Kinetic Analysis of Protein Stability Reveals Age-Dependent Degradation.

Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts. Within complexes, NED proteins have larger interaction interfaces and assemble earlier than ED subunits. Amplifying genes encoding NED proteins increases their initial degradation. Consistently, decay profiles can predict protein level attenuation in aneuploid cells. Together, our data show that non-exponential degradation is common, conserved, and has important consequences for complex formation and regulation of protein abundance.

A Field Guide to Eukaryotic Transposable Elements.

Transposable elements (TEs) are mobile DNA sequences that propagate within genomes. Through diverse invasion strategies, TEs have come to occupy a substantial fraction of nearly all eukaryotic genomes, and they represent a major source of genetic variation and novelty. Here we review the defining features of each major group of eukaryotic TEs and explore their evolutionary origins and relationships. We discuss how the unique biology of different TEs influences their propagation and distribution within and across genomes. Environmental and genetic factors acting at the level of the host species further modulate the activity, diversification, and fate of TEs, producing the dramatic variation in TE content observed across eukaryotes. We argue that cataloging TE diversity and dissecting the idiosyncratic behavior of individual elements are crucial to expanding our comprehension of their impact on the biology of genomes and the evolution of species.

Protein aggregation mediates stoichiometry of protein complexes in aneuploid cells.

Aneuploidy, a condition characterized by chromosome gains and losses, causes reduced fitness and numerous cellular stresses, including increased protein aggregation. Here, we identify protein complex stoichiometry imbalances as a major cause of protein aggregation in aneuploid cells. Subunits of protein complexes encoded on excess chromosomes aggregate in aneuploid cells, which is suppressed when expression of other subunits is coordinately altered. We further show that excess subunits are either degraded or aggregate and that protein aggregation is nearly as effective as protein degradation at lowering levels of excess proteins. Our study explains why proteotoxic stress is a universal feature of the aneuploid state and reveals protein aggregation as a form of dosage compensation to cope with disproportionate expression of protein complex subunits.

Transposable elements drive the evolution of metazoan zinc finger genes.

Cys2-His2 zinc finger genes (ZNFs) form the largest family of transcription factors in metazoans. ZNF evolution is highly dynamic and characterized by the rapid expansion and contraction of numerous subfamilies across the animal phylogeny. The forces and mechanisms underlying rapid ZNF evolution remain poorly understood, but there is growing evidence that, in tetrapods, the targeting and repression of lineage-specific transposable elements (TEs) plays a critical role in the evolution of the Krüppel-associated box ZNF (KZNF) subfamily. Currently, it is unknown whether this function and coevolutionary relationship is unique to KZNFs or is a broader feature of metazoan ZNFs. Here, we present evidence that genomic conflict with TEs has been a central driver of the diversification of ZNFs in animals. Sampling from 3221 genome assemblies, we show that the copy number of retroelements correlates with that of ZNFs across at least 750 million years of metazoan evolution. Using computational predictions, we show that ZNFs preferentially bind TEs in diverse animal species. We further investigate the largest ZNF subfamily found in cyprinid fish, which is characterized by a conserved sequence we dubbed the fish N-terminal zinc finger-associated (FiNZ) domain. Zebrafish possess approximately 700 FiNZ-ZNFs, many of which are evolving adaptively under positive selection. Like mammalian KZNFs, most zebrafish FiNZ-ZNFs are expressed at the onset of zygotic genome activation, and blocking their translation using morpholinos during early embryogenesis results in derepression of transcriptionally active TEs. Together, these data suggest that ZNF diversification has been intimately connected to TE expansion throughout animal evolution.

Co-translational assembly of protein complexes.

The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly.

Gag proteins encoded by endogenous retroviruses are required for zebrafish development.

Transposable elements (TEs) make up the bulk of eukaryotic genomes and examples abound of TE-derived sequences repurposed for organismal function. The process by which TEs become coopted remains obscure because most cases involve ancient, transpositionally inactive elements. Reports of active TEs serving beneficial functions are scarce and often contentious due to difficulties in manipulating repetitive sequences. Here we show that recently active TEs in zebrafish encode products critical for embryonic development. Knockdown and rescue experiments demonstrate that the endogenous retrovirus family BHIKHARI-1 (Bik-1) encodes a Gag protein essential for mesoderm development. Mechanistically, Bik-1 Gag associates with the cell membrane and its ectopic expression in chicken embryos alters cell migration. Similarly, depletion of BHIKHARI-2 Gag, a relative of Bik-1, causes defects in neural crest development in zebrafish. We propose an "addiction" model to explain how active TEs can be integrated into conserved developmental processes.

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia.

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits and demonstrate an accumulation of 'coat-less' vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

Most human cells have at least one small hair-like structure on their surface called a cilium. These structures can act as antennae and allow the cell to sense signals from the rest of the body. To do this, they contain proteins that differ from the rest of the cell. The content of cilia depends on regulated delivery of these proteins in and out of cilia by a process called the intraflagellar transport or IFT, which involves a large complex made of several proteins. This complex shuttles the cargo proteins back and forth between the base and the tip of the cilia. However, ciliary proteins are not produced in the cilia; instead, they are made in a different part of the cell and then they are transported to the ciliary base. At the point where they enter the cilia, they were thought to bind to the assembling IFT 'trains' and be transported across the ciliary gate to the positions where they are needed in cilia. One of the components of the IFT machinery is a protein called WDR35, also known as IFT121. If the gene that codes for this protein is faulty or missing, it results in severe disorders in both humans and mice including a range of potentially lethal skeletal dysplasias. Interestingly, without WDR35, cells cannot build functional cilia. The absence of this protein not only disrupts IFT, stopping certain ciliary proteins and their associated membranes from entering cilia; it also causes a 'traffic jam' with a pile-up of transport intermediates from the place in cell where they are made to the cilia. It is unclear why a mutation in one of the components of the IFT would have this effect, raising the question of whether WDR35, or IFTs a whole, has another role in bringing the cargo proteins into the cilia. To understand this phenomenon, Quidwai et al. analysed the structure of WDR35 and other IFT proteins and found that they are very similar to a protein complex called COPI, which is involved in transporting membrane proteins around the cell. When certain proteins are newly made, they are stored in small lipid bubbles ­ called vesicles ­ that then selectively move to where the proteins are needed. COPI coats these vesicles, helping them get to where they need to go in a process called vesicular transport. Quidwai et al. found that WDR35 and other IFT proteins are able to bind to specific types of lipid molecules, suggesting that they might be assisting in a form of vesicle transport too. Indeed, when mouse cells grown in the lab were genetically engineered so they could not produce WDR35, coatless vesicles accumulated around the base of the cilia. Adding back WDR35 to these mutant cells rescued these defects in vesicle transport to cilia as well as allowed functional cilia to be formed. These results provide evidence that WDR35, likely with other IFT proteins, acts as a COPI-like complex to deliver proteins to growing cilia. Further research will investigate the composition of these vesicles that transport proteins to cilia, and help pinpoint where they originate. Quidwai et al.'s findings not only shed light on how different genetic mutations found in patients with cilia dysfunction affect different steps of transporting proteins to and within cilia. They also increase our understanding of the cellular roadmap by which cells shuttle building blocks around in order to assemble these important 'antennae'.

A Graph-Based Approach for Detecting Sequence Homology in Highly Diverged Repeat Protein Families.

Reconstructing evolutionary relationships in repeat proteins is notoriously difficult due to the high degree of sequence divergence that typically occurs between duplicated repeats. This is complicated further by the fact that proteins with a large number of similar repeats are more likely to produce significant local sequence alignments than proteins with fewer copies of the repeat motif. Furthermore, biologically correct sequence alignments are sometimes impossible to achieve in cases where insertion or translocation events disrupt the order of repeats in one of the sequences being aligned. Combined, these attributes make traditional phylogenetic methods for studying protein families unreliable for repeat proteins, due to the dependence of such methods on accurate sequence alignment.We present here a practical solution to this problem, making use of graph clustering combined with the open-source software package HH-suite, which enables highly sensitive detection of sequence relationships. Carrying out multiple rounds of homology searches via alignment of profile hidden Markov models, large sets of related proteins are generated. By representing the relationships between proteins in these sets as graphs, subsequent clustering with the Markov cluster algorithm enables robust detection of repeat protein subfamilies.

The role of protein complexes in human genetic disease.

Many human genetic disorders are caused by mutations in protein-coding regions of DNA. Taking protein structure into account has therefore provided key insight into the molecular mechanisms underlying human genetic disease. Although most studies have focused on the intramolecular effects of mutations, the critical role of the assembly of proteins into complexes is being increasingly recognized. Here, we review multiple ways in which consideration of protein complexes can help us to understand and explain the effects of pathogenic mutations. First, we discuss disorders caused by mutations that perturb intersubunit interactions in homomeric and heteromeric complexes. Second, we address how protein complex assembly can facilitate a dominant-negative mechanism, whereby mutated subunits can disrupt the activity of wild-type protein. Third, we show how mutations that change protein expression levels can lead to damaging stoichiometric imbalances. Finally, we review how mutations affecting different subunits of the same heteromeric complex often cause similar diseases, whereas mutations in different interfaces of the same subunit can cause distinct phenotypes.

Experimental Characterization of Protein Complex Structure, Dynamics, and Assembly.

Experimental methods for the characterization of protein complexes have been instrumental in achieving our current understanding of the protein universe and continue to progress with each year that passes. In this chapter, we review some of the most important tools and techniques in the field, covering the important points in X-ray crystallography, cryo-electron microscopy, NMR spectroscopy, and mass spectrometry. Novel developments are making it possible to study large protein complexes at near-atomic resolutions, and we also now have the ability to study the dynamics and assembly pathways of protein complexes across a range of sizes.

Computational Modelling of Protein Complex Structure and Assembly.

Sequence and structure space are nowadays sufficiently large that we can use computational methods to model the structure of proteins based on sequence similarity alone. Not only useful as a standalone tool, homology modelling has also had a transformative effect on the ease with which we can solve crystal structures and electron density maps. Another technique-molecular dynamics-aims to model protein structures from first principles and, thanks to increases in computational power, is slowly becoming a viable tool for studying protein complexes. Finally, the prediction of protein assembly pathways from three-dimensional structures of complexes is also now becoming possible.

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens.

Protein complexes are assemblies of subunits that have co-evolved to execute one or many coordinated functions in the cellular environment. Functional annotation of mammalian protein complexes is critical to understanding biological processes, as well as disease mechanisms. Here, we used genetic co-essentiality derived from genome-scale RNAi- and CRISPR-Cas9-based fitness screens performed across hundreds of human cancer cell lines to assign measures of functional similarity. From these measures, we systematically built and characterized functional similarity networks that recapitulate known structural and functional features of well-studied protein complexes and resolve novel functional modules within complexes lacking structural resolution, such as the mammalian SWI/SNF complex. Finally, by integrating functional networks with large protein-protein interaction networks, we discovered novel protein complexes involving recently evolved genes of unknown function. Taken together, these findings demonstrate the utility of genetic perturbation screens alone, and in combination with large-scale biophysical data, to enhance our understanding of mammalian protein complexes in normal and disease states.

Evolution of condensin and cohesin complexes driven by replacement of Kite by Hawk proteins.

Mitotic chromosome condensation, sister chromatid cohesion, and higher order folding of interphase chromatin are mediated by condensin and cohesin, eukaryotic members of the SMC (structural maintenance of chromosomes)-kleisin protein family. Other members facilitate chromosome segregation in bacteria [1]. A hallmark of these complexes is the binding of the two ends of a kleisin subunit to the apices of V-shaped Smc dimers, creating a tripartite ring capable of entrapping DNA (Figure 1A). In addition to creating rings, kleisins recruit regulatory subunits. One family of regulators, namely Kite dimers (Kleisin interacting winged-helix tandem elements), interact with Smc-kleisin rings from bacteria, archaea and the eukaryotic Smc5-6 complex, but not with either condensin or cohesin [2]. These instead possess proteins containing HEAT (Huntingtin/EF3/PP2A/Tor1) repeat domains whose origin and distribution have not yet been characterized. Using a combination of profile Hidden Markov Model (HMM)-based homology searches, network analysis and structural alignments, we identify a common origin for these regulators, for which we propose the name Hawks, i.e. HEAT proteins associated with kleisins.

Regulation, evolution and consequences of cotranslational protein complex assembly.

Most proteins assemble into complexes, which are involved in almost all cellular processes. Thus it is crucial for cell viability that mechanisms for correct assembly exist. The timing of assembly plays a key role in determining the fate of the protein: if the protein is allowed to diffuse into the crowded cellular milieu, it runs the risk of forming non-specific interactions, potentially leading to aggregation or other deleterious outcomes. It is therefore expected that strong regulatory mechanisms should exist to ensure efficient assembly. In this review we discuss the cotranslational assembly of protein complexes and discuss how it occurs, ways in which it is regulated, potential disadvantages of cotranslational interactions between proteins and the implications for the inheritance of dominant-negative genetic disorders.

Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.