Determining and interpreting protein lifetimes in mammalian tissues.

The orchestration of protein production and degradation, and the regulation of protein lifetimes, play a central role in the majority of biological processes. Recent advances in proteomics have enabled the estimation of protein half-lives for thousands of proteins in vivo. What is the utility of these measurements, and how can they be leveraged to interpret the proteome changes occurring during development, aging, and disease? This opinion article summarizes leading technical approaches and highlights their strengths and weaknesses. We also disambiguate frequently used terminology, illustrate recent mechanistic insights, and provide guidance for interpreting and validating protein turnover measurements. Overall, protein lifetimes, coupled to estimates of protein levels, are essential for obtaining a deep understanding of mammalian biology and the basic processes defining life itself.

Tuning protein half-life in mouse using sequence-defined biopolymers functionalized with lipids.

The use of biologics in the treatment of numerous diseases has increased steadily over the past decade due to their high specificities, low toxicity, and limited side effects. Despite this success, peptide- and protein-based drugs are limited by short half-lives and immunogenicity. To address these challenges, we use a genomically recoded organism to produce genetically encoded elastin-like polypeptide-protein fusions containing multiple instances of para-azidophenylalanine (pAzF). Precise lipidation of these pAzF residues generated a set of sequence-defined synthetic biopolymers with programmable binding affinity to albumin without ablating the activity of model fusion proteins, and with tunable blood serum half-lives spanning 5 to 94% of albumin's half-life in a mouse model. Our findings present a proof of concept for the use of genetically encoded bioorthogonal conjugation sites for multisite lipidation to tune protein stability in mouse serum. This work establishes a programmable approach to extend and tune the half-life of protein or peptide therapeutics and a technical foundation to produce functionalized biopolymers endowed with programmable chemical and biophysical properties with broad applications in medicine, materials science, and biotechnology.

The αSynuclein half-life conundrum.

αSynuclein (αSyn) misfolding and aggregation frequently precedes neuronal loss associated with Parkinson's Disease (PD) and other Synucleinopathies. The progressive buildup of pathological αSyn species results from alterations on αSyn gene and protein sequence, increased local concentrations, variations in αSyn interactome and protein network. Therefore, under physiological conditions, it is mandatory to regulate αSyn proteostasis as an equilibrium among synthesis, trafficking, degradation and extracellular release. In this frame, a crucial parameter is protein half-life. It provides indications of the turnover of a specific protein and depends on mRNA synthesis and translation regulation, subcellular localization, function and clearance by the designated degradative pathways. For αSyn, the molecular mechanisms regulating its proteostasis in neurons have been extensively investigated in various cellular models, either using biochemical or imaging approaches. Nevertheless, a converging estimate of αSyn half-life has not emerged yet. Here, we discuss the challenges in studying αSyn proteostasis under physiological and pathological conditions, the advantages and disadvantages of the experimental strategies proposed so far, and the relevance of determining αSyn half-life from a translational perspective.

Identifying E3 Ligase Substrates With Quantitative Degradation Proteomics.

Controlled protein degradation by the ubiquitin-proteasome pathway is critical for almost all cellular processes. E3 ubiquitin ligases are responsible for targeting proteins for ubiquitylation and subsequent proteasomal degradation with spatial and temporal precision. While studies have revealed various E3-substrate pairs involved in distinct biological processes, the complete substrate profiles of individual E3 ligases are largely unknown. Here we report a new approach to identify substrates of an E3 ligase for proteasomal degradation using unnatural amino acid incorporation pulse-chase proteomics (degradomics). Applying this approach, we determine the steady-state substrates of the C-terminal to LisH (CTLH) E3 ligase, a multi-component complex with poorly defined substrates. By comparing the proteome degradation profiles of active and inactive CTLH-expressing cells, we successfully identify previously known and new potential substrates of CTLH ligase. Altogether, degradomics can comprehensively identify degradation substrates of an E3 ligase, which can be adapted for other E3 ligases in various cellular contexts.

Modeling SILAC Data to Assess Protein Turnover in a Cellular Model of Diabetic Nephropathy.

Protein turnover rate is finely regulated through intracellular mechanisms and signals that are still incompletely understood but that are essential for the correct function of cellular processes. Indeed, a dysfunctional proteostasis often impacts the cell's ability to remove unfolded, misfolded, degraded, non-functional, or damaged proteins. Thus, altered cellular mechanisms controlling protein turnover impinge on the pathophysiology of many diseases, making the study of protein synthesis and degradation rates an important step for a more comprehensive understanding of these pathologies. In this manuscript, we describe the application of a dynamic-SILAC approach to study the turnover rate and the abundance of proteins in a cellular model of diabetic nephropathy. We estimated protein half-lives and relative abundance for thousands of proteins, several of which are characterized by either an altered turnover rate or altered abundance between diabetic nephropathic subjects and diabetic controls. Many of these proteins were previously shown to be related to diabetic complications and represent therefore, possible biomarkers or therapeutic targets. Beside the aspects strictly related to the pathological condition, our data also represent a consistent compendium of protein half-lives in human fibroblasts and a rich source of important information related to basic cell biology.

Deubiquitinating Enzyme USP12 Regulates the Pro-Apoptosis Protein Bax.

The Bax protein is a pro-apoptotic protein belonging to the Bcl-2 family, involved in inducing apoptosis at the mitochondrial level. Regulating the protein levels of Bax is essential to enhancing apoptosis. In the current study, we ascertained the presence of deubiquitinating enzymes (DUBs) associated with Bax by performing the yeast two-hybrid screening (Y2H). We determined that ubiquitin-specific protease 12 (USP12), one of the DUBs, is associated with Bax. The binding of USP12 to Bax shows the interaction as a DUB, which regulates ubiquitination on Bax. Taken together, we believe that USP12 regulates Bax by detaching ubiquitin on K63-linked chains, indicating that USP12 affects the cellular functions of Bax, but it is not related with proteasomal degradation. The half-life of the Bax protein was determined by performing the site-directed mutagenesis of putative ubiquitination sites on Bax (K128R, K189R, and K190R). Of these, Bax (K128R and K190R) showed less ubiquitination; therefore, we compared the half-life of Bax (WT) and Bax K mutant forms in vitro. Interestingly, Bax (K189R) showed a higher ubiquitination level and shorter half-life than Bax (WT), and the (K128R and K190R) mutant form has a longer half-life than Bax (WT).

Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers.

(1) Background: Bladder cancer is a malignant tumor mainly caused by exposure to environmental chemicals, with a high recurrence rate. NR1H4, also known as Farnesoid X Receptor (FXR), acts as a nuclear receptor that can be activated by binding with bile acids, and FXR is highly correlated with the progression of cancers. The aim of this study was to verify the role of FXR in bladder cancer cells. (2) Methods: A FXR overexpressed system was established to investigate the effect of cell viability, migration, adhesion, and angiogenesis in low-grade TSGH8301 and high-grade T24 cells. (3) Results: After FXR overexpression, the ability of migration, adhesion, invasion and angiogenesis of bladder cancer cells declined significantly. Focal adhesive complex, MMP2, MMP9, and angiogenic-related proteins were decreased, while FXR was overexpressed in bladder cancer cells. Moreover, FXR overexpression reduced vascular endothelial growth factor mRNA and protein expression and secretion in bladder cancer cells. After treatment with the proteosome inhibitor MG132, the migration, adhesion and angiogenesis caused by FXR overexpression were all reversed in bladder cancer cells. (4) Conclusions: These results may provide evidence on the role of FXR in bladder cancer, and thus may improve the therapeutic efficacy of urothelial carcinoma in the future.

Structure and dynamics at N- and C-terminal regions of intrinsically disordered human c-Myc PEST degron reveal a pH-induced transition.

We investigated the structure and Brownian rotational motion of the PEST region (201-268) from human c-Myc oncoprotein, whose overexpression/dysregulation is associated with various types of cancer. The 77-residue PEST fragment revealed a large Stokes radius (~3.1 nm) and CD spectrum highlighting abundance of disordered structure. Changes in structure/dynamics at two specific sites in PEST degron were observed using time-resolved fluorescence spectroscopy by labeling Cys9 near N-terminal with dansyl probe and inserting a Trp70 near C-terminal (PEST M1). Trp in PEST M1 at pH 3 was inaccessible to quencher, showed hindered segmental motion and slow global rotation (~30 ns) in contrast to N-terminal where the dansyl probe was free, exposed with fast global rotation (~5 ns). Remarkably, this large monomeric structure at acidic pH was retained irrespective of ionic strength (0.03-0.25 M) and partially so in presence of 6 M Gdn.HCl. With gradual increase in pH, a structural transition (~pH 4.8) into a more exposed and freely rotating Trp was noticeable. Interestingly, the induced structure at C-terminal also influenced the dynamics of dansyl probe near N-terminal, which otherwise remained unstructured at pH > 5. FRET measurements confirmed a 11 Å decrease in distance between dansyl and indole at pH 4 compared to pH 9, coinciding with enhanced ANS binding and increase in strand/helix population in both PEST fragments. The protonation of glutamate/aspartate residues in C-terminal region of PEST is implicated in this disorder-order transition. This may have a bearing on the role of PEST in endocytic trafficking of eukaryotic proteins.

A molecular toolbox for studying protein degradation in mammalian cells.

Protein degradation is a crucial regulatory process in maintaining cellular proteostasis. The selective degradation of intracellular proteins controls diverse cellular and biochemical processes in all kingdoms of life. Targeted protein degradation is implicated in controlling the levels of regulatory proteins as well as eliminating misfolded and any otherwise abnormal proteins. Deregulation of protein degradation is concomitant with the progression of various neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. Thus, methods of measuring metabolic half-lives of proteins greatly influence our understanding of the diverse functions of proteins in mammalian cells including neuronal cells. Historically, protein degradation rates have been studied via exploiting methods that estimate overall protein degradation or focus on few individual proteins. Notably, with the recent technical advances and developments in proteomic and imaging techniques, it is now possible to measure degradation rates of a large repertoire of defined proteins and analyze the degradation profile in a detailed spatio-temporal manner, with the aim of determining proteome-wide protein stabilities upon different physiological conditions. Herein, we discuss some of the classical and novel methods for determining protein degradation rates highlighting the crucial role of some state of art approaches in deciphering the global impact of dynamic nature of targeted degradation of cellular proteins. This article is part of the Special Issue "Proteomics".

Proteome Dynamics from Heavy Water Metabolic Labeling and Peptide Tandem Mass Spectrometry.

Protein homeostasis (proteostasis) is a result of a dynamic equilibrium between protein synthesis and degradation. It is important for healthy cell/organ functioning and is often associated with diseases such as neurodegenerative diseases and non-Alcoholic Fatty Liver disease. Heavy water metabolic labeling, combined with liquid-chromatography and mass spectrometry (LC-MS), is a powerful approach to study proteostasis in vivo in high throughput. Traditionally, intact peptide signals are used to estimate stable isotope incorporation in time-course experiments. The time-course of label incorporation is used to extract protein decay rate constant (DRC). Intact peptide signals, computed from integration in chromatographic time and mass-to-charge ratio (m/z) domains, usually, provide an accurate estimate of label incorporation. However, sample complexity (co-elution), limited dynamic range, and low signal-to-noise ratio (S/N) may adversely interfere with the peptide signals. These artifacts complicate the DRC estimations by distorting peak shape in chromatographic time and m/z domains. Fragment ions, on the other hand, are less prone to these artifacts and are potentially well suited in aiding DRC estimations. Here, we show that the label incorporation encoded into the isotope distributions of fragment ions reflect the isotope enrichment during the metabolic labeling with heavy water. We explore the label incorporation statistics for devising practical approaches for DRC estimations.

Genetically Encoding a Lipidated Amino Acid for Extension of Protein Half-Life in†vivo.

Protein therapeutics are increasingly used to treat various diseases, yet they often suffer from short serum half-lives. An emerging strategy to extend lifetime in vivo is to attach fatty acids onto proteins to increase their binding to human serum albumin (HSA). Herein, the genetic encoding of ϵ-N-heptanoyl-l-lysine (HepoK) is reported, which introduces a fatty-acid-containing amino acid into proteins with exquisite site-specificity and homogeneity, overcoming issues associated with existing chemical conjugation methods. The expression in E .coli and purification of HepoK-incorporated glucagon-like peptide-1 (GLP1) is demonstrated. GLP1(HepoK) showed stronger binding to HSA than GLP1(WT), without impairing the stimulation of the GLP1 receptor in cells. Moreover, GLP1(HepoK) decreased blood glucose level to the same level as GLP1(WT) in mice, showing longer-lasting effects than GLP1(WT). HepoK incorporation will also be useful for investigating the function of protein lipidation.

d2ome, Software for in Vivo Protein Turnover Analysis Using Heavy Water Labeling and LC-MS, Reveals Alterations of Hepatic Proteome Dynamics in a Mouse Model of NAFLD.

Metabolic labeling with heavy water followed by LC-MS is a high throughput approach to study proteostasis in vivo. Advances in mass spectrometry and sample processing have allowed consistent detection of thousands of proteins at multiple time points. However, freely available automated bioinformatics tools to analyze and extract protein decay rate constants are lacking. Here, we describe d2ome-a robust, automated software solution for in vivo protein turnover analysis. d2ome is highly scalable, uses innovative approaches to nonlinear fitting, implements Grubbs' outlier detection and removal, uses weighted-averaging of replicates, applies a data dependent elution time windowing, and uses mass accuracy in peak detection. Here, we discuss the application of d2ome in a comparative study of protein turnover in the livers of normal vs Western diet-fed LDLR-/- mice (mouse model of nonalcoholic fatty liver disease), which contained 256 LC-MS experiments. The study revealed reduced stability of 40S ribosomal protein subunits in the Western diet-fed mice.

DNA-binding and repressor function are prerequisites for the turnover of the tomato heat stress transcription factor HsfB1.

HsfB1 is a central regulator of heat stress (HS) response and functions dually as a transcriptional co-activator of HsfA1a and a general repressor in tomato. HsfB1 is efficiently synthesized during the onset of HS and rapidly removed in the course of attenuation during the recovery phase. Initial results point to a complex regime modulating HsfB1 abundance involving the molecular chaperone Hsp90. However, the molecular determinants affecting HsfB1 stability needed to be established. We provide experimental evidence that DNA-bound HsfB1 is efficiently targeted for degradation when active as a transcriptional repressor. Manipulation of the DNA-binding affinity by mutating the HsfB1 DNA-binding domain directly influences the stability of the transcription factor. During HS, HsfB1 is stabilized, probably due to co-activator complex formation with HsfA1a. The process of HsfB1 degradation involves nuclear localized Hsp90. The molecular determinants of HsfB1 turnover identified in here are so far seemingly unique. A mutational switch of the R/KLFGV repressor motif's arginine and lysine implies that the abundance of other R/KLFGV type Hsfs, if not other transcription factors as well, might be modulated by a comparable mechanism. Thus, we propose a versatile mechanism for strict abundance control of the stress-induced transcription factor HsfB1 for the recovery phase, and this mechanism constitutes a form of transcription factor removal from promoters by degradation inside the nucleus.

A strategy based on thermal flexibility to design triosephosphate isomerase proteins with increased or decreased kinetic stability.

Kinetic stability of proteins determines their susceptibility to irreversibly unfold in a time-dependent process, and therefore its half-life. A residue displacement analysis of temperature-induced unfolding molecular dynamics simulations was recently employed to define the thermal flexibility of proteins. This property was found to be correlated with the activation energy barrier (Eact) separating the native from the transition state in the denaturation process. The Eact was determined from the application of a two-state irreversible model to temperature unfolding experiments using differential scanning calorimetry (DSC). The contribution of each residue to the thermal flexibility of proteins is used here to propose multiple mutations in triosephosphate isomerase (TIM) from Trypanosoma brucei (TbTIM) and Trypanosoma cruzi (TcTIM), two parasites closely related by evolution. These two enzymes, taken as model systems, have practically identical structure but large differences in their kinetic stability. We constructed two functional TIM variants with more than twice and less than half the activation energy of their respective wild-type reference structures. The results show that the proposed strategy is able to identify the crucial residues for the kinetic stability in these enzymes. As it occurs with other protein properties reflecting their complex behavior, kinetic stability appears to be the consequence of an extensive network of inter-residue interactions, acting in a concerted manner. The proposed strategy to design variants can be used with other proteins, to increase or decrease their functional half-life.

Histone deacetylase inhibitors interfere with angiogenesis by decreasing endothelial VEGFR-2 protein half-life in part via a VE-cadherin-dependent mechanism.

Recent evidence suggests that histone deacetylase inhibitors (HDACi) may mediate part of their antitumor effects by interfering with tumor angiogenesis. As signalling via the vascular endothelial growth factor receptor-2 (VEGFR-2) pathway is critical for angiogenic responses during tumor progression, we explored whether established antitumor effects of HDACi are partly mediated through diminished endothelial VEGFR-2 expression. We therefore examined the potential impact of three different HDACi, trichostatin A (TSA), sodium butyrate (But) and valproic acid (VPA), on VEGFR-2 protein expression. TSA, VPA and But significantly inhibit VEGFR-2 protein expression in endothelial cells. Pertinent to these data, VEGFR-2 protein half-life is shown to be decreased in response to HDACi. Recently, it could be demonstrated that expression of VE-cadherin influences VEGFR-2 protein half-life. In our experiments, VEGFR-2 downregulation was accompanied by HDACi-induced VE-cadherin suppression. Interestingly, siRNA-mediated knockdown of VE-cadherin led to a pronounced loss of VEGFR-2 expression on the protein as well as on the mRNA level, implicating that VE-cadherin not only influences VEGFR-2 protein half-life but also the transcriptional level. To further distinguish which of the eight different histone deacetylases are responsible for the regulation of VEGFR-2 expression, specific HDAC genes were silenced by transfecting respective siRNAs. These studies revealed that HDACs 1, 4, 5 and 6 are preferentially involved in VEGFR-2 expression. Therefore, these results provide an explanation for the anti-angiogenic action of HDAC inhibitors via a VE-cadherin, HDAC 1 and HDACs 4-6-mediated suppression of VEGFR-2 expression and might be of importance in the development of new anti-angiogenic drugs.

Short-lived Her proteins drive robust synchronized oscillations in the zebrafish segmentation clock.

Oscillations are prevalent in natural systems. A gene expression oscillator, called the segmentation clock, controls segmentation of precursors of the vertebral column. Genes belonging to the Hes/her family encode the only conserved oscillating genes in all analyzed vertebrate species. Hes/Her proteins form dimers and negatively autoregulate their own transcription. Here, we developed a stochastic two-dimensional multicellular computational model to elucidate how the dynamics, i.e. period, amplitude and synchronization, of the segmentation clock are regulated. We performed parameter searches to demonstrate that autoregulatory negative-feedback loops of the redundant repressor Her dimers can generate synchronized gene expression oscillations in wild-type embryos and reproduce the dynamics of the segmentation oscillator in different mutant conditions. Our model also predicts that synchronized oscillations can be robustly generated as long as the half-lives of the repressor dimers are shorter than 6 minutes. We validated this prediction by measuring, for the first time, the half-life of Her7 protein as 3.5 minutes. These results demonstrate the importance of building biologically realistic stochastic models to test biological models more stringently and make predictions for future experimental studies.

Protein turnover analysis in Salmonella Typhimurium during infection by dynamic SILAC, Topograph, and quantitative proteomics.

Protein turnover affects protein abundance and phenotypes. Comprehensive investigation of protein turnover dynamics has the potential to provide substantial information about gene expression. Here we report a large-scale protein turnover study in Salmonella Typhimurium during infection by quantitative proteomics. Murine macrophage-like RAW 264.7 cells were infected with SILAC labeled Salmonella. Bacterial cells were extracted after 0, 30, 60, 120, and 240 min. Mass spectrometry analyses yielded information about Salmonella protein turnover dynamics and a software program named Topograph was used for the calculation of protein half lives. The half lives of 311 proteins from intracellular Salmonella were obtained. For bacteria cultured in control medium (DMEM), the half lives for 870 proteins were obtained. The calculated median of protein half lives was 69.13 and 99.30 min for the infection group and the DMEM group, respectively, indicating an elevated protein turnover at the initial stage of infection. Gene ontology analyses revealed that a number of protein functional groups were significantly regulated by infection, including proteins involved in ribosome, periplasmic space, cellular amino acid metabolic process, ion binding, and catalytic activity. The half lives of proteins involved in purine metabolism pathway were found to be significantly shortened during infection.

Deciphering the impact of parameters influencing transgene expression kinetics after repeated cell transduction with integration-deficient retroviral vectors.

Lentiviral and gammaretroviral vectors are state-of-the-art tools for transgene expression within target cells. The integration of these vectors can be deliberately suppressed to derive a transient gene expression system based on extrachromosomal circular episomes with intact coding regions. These episomes can be used to deliver DNA templates and to express RNA or protein. Importantly, transient gene transfer avoids the genotoxic side effects of integrating vectors. Restricting their applicability, episomes are rapidly lost upon dilution in dividing target cells. Addressing this limitation, we could establish comparably stable percentages of transgene-positive cells over prolonged time periods in proliferating cells by repeated transductions. Flow cytometry was applied for kinetic analyses to decipher the impact of individual parameters on the kinetics of fluoroprotein expression after episomal retransduction and to visualize sequential and simultaneous transfer of heterologous fluoroproteins. Expression windows could be exactly timed by the number of transduction steps. The kinetics of signal loss was affected by the cell proliferation rate. The transfer of genes encoding fluoroproteins with different half-lives revealed a major impact of protein stability on temporal signal distribution and accumulation, determining optimal retransduction intervals. In addition, sequential transductions proved broad applicability in different cell types and using different envelope pseudotypes without receptor overload. Stable percentages of cells coexpressing multiple transgenes could be generated upon repeated coadministration of different episomal vectors. Alternatively, defined patterns of transgene expression could be recapitulated by sequential transductions. Altogether, we established a methodology to control and adjust a temporally defined window of transgene expression using retroviral episomal vectors. Combined with the highly efficient cell entry of these vectors while avoiding integration, the developed technology is of great significance for a broad panel of applications, including transcription-factor-based induced cell fate conversion and controlled transfer of genetically encoded RNA- or protein-based drugs.

Systematic analysis of proteome turnover in an organoid model of pancreatic cancer by dSILO.

The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.

Numbers of Exchangeable Hydrogens from LC-MS Data of Heavy Water Metabolically Labeled Samples.

Labeling with deuterium oxide (D2O) has emerged as one of the preferred approaches for measuring the synthesis of individual proteins in vivo. In these experiments, the synthesis rates of proteins are determined by modeling mass shifts in peptides during the labeling period. This modeling depends on a theoretical maximum enrichment determined by the number of labeling sites (NEH) of each amino acid in the peptide sequence. Currently, NEH is determined from one set of published values. However, it has been demonstrated that NEH can differ between species and potentially tissues. The goal of this work was to determine the number of NEH for each amino acid within a given experiment to capture the conditions unique to that experiment. We used four methods to compute the NEH values. To test these approaches, we used two publicly available data sets. In a de novo approach, we compute NEH values and the label enrichment from the abundances of three mass isotopomers. The other three methods use the complete isotope profiles and body water enrichment in deuterium as an input parameter. They determine the NEH values by (1) minimizing the residual sum of squares, (2) from the mole percent excess of labeling, and (3) the time course profile of the depletion of the relative isotope abundance of monoisotope. In the test samples, the method using residual sum of squares performed the best. The methods are implemented in a tool for determining the NEH for each amino acid within a given experiment to use in the determination of protein synthesis rates using D2O.