the first report linking membrane trafficking proteins to dystroglycanopathy and suggests that these genes should be considered in the diagnostic evaluation of patients with congenital muscular dystrophy and dystroglycanopathy.