An enzyme in the test tube,and a transcription factor in the cell: Moonlighting proteins and cellular factors that affect their behavior

In the cell, expression levels, allosteric modulators, post‐translational modifications, sequestration, and other factors can affect the level of protein function. For moonlighting proteins, cellular factors like these can also affect the kind of protein function. This minireview discusses examples of moonlighting proteins that illustrate how a single protein can have different functions in different cell types, in different intracellular locations, or under varying cellular conditions. This variability in the kind of protein activity, added to the variability in the amount of protein activity, contributes to the difficulty in predicting the behavior of proteins in the cell.     

西伯利亚蓼谷胱甘肽转移酶和半胱氨酸合成酶基因在酿酒酵母中的共表达

谷胱甘肽转移酶和半胱氨酸合成酶在清除活性氧（reactive oxygen species,ROS）中起重要作用。采用0．36mol·L^-1 NaHCO3对西伯利亚蓼（Polygonum sibiricum）进行胁迫处理,荧光定量PCR分析表明这2个基因的表达受盐胁迫强烈诱导。为了分析2个基因是否具有抗盐能力以及其相互协同能力,从cDNA文库中获得谷胱甘肽转移酶（GST）和半胱氨酸合成酶（Cs）2个基因,分别将GST、CS和GST＋CS转入酿酒酵母（Saccharomyces cerevisiae）中,并分别命名转基因酵母为ty-gst、tycs和ty-gc。在1mol·L^-1 Na2C03和5mol·L^-1 NaCl胁迫处理下,转基因酵母（ty-gst、ty-cs和ty-gc）的耐盐能力均明显高于野生型酵母（㈣,而三者之间并无显著差别。在0．4mol·L^-1 NaCl胁迫处理下,转基因酵母（ty-gst、ty-cs和ty-gc）的抗氧化酶类相关基因SOD1、SOD2、GPX1和GPX3的表达量均低于野生型酵母（对照）（wy）,而CTA7表达量均高于野生型酵母（对照）（wy）。转基因酵母ty-cs在0．4mol·L^-1 NaCl胁迫处理前后其超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）和谷胱甘肽过氧化物酶（glutathione peroxJdase,GPX）的活性均表现为最高。     

Cysteine Racemization in Peptide Synthesis: A New and Easy Detection Method

A new method has been developed for the rapid determination of D-cysteine contents in synthetic peptides. It is based on the reduction of cystine residues, when present, with tris- alkylphosphines, selective derivatization of the cysteine residues with 4-vinylpyridine, followed by acid hydrolysis of the (4-pyridylethyl)cysteine –peptides. Baseline enantiomeric resolution of theD ,L -S-β-(4-pyridylethyl)cysteine, and thus quantification ofD - enantiomer contents at levels ≤1%, is easily achieved by capillary zone electrophoresis exploiting the host–guest complexation principle with crown ethers or by gas chromatography on chiral glass capillary columns upon conventional derivatization of the hydrolysate. The acid-stability of the (4-pyridylethyl)cysteine derivative prevents racemization via thiazoline intermediates and allows for standardization of the acid hydrolysis-dependent racemization.     

ExoCarta: A compendium of exosomal proteins and RNA

Exosomes, membrane microvesicles (40–100 nm) secreted by most cell types, can be isolated in several ways while characterizing them is heavily based on electron microscopy and, most importantly, the identification of exosome marker proteins. Researchers rely on the identification of certain exosomal marker proteins including Alix, CD9 and CD63 to confirm the presence of exosomes in their preparations. An evolutionary‐conserved set of protein molecules have been identified in most exosomes studied to date. However, with the complexity of tissue/cell type‐specific proteins being incorporated in the exosomes, some of these so‐called exosomal markers are not always present in all the exosomes. The presence of tissue/cell type‐specific proteins in exosomes allows researchers to isolate them using immunoaffinity capture methods. A compendium for exosomal proteomes will aid researchers in identifying proteins that were more commonly found in various exosomes (exosome markers) and those that are specific to certain tissue/cell type‐derived exosomes. Here, we describe ExoCarta, a compendium for proteins and RNA molecules identified in exosomes. ExoCarta is first of its kind and the resource is freely available to the scientific community through the web ( http://exocarta.ludwig.edu.au ). We believe that this community resource will be of great biological importance for any future exosome analyses.     

The next wave of interactomics: Mapping the SLiM-based interactions of the intrinsically disordered proteome

Short linear motifs (SLiMs) are a unique and ubiquitous class of protein interaction modules that perform key regulatory functions and drive dynamic complex formation. For decades, interactions mediated by SLiMs have accumulated through detailed low-throughput experiments. Recent methodological advances have opened this previously underexplored area of the human interactome to high-throughput protein–protein interaction discovery. In this article, we discuss that SLiM-based interactions represent a significant blind spot in the current interactomics data, introduce the key methods that are illuminating the elusive SLiM-mediated interactome of the human cell on a large scale, and discuss the implications for the field.     

hu.MAP 2.0: integration of over 15,000 proteomic experiments builds a global compendium of human multiprotein assemblies

A general principle of biology is the self‐assembly of proteins into functional complexes. Characterizing their composition is, therefore, required for our understanding of cellular functions. Unfortunately, we lack knowledge of the comprehensive set of identities of protein complexes in human cells. To address this gap, we developed a machine learning framework to identify protein complexes in over 15,000 mass spectrometry experiments which resulted in the identification of nearly 7,000 physical assemblies. We show our resource, hu.MAP 2.0, is more accurate and comprehensive than previous state of the art high‐throughput protein complex resources and gives rise to many new hypotheses, including for 274 completely uncharacterized proteins. Further, we identify 253 promiscuous proteins that participate in multiple complexes pointing to possible moonlighting roles. We have made hu.MAP 2.0 easily searchable in a web interface (http://humap2.proteincomplexes.org/), which will be a valuable resource for researchers across a broad range of interests including systems biology, structural biology, and molecular explanations of disease.     

Role of the Liver in Regulation of Body Cysteine and Taurine Levels: A Brief Review

The first-pass metabolism of dietary sulfur amino acids by the liver and the robust upregulation of hepatic cysteine dioxygenase activity in response to an increase in dietary protein or sulfur amino acid level gives the liver a primary role in the removal of excess cysteine and in the synthesis of taurine. Hepatic taurine synthesis is largely restricted by the low availability of cysteinesulfinate as substrate for cysteinesulfinate decarboxylase, and taurine production is increased when cysteinesulfinate increases in response to an increase in the hepatic cysteine concentration and the associated increase in cysteine dioxygenase activity. The upregulation of cysteine dioxygenase in the presence of cysteine is a consequence of diminished ubiquitination of cysteine dioxygenase and a slower rate of degradation by the 26S proteasome.     

Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.

The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.     

The life cycle and pathogenesis of human cytomegalovirus infection: lessons from proteomics

Viruses have coevolved with their hosts, acquiring strategies to subvert host cellular pathways for effective viral replication and spread. Human cytomegalovirus (HCMV), a widely-spread β-herpesvirus, is a major cause of birth defects and opportunistic infections in HIV-1/AIDS patients. HCMV displays an intricate system-wide modulation of the human cell proteome. An impressive array of virus–host protein interactions occurs throughout the infection. To investigate the virus life cycle, proteomics has recently become a significant component of virology studies. Here, we review the mass spectrometry-based proteomics approaches used in HCMV studies, as well as their contribution to understanding the HCMV life cycle and the virus-induced changes to host cells. The importance of the biological insights gained from these studies clearly demonstrate the impact that proteomics has had and can continue to have on understanding HCMV biology and identifying new therapeutic targets.     

Structures of designed armadillo repeat proteins binding to peptides fused to globular domains

Designed armadillo repeat proteins (dArmRP) are α‐helical solenoid repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term “peptide” not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N‐terminus of Green Fluorescent Protein or to the C‐terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein–peptide interactions are characterized, and should always be correlated with measurements in solution.     

Modular peptide binding: From a comparison of natural binders to designed armadillo repeat proteins

Several binding scaffolds that are not based on immunoglobulins have been designed as alternatives to traditional monoclonal antibodies. Many of them have been developed to bind to folded proteins, yet cellular networks for signaling and protein trafficking often depend on binding to unfolded regions of proteins. This type of binding can thus be well described as a peptide–protein interaction. In this review, we compare different peptide-binding scaffolds, highlighting that armadillo repeat proteins (ArmRP) offer an attractive modular system, as they bind a stretch of extended peptide in a repeat-wise manner. Instead of generating each new binding molecule by an independent selection, preselected repeats – each complementary to a piece of the target peptide – could be designed and assembled on demand into a new protein, which then binds the prescribed complete peptide. Stacked armadillo repeats (ArmR), each typically consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure which enables binding of peptides in extended conformation. A consensus-based design approach, complemented with molecular dynamics simulations and rational engineering, resulted in well-expressed monomeric proteins with high stability. Peptide binders were selected and several structures were determined, forming the basis for the future development of modular peptide-binding scaffolds.     

Synthesis and biochemical evaluation of highly enantiomerically pure (R,R)- and (S,S)-alexidine

Alexidine is in everyday human use as oral disinfectant and contact lens disinfectant. It is used as a mixture of stereoisomers. Since all of alexidine’s known biological targets are chiral, the biological activity of any of its chiral stereoisomers could be significantly higher than that of the mixture of stereoisomers. This makes a synthetic methodology for obtaining the individual enantiomers of the chiral diastereoisomer highly desirable. Here, we describe the first synthesis of both enantiomers of alexidine in high enantiomeric purity, and demonstrate their activity against the protein–protein interaction between the anti-apoptotic protein Bcl-x_L and the pro-apoptotic protein Bak.     

Rabphilin-3A: A Multifunctional Regulator of Synaptic Vesicle Traffic

We have investigated the function of the synaptic vesicle protein Rabphilin-3A in neurotransmitter release at the squid giant synapse. Presynaptic microinjection of recombinant Rabphilin-3A reversibly inhibited the exocytotic release of neurotransmitter. Injection of fragments of Rabphilin-3A indicate that at least two distinct regions of the protein inhibit neurotransmitter release: the NH₂-terminal region that binds Rab3A and is phosphorylated by protein kinases and the two C2 domains that interact with calcium, phospholipid, and β-adducin. Each of the inhibitory fragments and the full-length protein had separate effects on presynaptic morphology, suggesting that individual domains were inhibiting a subset of the reactions in which the full-length protein participates. In addition to inhibiting exocytosis, constructs containing the NH₂ terminus of Rabphilin-3A also perturbed the endocytotic pathway, as indicated by changes in the membrane areas of endosomes, coated vesicles, and the plasma membrane. These results indicate that Rabphilin-3A regulates synaptic vesicle traffic and appears to do so at distinct stages of both the exocytotic and endocytotic pathways.     

S100-annexin complexes: some insights from structural studies

Several annexins have been shown to bind proteins that belong to the S100 calcium-binding protein family. The two best-characterized complexes are annexin II with p11 and annexin I with S100C, the former of which has been implicated in membrane fusion processes. We have solved the crystal structures of the complexes of p11 with annexin II N-terminus and of S100C with annexin I N-terminus. Using these structural results, as well as electron microscopy observations of liposome junctions formed in the presence of such complexes (Lambert et al., 1997 J Mol Biol 272, 42-55), we propose a computer generated model for the entire annexin II/p11 complex.     

Conformational insights on the molecular recognition processes of carbohydrate molecules by proteins and enzymes: A 3D view by using NMR

This review focuses, in a non-exhaustive manner, on the essential structural and conformational features of protein–carbohydrate interactions and on some applications of NMR spectroscopy to deal with this topic from different levels of complexity.     

Chemical cross-linking of the chloroplast localized small heat-shock protein, Hsp21, and the model substrate citrate synthase

The molecular mechanism whereby the small heat-shock protein (sHsp) chaperones interact with and prevent aggregation of other proteins is not fully understood. We have characterized the sHsp-substrate protein interaction at normal and increased temperatures utilizing a model substrate protein, citrate synthase (CS), widely used in chaperone assays, and a dodecameric plant sHsp, Hsp21, by chemical cross-linking with 3,3'-Dithiobis[sulfosuccinimidylpropionate] (DTSSP) and mass spectrometric peptide mapping. In the absence of CS, the cross-linker captured Hsp21 in dodecameric form, even at increased temperature (47 degrees C). In the presence of equimolar amounts of CS, no Hsp21 dodecamer was captured, indicating a substrate-induced Hsp21 dodecamer dissociation by equimolar amounts of CS. Cross-linked Hsp21-Hsp21 dipeptides indicated an exposure of the Hsp21 C-terminal tails and substrate-binding sites normally covered by the C terminus. Cross-linked Hsp21-CS dipeptides mapped to several sites on the surface of the CS dimer, indicating that there are numerous weak and short-lived interactions between Hsp21 and CS, even at normal temperatures. The N-terminal arms especially interacted with a motif in the CS dimer, which is absent in thermostable forms of CS. The cross-linking data suggest that the presence of substrate rather than temperature influences the conformation of Hsp21.     

qNABpredict: Quick,accurate, and taxonomy-aware sequence-based prediction of content of nucleic acid binding amino acids

Protein sequence-based predictors of nucleic acid (NA)-binding include methods that predict NA-binding proteins and NA-binding residues. The residue-level tools produce more details but suffer high computational cost since they must predict every amino acid in the input sequence and rely on multiple sequence alignments. We propose an alternative approach that predicts content (fraction) of the NA-binding residues, offering more information than the protein-level prediction and much shorter runtime than the residue-level tools. Our first-of-its-kind content predictor, qNABpredict, relies on a small, rationally designed and fast-to-compute feature set that represents relevant characteristics extracted from the input sequence and a well-parametrized support vector regression model. We provide two versions of qNABpredict, a taxonomy-agnostic model that can be used for proteins of unknown taxonomic origin and more accurate taxonomy-aware models that are tailored to specific taxonomic kingdoms: archaea, bacteria, eukaryota, and viruses. Empirical tests on a low-similarity test dataset show that qNABpredict is 100 times faster and generates statistically more accurate content predictions when compared to the content extracted from results produced by the residue-level predictors. We also show that qNABpredict's content predictions can be used to improve results generated by the residue-level predictors. We release qNABpredict as a convenient webserver and source code at http://biomine.cs.vcu.edu/servers/qNABpredict/ . This new tool should be particularly useful to predict details of protein–NA interactions for large protein families and proteomes.     

Analytical Guidelines for co-fractionation Mass Spectrometry Obtained through Global Profiling of Gold Standard Saccharomyces cerevisiae Protein Complexes

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Highlights

• •Guidelines for studying protein complexes via co-fractionation mass spectrometry.
• •A novel procedure for profiling gold standard protein complexes in CF-MS data.
• •Recommendations for efficient CF-MS fractionation collection.
• •Scoring metric recommendations for precise and sensitive CF-MS data analysis.