ABSTRACT
Several mammalian genes have originated from the domestication of retrotransposons, selfish mobile elements related to retroviruses. Some of the proteins encoded by these genes have maintained virus-like features; including self-processing, capsid structure formation, and the generation of different isoforms through -1 programmed ribosomal frameshifting. Using quantitative approaches in molecular evolution and biophysical analyses, we studied 28 retrotransposon-derived genes, with a focus on the evolution of virus-like features. By analyzing the rate of synonymous substitutions, we show that the -1 programmed ribosomal frameshifting mechanism in three of these genes (PEG10, PNMA3, and PNMA5) is conserved across mammals and originates alternative proteins. These genes were targets of positive selection in primates, and one of the positively selected sites affects a B-cell epitope on the spike domain of the PNMA5 capsid, a finding reminiscent of observations in infectious viruses. More generally, we found that retrotransposon-derived proteins vary in their intrinsically disordered region content and this is directly associated with their evolutionary rates. Most positively selected sites in these proteins are located in intrinsically disordered regions and some of them impact protein posttranslational modifications, such as autocleavage and phosphorylation. Detailed analyses of the biophysical properties of intrinsically disordered regions showed that positive selection preferentially targeted regions with lower conformational entropy. Furthermore, positive selection introduces variation in binary sequence patterns across orthologues, as well as in chain compaction. Our results shed light on the evolutionary trajectories of a unique class of mammalian genes and suggest a novel approach to study how intrinsically disordered region biophysical characteristics are affected by evolution.
Subject(s)
Evolution, Molecular , Retroelements , Animals , Selection, Genetic , Mammals/genetics , Mammals/virology , Intrinsically Disordered Proteins/genetics , Frameshifting, Ribosomal , HumansABSTRACT
As the first line of defence against pathogens, cells mount an innate immune response, which varies widely from cell to cell. The response must be potent but carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here we characterize the innate immune response's transcriptional divergence between species and variability in expression among cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we map the architecture of the innate immune response. Transcriptionally diverging genes, including those that encode cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes that are involved in the regulation of this response, such as those that encode transcription factors and kinases, are conserved between species and display low cell-to-cell variability in expression. We suggest that this expression pattern, which is observed across species and conditions, has evolved as a mechanism for fine-tuned regulation to achieve an effective but balanced response.
Subject(s)
Cells/metabolism , Evolution, Molecular , Immunity, Innate/genetics , Immunity, Innate/immunology , Organ Specificity/genetics , Species Specificity , Transcription, Genetic/genetics , Animals , Cells/cytology , Cytokines/genetics , Humans , Promoter Regions, Genetic/geneticsABSTRACT
BACKGROUND: Gene duplication is thought to be a central process in evolution to gain new functions. The factors that dictate gene retention following duplication as well paralog gene divergence in sequence, expression and function have been extensively studied. However, relatively little is known about the evolution of promoter regions of gene duplicates and how they influence gene duplicate divergence. Here, we focus on promoters of paralog genes, comparing their similarity in sequence, in the sets of transcription factors (TFs) that bind them, and in their overall promoter architecture. RESULTS: We observe that promoters of recent duplications display higher sequence similarity between them and that sequence similarity rapidly declines between promoters of more ancient paralogs. In contrast, similarity in cis-regulation, as measured by the set of TFs that bind promoters of both paralogs, does not simply decrease with time from duplication and is instead related to promoter architecture-paralogs with CpG Islands (CGIs) in their promoters share a greater fraction of TFs, while CGI-less paralogs are more divergent in their TF binding set. Focusing on recent duplication events and partitioning them by their duplication mechanism enables us to uncover promoter properties associated with gene retention, as well as to characterize the evolution of promoters of newly born genes: In recent retrotransposition-mediated duplications, we observe asymmetry in cis-regulation of paralog pairs: Retrocopy genes are lowly expressed and their promoters are bound by fewer TFs and are depleted of CGIs, in comparison with the original gene copy. Furthermore, looking at recent segmental duplication regions in primates enable us to compare successful retentions versus loss of duplicates, showing that duplicate retention is associated with fewer TFs and with CGI-less promoter architecture. CONCLUSIONS: In this work, we profiled promoters of gene duplicates and their inter-paralog divergence. We also studied how their characteristics are associated with duplication time and duplication mechanism, as well as with the fate of these duplicates. These results underline the importance of cis-regulatory mechanisms in shaping the evolution of new genes and their fate following duplication.
Subject(s)
Evolution, Molecular , Gene Duplication , Animals , Promoter Regions, Genetic , Transcription Factors , Mammals/geneticsABSTRACT
Bulk and single-cell DNA sequencing has enabled reconstructing clonal substructures of somatic tissues from frequency and cooccurrence patterns of somatic variants. However, approaches to characterize phenotypic variations between clones are not established. Here we present cardelino (https://github.com/single-cell-genetics/cardelino), a computational method for inferring the clonal tree configuration and the clone of origin of individual cells assayed using single-cell RNA-seq (scRNA-seq). Cardelino flexibly integrates information from imperfect clonal trees inferred based on bulk exome-seq data, and sparse variant alleles expressed in scRNA-seq data. We apply cardelino to a published cancer dataset and to newly generated matched scRNA-seq and exome-seq data from 32 human dermal fibroblast lines, identifying hundreds of differentially expressed genes between cells from different somatic clones. These genes are frequently enriched for cell cycle and proliferation pathways, indicating a role for cell division genes in somatic evolution in healthy skin.
Subject(s)
Fibroblasts/metabolism , Gene Expression Profiling/methods , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Software , Algorithms , Cell Cycle , Cell Proliferation , Humans , Melanoma , Mutation , TranscriptomeABSTRACT
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.
Subject(s)
Cellular Reprogramming/physiology , Induced Pluripotent Stem Cells/physiology , Models, Biological , Animals , Cell Line , Cells, Cultured , Cellular Reprogramming/genetics , DNA-Binding Proteins/genetics , Embryonic Stem Cells , Female , Gene Expression Regulation , HEK293 Cells , Humans , Kruppel-Like Factor 4 , Male , Mice , Transcription Factors/geneticsABSTRACT
Nodamura Virus (NoV) is a nodavirus originally isolated from insects that can replicate in a wide variety of hosts, including mammals. Because of their simplicity and ability to replicate in many diverse hosts, NoV, and the Nodaviridae in general, provide a unique window into the evolution of viruses and host-virus interactions. Here we show that the C-terminus of the viral polymerase exhibits extreme structural and evolutionary flexibility. Indeed, fewer than 10 positively charged residues from the 110 amino acid-long C-terminal region of protein A are required to support RNA1 replication. Strikingly, this region can be replaced by completely unrelated protein sequences, yet still produce a functional replicase. Structure predictions, as well as evolutionary and mutational analyses, indicate that the C-terminal region is structurally disordered and evolves faster than the rest of the viral proteome. Thus, the function of an intrinsically unstructured protein region can be independent of most of its primary sequence, conferring both functional robustness and sequence plasticity on the protein. Our results provide an experimental explanation for rapid evolution of unstructured regions, which enables an effective exploration of the sequence space, and likely function space, available to the virus.
Subject(s)
Nodaviridae/genetics , Nodaviridae/physiology , Staphylococcal Protein A/analysis , Viral Proteins/analysis , Amino Acid Sequence , Base Sequence , Cells, Cultured , DNA, Viral/genetics , Mutation/genetics , Replicon/genetics , Staphylococcal Protein A/genetics , Viral Proteins/genetics , Virus Replication/genetics , Virus Replication/physiologyABSTRACT
Entropic stabilization of native protein structures typically relies on strategies that serve to decrease the entropy of the unfolded state. Here we report, using a combination of experimental and computational approaches, on enhanced thermodynamic stability conferred by an increase in the configurational entropy of the folded state. The enhanced stability is observed upon modifications of a loop region in the enzyme acylphosphatase and is achieved despite significant enthalpy losses. The modifications that lead to increased stability, as well as those that result in destabilization, however, strongly compromise enzymatic activity, rationalizing the preservation of the native loop structure even though it does not provide the protein with maximal stability or kinetic foldability.
Subject(s)
Acid Anhydride Hydrolases/chemistry , Protein Stability , Acid Anhydride Hydrolases/genetics , Biophysical Phenomena , Computer Simulation , Entropy , Enzyme Stability , Humans , Models, Molecular , Molecular Dynamics Simulation , Mutagenesis, Insertional , Mutagenesis, Site-Directed , Mutant Proteins/chemistry , Mutant Proteins/genetics , Protein Folding , Thermodynamics , AcylphosphataseABSTRACT
The 26S proteasome is the end point of the ubiquitin- and ATP-dependent degradation pathway. The 26S proteasome complex (26S PC) integrity and function has been shown to be highly dependent on ATP and its homolog nucleotides. We report here that the redox molecule NADH binds the 26S PC and is sufficient in maintaining 26S PC integrity even in the absence of ATP. Five of the 19S proteasome complex subunits contain a putative NADH binding motif (GxGxxG) including the AAA-ATPase subunit, Psmc1 (Rpt2). We demonstrate that recombinant Psmc1 binds NADH via the GxGxxG motif. Introducing the ΔGxGxxG Psmc1 mutant into cells results in reduced NADH-stabilized 26S proteasomes and decreased viability following redox stress induced by the mitochondrial inhibitor rotenone. The newly identified NADH binding of 26S proteasomes advances our understanding of the molecular mechanisms of protein degradation and highlights a new link between protein homeostasis and the cellular metabolic/redox state.
Subject(s)
NADP/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Amino Acid Motifs , Animals , Enzyme Stability/physiology , HEK293 Cells , Humans , Male , Mice , Mice, Inbred ICR , NADP/genetics , NIH 3T3 Cells , Oxidation-Reduction , Proteasome Endopeptidase Complex/genetics , Protein Binding/physiologyABSTRACT
Protein ubiquitination controls the cellular fate of numerous eukaryotic proteins. Despite its importance, many fundamental questions remain regarding its mechanism. One such question is how ubiquitination alters the biophysical properties of the modified protein and whether these alterations are significant in the cellular context. In this study, we investigate the effects of ubiquitination on the folding thermodynamics and mechanism of various substrates using computational tools and find that ubiquitination changes the thermal stability of modified proteins in a manner relevant to cellular processes. These changes depend on the substrate modification site and on the type of ubiquitination. Ubiquitination of the substrate Ubc7 at the residues that are modified in vivo prior to proteasomal degradation uniquely results in significant thermal destabilization and a local unwinding near the modification site, which indicates that ubiquitination possibly facilitates the unfolding process and improves substrate degradation efficiency. With respect to the substrate p19(4inkd), our results support a synergetic effect of ubiquitination and phosphorylation on the degradation process via enhanced thermal destabilization. Our study implies that, in addition to its known role as a recognition signal, the ubiquitin attachment may be directly involved in the cellular process it regulates by changing the biophysical properties of the substrate.
Subject(s)
Proteins/chemistry , Proteins/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Biophysical Phenomena , Cyclin-Dependent Kinase Inhibitor p19/chemistry , Cyclin-Dependent Kinase Inhibitor p19/metabolism , Models, Molecular , Phosphorylation , Protein Folding , Protein Stability , Thermodynamics , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Conjugating Enzymes/metabolism , UbiquitinationABSTRACT
Epithelial cells that participated in wound repair elicit a more efficient response to future injuries, which is believed to be locally restricted. Here we show that cell adaptation resulting from a localized tissue damage has a wide spatial impact at a scale not previously appreciated. We demonstrate that a specific stem cell population, distant from the original injury, originates long-lasting wound memory progenitors residing in their own niche. Notably, these distal memory cells have not taken part in the first healing but become intrinsically pre-activated through priming. This cell state, maintained at the chromatin and transcriptional level, leads to an enhanced wound repair that is partially recapitulated through epigenetic perturbation. Importantly wound memory has long-term harmful consequences, exacerbating tumourigenesis. Overall, we show that sub-organ-scale adaptation to injury relies on spatially organized memory-dedicated progenitors, characterized by an actionable cell state that establishes an epigenetic field cancerization and predisposes to tumour onset.
Subject(s)
Epithelial Cells , Wound Healing , Wound Healing/physiology , Epithelial Cells/physiology , Chromatin/genetics , Stem Cells/physiologyABSTRACT
Bats host a range of disease-causing viruses without displaying clinical symptoms. The mechanisms behind this are a continuous source of interest. Here, we studied the antiviral response in the Egyptian fruit bat and Kuhl's pipistrelle, representing two subordinal clades. We profiled the antiviral response in fibroblasts using RNA sequencing and compared bat with primate and rodent responses. Both bats upregulate similar genes; however, a subset of these genes is transcriptionally divergent between them. These divergent genes also evolve rapidly in sequence, have specific promoter architectures, and are associated with programs underlying tolerance and resistance. Finally, we characterized antiviral genes that expanded in bats, with duplicates diverging in sequence and expression. Our study reveals a largely conserved antiviral program across bats and points to a set of genes that rapidly evolve through multiple mechanisms. These can contribute to bat adaptation to viral infection and provide directions to understanding the mechanisms behind it.
ABSTRACT
Common genetic variants across individuals modulate the cellular response to pathogens and are implicated in diverse immune pathologies, yet how they dynamically alter the response upon infection is not well understood. Here, we triggered antiviral responses in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-sequencing. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), a statistical approach designed to identify nonlinear dynamic genetic effects across transcriptional trajectories of cells. This approach identified 1,275 expression quantitative trait loci (local false discovery rate 10%) that manifested during the responses, many of which were colocalized with susceptibility loci identified by genome-wide association studies of infectious and autoimmune diseases, including the OAS1 splicing quantitative trait locus in a COVID-19 susceptibility locus. In summary, our analytical approach provides a unique framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.
Subject(s)
Autoimmune Diseases , COVID-19 , Pentaerythritol Tetranitrate , Humans , Genome-Wide Association Study , Immunity, InnateABSTRACT
Repeat proteins have unique elongated structures that, unlike globular proteins, are quite modular. Despite their simple one-dimensional structure, repeat proteins exhibit intricate folding behavior with a complexity similar to that of globular proteins. Therefore, repeat proteins allow one to quantify fundamental aspects of the biophysics of protein folding. One important feature of repeat proteins is the interfaces between the repeating units. In particular, the distribution of stabilities within and between the repeats was previously suggested to affect their folding characteristics. In this study, we explore how the interface affects folding kinetics and cooperativity by investigating two families of repeat proteins, namely, the Ankyrin and tetratricopeptide repeat proteins, which differ in the number of interfacial contacts that are formed between their units as well as in their folding behavior. By using simple topology-based models, we show that modulating the energetic strength of the interface relative to that of the repeat itself can drastically change the protein stability, folding rate, and cooperativity. By further dissecting the interfacial contacts into several subsets, we isolated the effects of each of these groups on folding kinetics. Our study highlights the importance of interface connectivity in determining the folding behavior.
Subject(s)
Molecular Dynamics Simulation , Protein Folding , Ankyrins/chemistry , Kinetics , Protein Stability , Protein Structure, TertiaryABSTRACT
Evolutionary changes in host-virus interactions can alter the course of infection, but the biophysical and regulatory constraints that shape interface evolution remain largely unexplored. Here, we focus on viral mimicry of host-like motifs that allow binding to host domains and modulation of cellular pathways. We observe that motifs from unrelated viruses preferentially target conserved, widely expressed, and highly connected host proteins, enriched with regulatory and essential functions. The interface residues within these host domains are more conserved and bind a larger number of cellular proteins than similar motif-binding domains that are not known to interact with viruses. In contrast, rapidly evolving viral-binding human proteins form few interactions with other cellular proteins and display high tissue specificity, and their interfaces have few inter-residue contacts. Our results distinguish between conserved and rapidly evolving host-virus interfaces and show how various factors limit host capacity to evolve, allowing for efficient viral subversion of host machineries.
Subject(s)
Proteins , Viruses , Amino Acid Motifs , Humans , Proteins/metabolism , Viruses/metabolismABSTRACT
When challenged with an invading pathogen, the host-defense response is engaged to eliminate the pathogen (resistance) and to maintain health in the presence of the pathogen (disease tolerance). However, the identification of distinct molecular programs underpinning disease tolerance and resistance remained obscure. We exploited transcriptional and physiological monitoring across 33 mouse strains, during in vivo influenza virus infection, to identify two host-defense gene programs-one is associated with hallmarks of disease tolerance and the other with hallmarks of resistance. Both programs constitute generic responses in multiple mouse and human cell types. Our study describes the organizational principles of these programs and validates Arhgdia as a regulator of disease-tolerance states in epithelial cells. We further reveal that the baseline disease-tolerance state in peritoneal macrophages is associated with the pathophysiological response to injury and infection. Our framework provides a paradigm for the understanding of disease tolerance and resistance at the molecular level.
Subject(s)
Influenza, Human , Orthomyxoviridae Infections , Mice , Humans , Animals , Influenza, Human/genetics , Host-Pathogen Interactions/genetics , Orthomyxoviridae Infections/genetics , Epithelial Cells/metabolismABSTRACT
Dengue virus (DENV) cycles between mosquito and mammalian hosts. To examine how DENV populations adapt to these different host environments, we used serial passage in human and mosquito cell lines and estimated fitness effects for all single-nucleotide variants in these populations using ultra-deep sequencing. This allowed us to determine the contributions of beneficial and deleterious mutations to the collective fitness of the population. Our analysis revealed that the continuous influx of a large burden of deleterious mutations counterbalances the effect of rare, host-specific beneficial mutations to shape the path of adaptation. Beneficial mutations preferentially map to intrinsically disordered domains in the viral proteome and cluster to defined regions in the genome. These phenotypically redundant adaptive alleles may facilitate host-specific DENV adaptation. Importantly, the evolutionary constraints described in our simple system mirror trends observed across DENV and Zika strains, indicating it recapitulates key biophysical and biological constraints shaping long-term viral evolution.
Viruses are constantly evolving as a result of mutations in their genetic material and environmental pressures. Viruses switching between insects and mammals face unique evolutionary pressures because they must retain their ability to infect both types of organisms. Yet, the mutations in a virus that may be beneficial in an insect may be different from the ones that may be beneficial in a mammal. Mutations in one host may be even harmful in the other. To learn more about how such viruses thrive as they switch between hosts, Dolan, Taguwa et al. studied the dengue virus, which causes over 390 million infections and over 10,000 deaths each year around the globe. They compared the mutations that occurred as the virus multiplied in human and mosquito cells grown in a laboratory. In the experiments, they used a method called ultra-deep RNA sequencing to identify every change that occurred in the genetic material of the virus each time it multiplied. They determined whether the mutations were beneficial or harmful based on whether they became more common suggesting they helped the virus survive or whether they did not persist because they were likely harmful or even fatal to the virus. The experiments showed that many harmful mutations constantly occur in the virus, in both human and mosquito cells. Beneficial changes happen rarely, and those that do are usually only helpful in one type of cell. Fatal mutations tended to occur in parts of the genetic material that encodes regions in the viral proteins that must remain the same. These structural elements appear to be essential to the virus's survival and unable to undergo change, which makes them good targets for antiviral drugs or vaccines. The techniques used in the study may be useful for investigating other viruses and for understanding the evolutionary constraints on viruses more generally. This may help scientists develop antiviral drugs or vaccines that will remain effective even as viruses continue to evolve and mutate.
Subject(s)
Dengue Virus/physiology , Evolution, Molecular , Genetic Fitness , Genotype , Aedes/virology , Animals , Cell Line , Humans , Serial PassageABSTRACT
The skin confers biophysical and immunological protection through a complex cellular network established early in embryonic development. We profiled the transcriptomes of more than 500,000 single cells from developing human fetal skin, healthy adult skin, and adult skin with atopic dermatitis and psoriasis. We leveraged these datasets to compare cell states across development, homeostasis, and disease. Our analysis revealed an enrichment of innate immune cells in skin during the first trimester and clonal expansion of disease-associated lymphocytes in atopic dermatitis and psoriasis. We uncovered and validated in situ a reemergence of prenatal vascular endothelial cell and macrophage cellular programs in atopic dermatitis and psoriasis lesional skin. These data illustrate the dynamism of cutaneous immunity and provide opportunities for targeting pathological developmental programs in inflammatory skin diseases.
Subject(s)
Dermatitis, Atopic/embryology , Dermatitis, Atopic/pathology , Psoriasis/embryology , Psoriasis/pathology , Skin/embryology , Animals , Atlases as Topic , Cell Movement , Datasets as Topic , Dendritic Cells/immunology , Dermatitis, Atopic/immunology , Dermatologic Agents/pharmacology , Humans , Immunity, Innate/genetics , Methotrexate/pharmacology , Mice , Phagocytes/immunology , Psoriasis/immunology , Single-Cell Analysis , Skin/cytology , Skin/immunology , T-Lymphocytes/immunology , TranscriptomeABSTRACT
Here, using single-cell RNA sequencing, we examine the stromal compartment in murine melanoma and draining lymph nodes (LNs) at points across tumor development, providing data at http://www.teichlab.org/data/. Naive lymphocytes from LNs undergo activation and clonal expansion within the tumor, before PD1 and Lag3 expression, while tumor-associated myeloid cells promote the formation of a suppressive niche. We identify three temporally distinct stromal populations displaying unique functional signatures, conserved across mouse and human tumors. Whereas "immune" stromal cells are observed in early tumors, "contractile" cells become more prevalent at later time points. Complement component C3 is specifically expressed in the immune population. Its cleavage product C3a supports the recruitment of C3aR+ macrophages, and perturbation of C3a and C3aR disrupts immune infiltration, slowing tumor growth. Our results highlight the power of scRNA-seq to identify complex interplays and increase stromal diversity as a tumor develops, revealing that stromal cells acquire the capacity to modulate immune landscapes from early disease.
Subject(s)
Melanoma/immunology , Sequence Analysis, RNA/methods , Stromal Cells/metabolism , Tumor Microenvironment/immunology , Animals , Humans , MiceABSTRACT
The complexity of the mechanisms by which proteins fold has been shown by many studies to be governed by their native-state topologies. This was manifested in the ability of the native topology-based model to capture folding mechanisms and the success of folding rate predictions based on various topological measures, such as the contact order. However, while the finer details of topological complexity have been thoroughly examined and related to folding kinetics, simpler characteristics of the protein, such as its overall shape, have been largely disregarded. In this study, we investigated the folding of proteins with an unusual elongated geometry that differs substantially from the common globular structure. To study the effect of the elongation degree on the folding kinetics, we used repeat proteins, which become more elongated as they include more repeating units. Some of these have apparently anomalous experimental folding kinetics, with rates that are often less than expected on the basis of rates for globular proteins possessing similar topological complexity. Using experimental folding rates and a larger set of rates obtained from simulations, we have shown that as the protein becomes increasingly elongated, its folding kinetics becomes slower and deviates more from the rate expected on the basis of topology measures fitted for globular proteins. The observed slow kinetics is a result of a more complex pathway in which stable intermediates composed of several consecutive repeats can appear. We thus propose a novel measure, an elongation-sensitive contact order, that takes into account both the extent of elongation and the topological complexity of the protein. This new measure resolves the apparent discrimination between the folding of globular and elongated repeat proteins. Our study extends the current capabilities of folding-rate predictions by unifying the kinetics of repeat and globular proteins.