Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis

The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.     

Structural disorder throws new light on moonlighting

A basic mechanism by which individual proteins can increase network complexity is moonlighting, whereby a given protein fulfils more than one function. Traditionally, this phenomenon is attributed to separate binding surfaces of globular, folded proteins but we suggest that intrinsically unstructured proteins (IUPs) might provide radically different mechanisms. Eleven IUPs have been identified that suggest that the structural malleability of IUPs gives rise to unprecedented cases of moonlighting by eliciting opposing (inhibiting and activating) action on different partners or even the same partner molecule. Unlike classical cases, these proteins use the same region or overlapping interaction surfaces to exert distinct effects and employ non-conventional mechanisms to switch function, enabled by their capacity to adopt different conformations upon binding. Owing to the apparent functional benefits, we expect to see many more examples of this parsimonious use of protein material in complex metabolic networks.     

Co‐evolution‐driven switch of J‐protein specificity towards an Hsp70 partner

Molecular mechanisms by which protein–protein interactions are preserved or lost after gene duplication are not understood. Taking advantage of the well–studied yeast mtHsp70:J–protein molecular chaperone system, we considered whether changes in partner proteins accompanied specialization of gene duplicates. Here, we report that existence of the Hsp70 Ssq1, which arose by duplication of the gene encoding multifunction mtHsp70 and specializes in iron–sulphur cluster biogenesis, correlates with functional and structural changes in the J domain of its J–protein partner Jac1. All species encoding this shorter alternative version of the J domain share a common ancestry, suggesting that all short JAC1 proteins arose from a single deletion event. Construction of a variant that extended the length of the J domain of a ‘short’ Jac1 enhanced its ability to partner with multifunctional Hsp70. Our data provide a causal link between changes in the J protein partner and specialization of duplicate Hsp70.     

Redox modulation of cellular signaling and metabolism through reversible oxidation of methionine sensors in calcium regulatory proteins

Adaptive responses associated with environmental stressors are critical to cell survival. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met(144) or Met(145) results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylation-dependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in response to beta-adrenergic signaling in the heart. We suggest that under acute conditions, such as inflammation or ischemia, these types of mechanisms ensure minimal nonspecific cellular damage, allowing for rapid restoration of cellular function through repair of oxidized methionines by methionine sulfoxide reductases and degradation pathways after restoration of normal cellular redox conditions.     

On protein partitioning in two-phase aqueous polymer systems.

The partitioning of proteins between the coexisting phases of two-phase aqueous polymer systems reflects an intricate and delicate balance of interactions between proteins, polymers, salts and water. Experimental investigations have suggested that a large number of factors influence protein partitioning, including the types of polymers, their molecular weight and concentration; the protein sizes, conformation and composition; salt type and concentration, and solution pH; and the presence of ligands attached to the polymer which may interact with surface sites of the protein. Complementary modelling attempts have been successful in illuminating several molecular-level mechanisms influencing protein partitioning using lattice-model techniques, viral expansions and a scaling-thermodynamic approach. In spite of these experimental and modelling approaches, many of the physical phenomena associated with these complex systems are not well understood. Notably, the precise nature of the protein-polymer interactions and the potent effect of inorganic salts on the partitioning of proteins in these systems remains poorly understood.     

Off the beaten paths: alternative and crosstalk regulation of Rho GTPases

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.     

Regulation of alternative splicing by reversible protein phosphorylation

The vast majority of human protein-coding genes are subject to alternative splicing, which allows the generation of more than one protein isoform from a single gene. Cells can change alternative splicing patterns in response to a signal, which creates protein variants with different biological properties. The selection of alternative splice sites is governed by the dynamic formation of protein complexes on the processed pre-mRNA. A unique set of these splicing regulatory proteins assembles on different pre-mRNAs, generating a "splicing" or "messenger ribonucleoprotein code" that determines exon recognition. By influencing protein/protein and protein/RNA interactions, reversible protein phosphorylation modulates the assembly of regulatory proteins on pre-mRNA and therefore contributes to the splicing code. Studies of the serine/arginine-rich protein class of regulators identified different kinases and protein phosphatase 1 as the molecules that control reversible phosphorylation, which controls not only splice site selection, but also the localization of serine/arginine-rich proteins and mRNA export. The involvement of protein phosphatase 1 explains why second messengers like cAMP and ceramide that control the activity of this phosphatase influence alternative splicing. The emerging mechanistic links between splicing regulatory proteins and known signal transduction pathways now allow in detail the understanding how cellular signals modulate gene expression by influencing alternative splicing. This knowledge can be applied to human diseases that are caused by the selection of wrong splice sites.     

A two-hybrid dual bait system to discriminate specificity of protein interactions.

Biological regulatory systems require the specific organization of proteins into multicomponent complexes. Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins. An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific. We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (lambda cI). Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters. The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background. These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners.     

How to make a biological switch

Some biological regulatory systems must "remember" a state for long periods of time. A simple type of system that can accomplish this task is one in which two regulatory elements negatively regulate one another. For example, two repressor proteins might control one another's synthesis. Qualitative reasoning suggests that such a system will have two stable states, one in which the first element is "on" and the second "off", and another in which these states are reversed. Quantitative analysis shows that the existence of two stable steady states depends on the details of the system. Among other things, the shapes of functions describing the effect of one regulatory element on the other must meet certain criteria in order for two steady states to exist. Many biologically reasonable functions do not meet these criteria. In particular, repression that is well described by a Michaelis-Menten-type equation cannot lead to a working switch. However, functions describing positive cooperativity of binding, non-additive effects of multiple operator sites, or depletion of free repressor can lead to working switches.     

Caught in self-interaction: evolutionary and functional mechanisms of protein homooligomerization

Many soluble and membrane proteins form homooligomeric complexes in a cell which are responsible for the diversity and specificity of many pathways, may mediate and regulate gene expression, activity of enzymes, ion channels, receptors, and cell adhesion processes. The evolutionary and physical mechanisms of oligomerization are very diverse and its general principles have not yet been formulated. Homooligomeric states may be conserved within certain protein subfamilies and might be important in providing specificity to certain substrates while minimizing interactions with other unwanted partners. Moreover, recent studies have led to a greater awareness that transitions between different oligomeric states may regulate protein activity and provide the switch between different pathways. In this paper we summarize the biological importance of homooligomeric assemblies, physico-chemical properties of their interfaces, experimental and computational methods for their identification and prediction. We particularly focus on homooligomer evolution and describe the mechanisms to develop new specificities through the formation of different homooligomeric complexes. Finally, we discuss the possible role of oligomeric transitions in the regulation of protein activity and compile a set of experimental examples with such regulatory mechanisms.     

Antiviral protein Ski8 is a direct partner of Spo11 in meiotic DNA break formation, independent of its cytoplasmic role in RNA metabolism

Meiotic recombination initiates with double-strand breaks (DSBs) catalyzed by Spo11 in conjunction with accessory proteins whose roles are not understood. Two-hybrid analysis reveals a network of interactions connecting the yeast DSB proteins to one another. Of these proteins, Ski8 was known to function in cytoplasmic RNA metabolism, suggesting that its role in recombination might be indirect. However, obligate partners of Ski8 in RNA metabolism are dispensable for recombination and Ski8 relocalizes to the nucleus and associates with chromosomes specifically during meiosis. Interaction of Ski8 with Spo11 is essential for DSB formation and Ski8 relocalization. Thus, Ski8 plays distinct roles in RNA metabolism and, as a direct partner of Spo11, in DSB formation. Ski8 works with Spo11 to recruit other DSB proteins to meiotic chromosomes, implicating Ski8 as a scaffold protein mediating assembly of a multiprotein complex essential for DSB formation.