Single-cell expression predicts neuron-specific protein homeostasis networks.

The protein homeostasis network keeps proteins in their correct shapes and avoids unwanted aggregation. In turn, the accumulation of aberrantly misfolded proteins has been directly associated with the onset of ageing-associated neurodegenerative diseases such as Alzheimer's and Parkinson's. However, a detailed and rational understanding of how protein homeostasis is achieved in health, and how it can be targeted for therapeutic intervention in diseases remains missing. Here, large-scale single-cell expression data from the Allen Brain Map are analysed to investigate the transcription regulation of the core protein homeostasis network across the human brain. Remarkably, distinct expression profiles suggest specialized protein homeostasis networks with systematic adaptations in excitatory neurons, inhibitory neurons and non-neuronal cells. Moreover, several chaperones and Ubiquitin ligases are found transcriptionally coregulated with genes important for synapse formation and maintenance, thus linking protein homeostasis to the regulation of neuronal function. Finally, evolutionary analyses highlight the conservation of an elevated interaction density in the chaperone network, suggesting that one of the most exciting aspects of chaperone action may yet be discovered in their collective action at the systems level. More generally, our work highlights the power of computational analyses for breaking down complexity and gaining complementary insights into fundamental biological problems.

Functional network motifs defined through integration of protein-protein and genetic interactions.

Cells are enticingly complex systems. The identification of feedback regulation is critically important for understanding this complexity. Network motifs defined as small graphlets that occur more frequently than expected by chance have revolutionized our understanding of feedback circuits in cellular networks. However, with their definition solely based on statistical over-representation, network motifs often lack biological context, which limits their usefulness. Here, we define functional network motifs (FNMs) through the systematic integration of genetic interaction data that directly inform on functional relationships between genes and encoded proteins. Occurring two orders of magnitude less frequently than conventional network motifs, we found FNMs significantly enriched in genes known to be functionally related. Moreover, our comprehensive analyses of FNMs in yeast showed that they are powerful at capturing both known and putative novel regulatory interactions, thus suggesting a promising strategy towards the systematic identification of feedback regulation in biological networks. Many FNMs appeared as excellent candidates for the prioritization of follow-up biochemical characterization, which is a recurring bottleneck in the targeting of complex diseases. More generally, our work highlights a fruitful avenue for integrating and harnessing genomic network data.

Inferring translational heterogeneity from Saccharomyces cerevisiae ribosome profiling.

Translation of mRNAs into proteins by the ribosome is the most important step of protein biosynthesis. Accordingly, translation is tightly controlled and heavily regulated to maintain cellular homeostasis. Ribosome profiling (Ribo-seq) has revolutionized the study of translation by revealing many of its underlying mechanisms. However, equally many aspects of translation remain mysterious, in part also due to persisting challenges in the interpretation of data obtained from Ribo-seq experiments. Here, we show that some of the variability observed in Ribo-seq data has biological origins and reflects programmed heterogeneity of translation. Through a comparative analysis of Ribo-seq data from Saccharomyces cerevisiae, we systematically identify short 3-codon sequences that are differentially translated (DT) across mRNAs, that is, identical sequences that are translated sometimes fast and sometimes slowly beyond what can be attributed to variability between experiments. Remarkably, the thus identified DT sequences link to mechanisms known to regulate translation elongation and are enriched in genes important for protein and organelle biosynthesis. Our results thus highlight examples of translational heterogeneity that are encoded in the genomic sequences and tuned to optimizing cellular homeostasis. More generally, our work highlights the power of Ribo-seq to understand the complexities of translation regulation.

Altered rRNA processing disrupts nuclear RNA homeostasis via competition for the poly(A)-binding protein Nab2.

RNA-binding proteins (RBPs) are key mediators of RNA metabolism. Whereas some RBPs exhibit narrow transcript specificity, others function broadly across both coding and non-coding RNAs. Here, in Saccharomyces cerevisiae, we demonstrate that changes in RBP availability caused by disruptions to distinct cellular processes promote a common global breakdown in RNA metabolism and nuclear RNA homeostasis. Our data shows that stabilization of aberrant ribosomal RNA (rRNA) precursors in an enp1-1 mutant causes phenotypes similar to RNA exosome mutants due to nucleolar sequestration of the poly(A)-binding protein (PABP) Nab2. Decreased nuclear PABP availability is accompanied by genome-wide changes in RNA metabolism, including increased pervasive transcripts levels and snoRNA processing defects. These phenotypes are mitigated by overexpression of PABPs, inhibition of rDNA transcription, or alterations in TRAMP activity. Our results highlight the need for cells to maintain poly(A)-RNA levels in balance with PABPs and other RBPs with mutable substrate specificity across nucleoplasmic and nucleolar RNA processes.

Programmed Trade-offs in Protein Folding Networks.

Molecular chaperones as specialized protein quality control enzymes form the core of cellular protein homeostasis. How chaperones selectively interact with their substrate proteins thus allocate their overall limited capacity remains poorly understood. Here, I present an integrated analysis of sequence and structural determinants that define interactions of protein domains as the basic protein folding unit with the Saccharomyces cerevisiae Hsp70 Ssb. Structural homologs of single-domain proteins that differentially interact with Ssb for de novo folding were found to systematically differ in complexity of their folding landscapes, selective use of nonoptimal codons, and presence of short discriminative sequences, thus highlighting pervasive trade-offs in chaperone-assisted protein folding landscapes. However, short discriminative sequences were found to contribute by far the strongest signal toward explaining Ssb interactions. This observation suggested that some chaperone interactions may be directly programmed in the amino acid sequences rather than responding to folding challenges, possibly for regulatory advantages.

Improvement of the memory function of a mutual repression network in a stochastic environment by negative autoregulation.

BACKGROUND: Cellular memory is a ubiquitous function of biological systems. By generating a sustained response to a transient inductive stimulus, often due to bistability, memory is central to the robust control of many important biological processes. However, our understanding of the origins of cellular memory remains incomplete. Stochastic fluctuations that are inherent to most biological systems have been shown to hamper memory function. Yet, how stochasticity changes the behavior of genetic circuits is generally not clear from a deterministic analysis of the network alone. Here, we apply deterministic rate equations, stochastic simulations, and theoretical analyses of Fokker-Planck equations to investigate how intrinsic noise affects the memory function in a mutual repression network. RESULTS: We find that the addition of negative autoregulation improves the persistence of memory in a small gene regulatory network by reducing stochastic fluctuations. Our theoretical analyses reveal that this improved memory function stems from an increased stability of the steady states of the system. Moreover, we show how the tuning of critical network parameters can further enhance memory. CONCLUSIONS: Our work illuminates the power of stochastic and theoretical approaches to understanding biological circuits, and the importance of considering stochasticity when designing synthetic circuits with memory function.

Pervasive convergent evolution and extreme phenotypes define chaperone requirements of protein homeostasis.

Maintaining protein homeostasis is an essential requirement for cell and organismal viability. An elaborate regulatory system within cells, the protein homeostasis network, safeguards that proteins are correctly folded and functional. At the heart of this regulatory system lies a class of specialized protein quality control enzymes called chaperones that are tasked with assisting proteins in their folding, avoiding aggregation and degradation. Failure and decline of protein homeostasis are directly associated with conditions of aging and aging-related neurodegeneration. However, it is not clear what tips the balance of protein homeostasis and leads to onset of aging and diseases. Here, using a comparative genomics approach we report general principles of maintaining protein homeostasis across the eukaryotic tree of life. Expanding a previous study of 16 eukaryotes to the quantitative analysis of 216 eukaryotic genomes, we find a strong correlation between the composition of eukaryotic chaperone networks and genome complexity that is distinct for different species kingdoms. Organisms with pronounced phenotypes clearly buck this trend. Northobranchius furzeri, the shortest-lived vertebrate and a widely used model for fragile protein homeostasis, is found to be chaperone limited while Heterocephalus glaber as the longest-lived rodent and thus an especially robust organism is characterized by above-average numbers of chaperones. Strikingly, the relative size of chaperone networks is found to generally correlate with longevity in Metazoa. Our results thus indicate that the balance in protein homeostasis may be a key variable in explaining organismal robustness.

Coping with stress by regulating tRNAs.

Stress conditions curtail the energetically costly process of messenger RNA translation. In this issue of Science Signaling, Torrent et al. report key evidence for a direct link between codon usage and translation regulation in response to stress.

Hsp90 shapes protein and RNA evolution to balance trade-offs between protein stability and aggregation.

Acquisition of mutations is central to evolution; however, the detrimental effects of most mutations on protein folding and stability limit protein evolvability. Molecular chaperones, which suppress aggregation and facilitate polypeptide folding, may alleviate the effects of destabilizing mutations thus promoting sequence diversification. To illuminate how chaperones can influence protein evolution, we examined the effect of reduced activity of the chaperone Hsp90 on poliovirus evolution. We find that Hsp90 offsets evolutionary trade-offs between protein stability and aggregation. Lower chaperone levels favor variants of reduced hydrophobicity and protein aggregation propensity but at a cost to protein stability. Notably, reducing Hsp90 activity also promotes clusters of codon-deoptimized synonymous mutations at inter-domain boundaries, likely to facilitate cotranslational domain folding. Our results reveal how a chaperone can shape the sequence landscape at both the protein and RNA levels to harmonize competing constraints posed by protein stability, aggregation propensity, and translation rate on successful protein biogenesis.

Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo.

The genetic code allows most amino acids a choice of optimal and nonoptimal codons. We report that synonymous codon choice is tuned to promote interaction of nascent polypeptides with the signal recognition particle (SRP), which assists in protein translocation across membranes. Cotranslational recognition by the SRP in vivo is enhanced when mRNAs contain nonoptimal codon clusters 35-40 codons downstream of the SRP-binding site, the distance that spans the ribosomal polypeptide exit tunnel. A local translation slowdown upon ribosomal exit of SRP-binding elements in mRNAs containing these nonoptimal codon clusters is supported experimentally by ribosome profiling analyses in yeast. Modulation of local elongation rates through codon choice appears to kinetically enhance recognition by ribosome-associated factors. We propose that cotranslational regulation of nascent-chain fate may be a general constraint shaping codon usage in the genome.

Interplay between chaperones and protein disorder promotes the evolution of protein networks.

Evolution is driven by mutations, which lead to new protein functions but come at a cost to protein stability. Non-conservative substitutions are of interest in this regard because they may most profoundly affect both function and stability. Accordingly, organisms must balance the benefit of accepting advantageous substitutions with the possible cost of deleterious effects on protein folding and stability. We here examine factors that systematically promote non-conservative mutations at the proteome level. Intrinsically disordered regions in proteins play pivotal roles in protein interactions, but many questions regarding their evolution remain unanswered. Similarly, whether and how molecular chaperones, which have been shown to buffer destabilizing mutations in individual proteins, generally provide robustness during proteome evolution remains unclear. To this end, we introduce an evolutionary parameter λ that directly estimates the rate of non-conservative substitutions. Our analysis of λ in Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens sequences reveals how co- and post-translationally acting chaperones differentially promote non-conservative substitutions in their substrates, likely through buffering of their destabilizing effects. We further find that λ serves well to quantify the evolution of intrinsically disordered proteins even though the unstructured, thus generally variable regions in proteins are often flanked by very conserved sequences. Crucially, we show that both intrinsically disordered proteins and highly re-wired proteins in protein interaction networks, which have evolved new interactions and functions, exhibit a higher λ at the expense of enhanced chaperone assistance. Our findings thus highlight an intricate interplay of molecular chaperones and protein disorder in the evolvability of protein networks. Our results illuminate the role of chaperones in enabling protein evolution, and underline the importance of the cellular context and integrated approaches for understanding proteome evolution. We feel that the development of λ may be a valuable addition to the toolbox applied to understand the molecular basis of evolution.

Principles of cotranslational ubiquitination and quality control at the ribosome.

Achieving efficient cotranslational folding of complex proteomes poses a challenge for eukaryotic cells. Nascent polypeptides that emerge vectorially from the ribosome often cannot fold stably and may be susceptible to misfolding and degradation. The extent to which nascent chains are subject to cotranslational quality control and degradation remains unclear. Here, we directly and quantitatively assess cotranslational ubiquitination and identify, at a systems level, the determinants and factors governing this process. Cotranslational ubiquitination occurs at very low levels and is carried out by a complex network of E3 ubiquitin ligases. Ribosome-associated chaperones and cotranslational folding protect the majority of nascent chains from premature quality control. Nonetheless, a number of nascent chains whose intrinsic properties hinder efficient cotranslational folding remain susceptible for cotranslational ubiquitination. We find that quality control at the ribosome is achieved through a tiered system wherein nascent polypeptides have a chance to fold before becoming accessible to ubiquitination.

The ribosome as a hub for protein quality control.

Cells face a constant challenge as they produce new proteins. The newly synthesized polypeptides must be folded properly to avoid aggregation. If proteins do misfold, they must be cleared to maintain a functional and healthy proteome. Recent work is revealing the complex mechanisms that work cotranslationally to ensure protein quality control during biogenesis at the ribosome. Indeed, the ribosome is emerging as a central hub in coordinating these processes, particularly in sensing the nature of the nascent protein chain, recruiting protein folding and translocation components, and integrating mRNA and nascent chain quality control. The tiered and complementary nature of these decision-making processes confers robustness and fidelity to protein homeostasis during protein synthesis.

The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis.

In eukaryotic cells a molecular chaperone network associates with translating ribosomes, assisting the maturation of emerging nascent polypeptides. Hsp70 is perhaps the major eukaryotic ribosome-associated chaperone and the first reported to bind cotranslationally to nascent chains. However, little is known about the underlying principles and function of this interaction. Here, we use a sensitive and global approach to define the cotranslational substrate specificity of the yeast Hsp70 SSB. We find that SSB binds to a subset of nascent polypeptides whose intrinsic properties and slow translation rates hinder efficient cotranslational folding. The SSB-ribosome cycle and substrate recognition is modulated by its ribosome-bound cochaperone, RAC. Deletion of SSB leads to widespread aggregation of newly synthesized polypeptides. Thus, cotranslationally acting Hsp70 meets the challenge of folding the eukaryotic proteome by stabilizing its longer, more slowly translated, and aggregation-prone nascent polypeptides.

Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding.

The choice of codons can influence local translation kinetics during protein synthesis. Whether codon preference is linked to cotranslational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale that incorporates the competition between tRNA supply and demand. Applying this scale to ten closely related yeast species, we uncover the evolutionary conservation of codon optimality in eukaryotes. This analysis reveals universal patterns of conserved optimal and nonoptimal codons, often in clusters, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Our analysis suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate cotranslational polypeptide folding, beyond its previously proposed role of adapting to the cost of expression. These findings establish how mRNA sequences are generally under selection to optimize the cotranslational folding of corresponding polypeptides.

The molecular architecture of the eukaryotic chaperonin TRiC/CCT.

TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.

Defining the specificity of cotranslationally acting chaperones by systematic analysis of mRNAs associated with ribosome-nascent chain complexes.

Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.

Derivation of a solubility condition for proteins from an analysis of the competition between folding and aggregation.

Failure in maintaining protein solubility in vivo impairs protein homeostasis and results in protein misfolding and aggregation, which are often associated with severe neurodegenerative and systemic disorders that include Alzheimer's and Parkinson's diseases and type II diabetes. In this work we formulate a model of the competition between folding and aggregation, and derive a condition on the solubility of proteins in terms of the stability of their folded states, their aggregation propensities and their degradation rates. From our model, the bistability between folding and aggregation emerges as an intrinsic aspect of protein homeostasis. The analysis of the conditions that determine such a bistability provides a rationalization of the recently observed relationship between the cellular abundance and the aggregation propensity of proteins. We then discuss how the solubility condition that we derive can help rationalise the correlation that has been reported between evolutionary rates and expression levels or proteins, as well as in vivo protein solubility and expression level measurements, and recently elucidated trends of proteome evolution.

Physicochemical principles that regulate the competition between functional and dysfunctional association of proteins.

To maintain protein homeostasis, a variety of quality control mechanisms, such as the unfolded protein response and the heat shock response, enable proteins to fold and to assemble into functional complexes while avoiding the formation of aberrant and potentially harmful aggregates. We show here that a complementary contribution to the regulation of the interactions between proteins is provided by the physicochemical properties of their amino acid sequences. The results of a systematic analysis of the protein-protein complexes in the Protein Data Bank (PDB) show that interface regions are more prone to aggregate than other surface regions, indicating that many of the interactions that promote the formation of functional complexes, including hydrophobic and electrostatic forces, can potentially also cause abnormal intermolecular association. We also show, however, that aggregation-prone interfaces are prevented from triggering uncontrolled assembly by being stabilized into their functional conformations by disulfide bonds and salt bridges. These results indicate that functional and dysfunctional association of proteins are promoted by similar forces but also that they are closely regulated by the presence of specific interactions that stabilize native states.

A relationship between mRNA expression levels and protein solubility in E. coli.

Each step in the process of gene expression, from the transcription of DNA into mRNA to the folding and posttranslational modification of proteins, is regulated by complex cellular mechanisms. At the same time, stringent conditions on the physicochemical properties of proteins, and hence on the nature of their amino acids, are imposed by the need to avoid aggregation at the concentrations required for optimal cellular function. A relationship is therefore expected to exist between mRNA expression levels and protein solubility in the cell. By investigating such a relationship, we formulate a method that enables the prediction of the maximal levels of mRNA expression in Escherichia coli with an accuracy of 83% and of the solubility of recombinant human proteins expressed in E. coli with an accuracy of 86%.