Tzachi Hagai

Deterministic direct reprogramming of somatic cells to pluripotency

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.

Ubiquitin not only serves as a tag but also assists degradation by inducing protein unfolding

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 p194inkd, 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.

Intrinsic Disorder in Ubiquitination Substrates

The ubiquitin-proteasome system is responsible for the degradation of numerous proteins in eukaryotes. Degradation is an essential process in many cellular pathways and involves the proteasome degrading a wide variety of unrelated substrates while retaining specificity in terms of its targets for destruction and avoiding unneeded proteolysis. How the proteasome achieves this task is the subject of intensive research. Many proteins are targeted for degradation by being covalently attached to a poly-ubiquitin chain. Several studies have indicated the importance of a disordered region for efficient degradation. Here, we analyze a data set of 482 in vivo ubiquitinated substrates and a subset in which ubiquitination is known to mediate degradation. We show that, in contrast to phosphorylation sites and other regulatory regions, ubiquitination sites do not tend to be located in disordered regions and that a large number of substrates are modified at structured regions. In degradation-mediated ubiquitination, there is a significant bias of ubiquitination sites to be in disordered regions; however, a significant number is still found in ordered regions. Moreover, in many cases, disordered regions are absent from ubiquitinated substrates or are located far away from the modified region. These surprising findings raise the question of how these proteins are successfully unfolded and ultimately degraded by the proteasome. They indicate that the folded domain must be perturbed by some additional factor, such as the p97 complex, or that ubiquitination may induce unfolding.

Folding of Elongated Proteins : Conventional or Anomalous?

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.

Stabilization of a protein conferred by an increase in folded state entropy

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.

Modulation of Folding Kinetics of Repeat Proteins: Interplay between Intra- and Interdomain Interactions

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.

Nonspecific yet decisive: Ubiquitination can affect the native-state dynamics of the modified protein

Ubiquitination is one of the most common post‐translational modifications of proteins, and mediates regulated protein degradation among other cellular processes. A fundamental question regarding the mechanism of protein ubiquitination is whether and how ubiquitin affects the biophysical nature of the modified protein. For some systems, it was shown that the position of ubiquitin within the attachment site is quite flexible and ubiquitin does not specifically interact with its substrate. Nevertheless, it was revealed that polyubiquitination can decrease the thermal stability of the modified protein in a site‐specific manner because of alterations of the thermodynamic properties of the folded and unfolded states. In this study, we used detailed atomistic simulations to focus on the molecular effects of ubiquitination on the native structure of the modified protein. As a model, we used Ubc7, which is an E2 enzyme whose in vivo ubiquitination process is well characterized and known to lead to degradation. We found that, despite the lack of specific direct interactions between the ubiquitin moiety and Ubc7, ubiquitination decreases the conformational flexibility of certain regions of the substrate Ubc7 protein, which reduces its entropy and thus destabilizes it. The strongest destabilizing effect was observed for systems in which Lys48‐linked tetra‐ubiquitin was attached to sites used for in vivo degradation. These results reveal how changes in the configurational entropy of the folded state may modulate the stability of the proteins native state. Overall, our results imply that ubiquitination can modify the biophysical properties of the attached protein in the folded state and that, in some proteins, different ubiquitination sites will lead to different biophysical outcomes. We propose that this destabilizing effect of polyubiquitin on the substrate is linked to the functions carried out by the modification, and in particular, regulatory control of protein half‐life through proteasomal degradation.

Folding of Conjugated Proteins

Abstract This review aims at discussing the molecular details of the folding mechanisms of conjugated proteins using computational tools. Almost all studies of protein folding focus on individual proteins and do not consider how interactions with posttranslational modifications and between domains might affect folding. However, different chemical conjugations may introduce a variety of effects on the protein biophysics. These effects depend both on the chemical characteristics of the protein substrate as well as on the chemical and physical properties of the attachment. We review the folding of various types of conjugated proteins, glycoproteins, proteins with tails, ubiquitinated proteins, and multidomain proteins, to explore the underlying biophysical principles of these complex folding processes and in particular to quantify the cross-talk between the protein and its conjugated polymer.

Cardelino: Integrating whole exomes and single-cell transcriptomes to reveal phenotypic impact of somatic variants

Decoding the clonal substructures of somatic tissues sheds light on cell growth, development and differentiation in health, ageing and disease. DNA-sequencing, either using bulk or using single-cell assays, has enabled the reconstruction of clonal trees from frequency and co-occurrence patterns of somatic variants. However, approaches to systematically characterize phenotypic and functional variations between individual clones are not established. Here we present cardelino (https://github.com/PMBio/cardelino), a computational method for inferring the clone of origin of individual cells that have been assayed using single-cell RNA-seq (scRNA-seq). After validating our model using simulations, we apply cardelino to matched scRNA-seq and exome sequencing 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 key role for cell division genes in non-neutral somatic evolution. Key findings A novel approach for integrating DNA-seq and single-cell RNA-seq data to reconstruct clonal substructure for single-cell transcriptomes. Evidence for non-neutral evolution of clonal populations in human fibroblasts. Proliferation and cell cycle pathways are commonly distorted in mutated clonal populations.