The ubiquitin-proteasome system and autophagy: Coordinated and independent activities

The living cell is an ever changing, responsive, and adaptive environment where proteins play key roles in all processes and functions. While the scientific community focused for a long time on the decoding of the information required for protein synthesis, little attention was paid to the mechanisms by which proteins are removed from the cell. We now realize that the timely and proper activity of proteins is regulated to a large extent by their degradation; that cellular coping with different physiological cues and stress conditions depends on different catabolic pathways; and that many pathological states result from improper protein breakdown.There are two major protein degradation systems in all eukaryotic cells—the ubiquitin- proteasome and the autophagy-lysosome. The two systems are highly regulated, and—via degradation of a broad array of proteins—are responsible for maintenance of protein homeostasis and adaptation to environmental changes. Each is comprised of numerous components responsible for its coordinated function, and together they encompass a considerable fraction of the entire genome.In this review, we shall discuss the common and diverse characteristics of the ubiquitin-proteasome system (UPS) and autophagy—their substructure, mechanisms of action, function and concerted regulation under varying pathophysiological conditions.     

Native Size-Exclusion Chromatography–Based Mass Spectrometry Reveals New Components of the Early Heat Shock Protein 90 Inhibition Response Among Limited Global Changes

The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery—suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these—the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex—as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459.     

A new Escherichia coli gene,fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

A program to find regions of similarity between homologous protein sequences using dotmatrix analysis

MATRIX is a program designed primarily to enable the user to visualize all regions of similarity between two proteins at a glance. The program helps the user to see where they are similar—at what relative positions in the amino acid sequences of the two proteins in question does the similarity exist; how they are similar—what functional characteristics the two similar sequences have in common; and to what extent they are similar—is the similarity significant, if so how significant relative to other similar sequences in the protein. This is achieved by constructing a diagram in which quantitative parameters of amino acids are used to compare every amino acid residue of the first protein with every amino acid residue of the second.Another function of the program is, given two sets of atomic coordinates—either of different proteins or for the same protein (for self-comparison)—to demonstrate which residues of the two proteins, when the two proteins are superimposed upon each other, appear in the same space (or are close to each other).     

Studying pressure denaturation of a protein by molecular dynamics simulations

Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This “unfolding‐up‐on‐squeezing” is counter‐intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results—that pressure denatured states are water‐swollen, and theoretical results—that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states—their water‐swollen nature, retention of secondary structure, and overall compactness—mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure‐dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately ?60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500–2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water‐swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties and pressure stability of proteins, and can be potentially extended for applications to protein complexes and assemblies. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Ubiquilin4 is an adaptor protein that recruits Ubiquilin1 to the autophagy machinery

Ubiquilins (Ubqlns)—a family of ubiquitin‐binding proteins—are involved in several protein degradation pathways and have been implicated in various neurodegenerative diseases. Ubqln1 regulates autophagosome maturation during autophagy‐mediated degradation. We now show that Ubqln4 mediates the interaction between Ubqln1 and the autophagy machinery by recruiting Ubqln1 to LC3. This targeting of Ubqln1 to autophagosomes requires the Ubqln4 UBL domain and the Ubqln1 UBA domain. This study identifies a new role for Ubqln4, expanding the role for Ubqlns in protein degradation.     

On the Regularities of the Polar Profiles of Proteins Related to Ebola Virus Infection and their Functional Domains

The number of fatalities and economic losses caused by the Ebola virus infection across the planet culminated in the havoc that occurred between August and November 2014. However, little is known about the molecular protein profile of this devastating virus. This work represents a thorough bioinformatics analysis of the regularities of charge distribution (polar profiles) in two groups of proteins and their functional domains associated with Ebola virus disease: Ebola virus proteins and Human proteins interacting with Ebola virus. Our analysis reveals that a fragment exists in each of these proteins—one named the “functional domain”—with the polar profile similar to the polar profile of the protein that contains it. Each protein is formed by a group of short sub-sequences, where each fragment has a different and distinctive polar profile and where the polar profile between adjacent short sub-sequences changes orderly and gradually to coincide with the polar profile of the whole protein. When using the charge distribution as a metric, it was observed that it effectively discriminates the proteins from their functional domains. As a counterexample, the same test was applied to a set of synthetic proteins built for that purpose, revealing that any of the regularities reported here for the Ebola virus proteins and human proteins interacting with Ebola virus were not present in the synthetic proteins. Our results indicate that the polar profile of each protein studied and its corresponding functional domain are similar. Thus, when building each protein from its functional domai—adding one amino acid at a time and plotting each time its polar profile—it was observed that the resulting graphs can be divided into groups with similar polar profiles.     

Pulmonary surfactant protein B carried by HDL predicts incident CVD in patients with type 1 diabetes

Atherosclerotic CVD is the major cause of death in patients with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome have been shown to associate with prevalent CVD in T1DM. We therefore sought to determine which proteins carried by HDL might predict incident CVD in patients with T1DM. Using targeted MS/MS, we quantified 50 proteins in HDL from 181 T1DM subjects enrolled in the prospective Coronary Artery Calcification in Type 1 Diabetes study. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). We found that only one HDL protein—SFTPB (pulmonary surfactant protein B)—predicted incident CVD in all the models tested. In a fully adjusted model that controlled for lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12–4.21, P = 0.022). In addition, plasma fractionation demonstrated that SFTPB is nearly entirely bound to HDL. Although previous studies have shown that high plasma levels of SFTPB associate with prevalent atherosclerosis only in smokers, we found that SFTPB predicted incident CVD in T1DM independently of smoking status and a wide range of confounding factors, including HDL-C, LDL-C, and triglyceride levels. Because SFTPB is almost entirely bound to plasma HDL, our observations support the proposal that SFTPB carried by HDL is a marker—and perhaps mediator—of CVD risk in patients with T1DM.     

Taste receptor proteins

The chemistry of taste has eluded widespread investigation until only recently. Of the four basic taste qualities — sweet, salty, sour and bitter, only sweet and to some lesser extent — bitter — have had inroads made on the molecular level. Carbon-14 labeled sugars are preferently bound to taste bud proteins versus other non-sensory proteins. The binding of the sugars is in good agreement with their relative sweetness and graded conformational changes in protein molecules are seen, by a number of methods, upon these interactions. The protein may occur in two forms — soluble and membrane bound, and the bound form can be solubilized. Unique proteins are present in taste buds and would thus differentiate them from other non-sensory proteins. The sweet-sensitive protein activity has been studied in a number of animals, but purification and characterization in all has not proceeded to the point wherein comparisons can be made. Bitter reception has been suggested as the property of a protein from pig tongues, and an enzyme — phosphodiesterase has been found to be both activated and inhibited by bitter compounds. Lipids from gustatory tissue have been suggested as candidates for receptors for salty, sour and bitter, but not sweet stimulants.     

Non‐allosteric enzyme switches possess larger effector‐induced changes in thermodynamic stability than their non‐switch analogs

The ability to regulate cellular protein activity offers a broad range of biotechnological and biomedical applications. Such protein regulation can be achieved by modulating the specific protein activity or through processes that regulate the amount of protein in the cell. We have previously demonstrated that the nonhomologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β‐lactamase (BLA) can result in genes that confer maltose‐dependent resistance to β‐lactam antibiotics even though the encoded proteins are not allosteric enzymes. We showed that these phenotypic switches—named based on their conferral of a switching phenotype to cells—resulted from a specific interaction with maltose in the cell that increased the switches cellular accumulation. Since phenotypic switches represent an important class of engineered proteins for basic science and biotechnological applications in vivo, we sought to elucidate the phenomena behind the increased accumulation and switching properties. Here, we demonstrate the key role for the linker region between the two proteins. Experimental evidence supports the hypothesis that in the absence of their effector, some phenotypic switches possess an increased rate of unfolding, decreased conformational stability, and increased protease susceptibility. These factors alone or in combination serve to decrease cellular accumulation. The effector functions to increase cellular accumulation by alleviating one or more of these defects. This perspective on the mechanism for phenotypic switching will aid the development of design rules for switch construction for applications and inform the study of the regulatory mechanisms of natural cellular proteins.     

Exit of proteins and fragments thereof from mitochondria is accelerated by the import of cytosolic synthesized proteins

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria. Incubation of ³⁵S-methionine labeled mitochondria from rat hepatocytes with proteins synthesized in a cell-free system, using messenger RNA from rat liver, dramatically increased the release of mitochondrial proteins and fragments thereof into the medium. Since the synthesized proteins include cytosolic precursors of mitochondrial proteins, our results strongly suggest that import of proteins from the cytosol into mitochondria influences the half-life of proteins in these organelles. The use of this simple approach — i.e. combining the study of protein import and exit with mitochondria — to further clarify intracellular protein turnover and its regulation is suggested.     

Cotranslational Folding of a Pentarepeat β-Helix Protein

It is becoming increasingly clear that many proteins start to fold cotranslationally before the entire polypeptide chain has been synthesized on the ribosome. One class of proteins that a priori would seem particularly prone to cotranslational folding is repeat proteins, that is, proteins that are built from an array of nearly identical sequence repeats. However, while the folding of repeat proteins has been studied extensively in vitro with purified proteins, only a handful of studies have addressed the issue of cotranslational folding of repeat proteins. Here, we have determined the structure and studied the cotranslational folding of a β-helix pentarepeat protein from the human pathogen Clostridium botulinum—a homolog of the fluoroquinolone resistance protein MfpA—using an assay in which the SecM translational arrest peptide serves as a force sensor to detect folding events. We find that cotranslational folding of a segment corresponding to the first four of the eight β-helix coils in the protein produces enough force to release ribosome stalling and that folding starts when this unit is ~ 35 residues away from the P-site, near the distal end of the ribosome exit tunnel. An additional folding transition is seen when the whole PENT moiety emerges from the exit tunnel. The early cotranslational formation of a folded unit may be important to avoid misfolding events in vivo and may reflect the minimal size of a stable β-helix since it is structurally homologous to the smallest known β-helix protein, a four-coil protein that is stable in solution.     

Exploring Electron Transfer Between Heme Proteins of Cytochrome c Super Family in Biosensors: A Molecular Mechanics Study

Abstract

Electron transfer between heme proteins with mediators plays an important role in the fabrication of sensitive bio-nano sensors. Heme protein Cytochrome c (pdb code—1HRC) was chosen as the mediator with Cytochrome c′ (pdb code—1A7V) as the probe protein for our investigation on the electron transfer process. We used the software GRAMM, HEX, and MACRODOX to build the protein complex with further evaluation by GROMACS potential. After molecular mechanics refinement by GROMACS the protein complexes were evaluated in terms of the following criteria: Hydrophobic packing, proximity of the hemes, hydrogen bonds, enthalpy and entropy of binding. The free energy was calculated for each complex to derive the feasible stable models. The combined electron transport of the chosen geometric models was evaluated to choose the possible models. Electrostatic potential was calculated using the program APBS around the heme in the presence and absence of other proteins. From our studies, we derived multiple feasible models and possible electronic path. These studies helped us to understand the relay mechanism between the two proteins and to design mutant proteins by rational site directed mutagenesis to enhance the redox potential and thereby improving the signal to noise ratio in amperometric bionano sensors.     

Mapping the protein–protein and genetic interactions of cancer to guide precision medicine

Massive efforts to sequence cancer genomes have compiled an impressive catalogue of cancer mutations, revealing the recurrent exploitation of a handful of ‘hallmark cancer pathways’. However, unraveling how sets of mutated proteins in these and other pathways hijack pro-proliferative signaling networks and dictate therapeutic responsiveness remains challenging. Here, we show that cancer driver protein–protein interactions are enriched for additional cancer drivers, highlighting the power of physical interaction maps to explain known, as well as uncover new, disease-promoting pathway interrelationships. We hypothesize that by systematically mapping the protein–protein and genetic interactions in cancer—thereby creating Cancer Cell Maps—we will create resources against which to contextualize a patient’s mutations into perturbed pathways/complexes and thereby specify a matching targeted therapeutic cocktail.     

Proteomic analysis of early lactation milk of the tammar wallaby (Macropus eugenii)

We report the successful use of 2D electrophoresis, MALDI MS/MS and chemical derivatisation protocols of guanidination and sulfonation to identify over 100 protein spots present in early marsupial milk (tammar wallaby) at 40 days lactation, where a limited translated genomic database is publicly available for cross species matching and protein identification. Of the proteins identified, 25 matched to 6 existing marsupial milk protein sequences in the NCBI database; another 6 were identified with high confidence to other mammals and have not previously been identified in marsupial milk. By using chemical derivatisation, the reliable identification of a further 81 proteins was achieved. The identified proteins could be grouped into three main functional categories — transport, nutrition and immune protection. All these proteins play a potential role in determining growth and immunological protection of the highly altricial marsupial young at 40 days after birth.     

UNDERSTANDING INTERCELLULAR INTERACTIONS AND CELL ADHESION: LESSONS FROM STUDIES ON PROTEIN—METAL INTERACTIONS

To understand cell—cell interactions and the interactions of cells to non-biological materials, studies on binding forces between cellular proteins and between proteins and non-biological material such as metal surfaces are essential. The adsorption of proteins to solid—water interfaces is a multifactorial and a multistep process. First steps are determined by long-range interactions where surface properties such as hydrophobicity, distribution of charged groups, ion concentrations and pH play important roles. In later steps structural rearrangements in the protein molecule and dehydration effects become more important making the adsorption process often irreversible. In the following we demonstrate that protein A and tubulin have a specific type of interaction to metal surfaces probably as an intermediate step in the adsorption process. The proteins were attached to the tip of a microfabricated cantilever in such a way that only one molecule interacts with the surface. By recording force—distance curves with an atomic force microscope the adhesion forces of single molecules binding to gold, titanium and indium—tinoxid surfaces were measured.     

Drosophila Protein interaction Map (DPiM)

Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein—especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex “map” provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.