Changes in protein fluxes and gene expression in non-injured muscle tissue distant from an acute myotoxic injury in male mice.

The response to acute myotoxic injury requires stimulation of local repair mechanisms in the damaged tissue. However, satellite cells in muscle distant from acute injury have been reported to enter a functional state between quiescence and active proliferation. Here, we asked whether protein flux rates are altered in muscle distant from acute local myotoxic injury and how they compare to changes in gene expression from the same tissue. Broad and significant alterations in protein turnover were observed across the proteome in the limb contralateral to injury during the first 10 days after. Interestingly, mRNA changes had almost no correlation with directly measured protein turnover rates. In summary, we show consistent and striking changes in protein flux rates in muscle tissue contralateral to myotoxic injury, with no correlation between changes in mRNA levels and protein synthesis rates. This work motivates further investigation of the mechanisms, including potential neurological factors, responsible for this distant effect. KEY POINTS: Previous literature demonstrates that stem cells of uninjured muscle respond to local necrotic muscle tissue damage and regeneration. We show that muscle tissue that was distant from a model of local necrotic damage had functional changes at both the gene expression and the protein turnover level. However, these changes in distant tissue were more pronounced during the earlier stages of tissue regeneration and did not correlate well with each other. The results suggest communication between directly injured tissue and non-affected tissues that are distant from injury, which warrants further investigation into the potential of this mechanism as a proactive measure for tissue regeneration from damage.

Elevated Temperature Effects on Protein Turnover Dynamics in Arabidopsis thaliana Seedlings Revealed by 15N-Stable Isotope Labeling and ProteinTurnover Algorithm.

Global warming poses a threat to plant survival, impacting growth and agricultural yield. Protein turnover, a critical regulatory mechanism balancing protein synthesis and degradation, is crucial for the cellular response to environmental changes. We investigated the effects of elevated temperature on proteome dynamics in Arabidopsis thaliana seedlings using 15N-stable isotope labeling and ultra-performance liquid chromatography-high resolution mass spectrometry, coupled with the ProteinTurnover algorithm. Analyzing different cellular fractions from plants grown under 22 °C and 30 °C growth conditions, we found significant changes in the turnover rates of 571 proteins, with a median 1.4-fold increase, indicating accelerated protein dynamics under thermal stress. Notably, soluble root fraction proteins exhibited smaller turnover changes, suggesting tissue-specific adaptations. Significant turnover alterations occurred with redox signaling, stress response, protein folding, secondary metabolism, and photorespiration, indicating complex responses enhancing plant thermal resilience. Conversely, proteins involved in carbohydrate metabolism and mitochondrial ATP synthesis showed minimal changes, highlighting their stability. This analysis highlights the intricate balance between proteome stability and adaptability, advancing our understanding of plant responses to heat stress and supporting the development of improved thermotolerant crops.

Proteome Birthdating Reveals Age-Selectivity of Protein Ubiquitination.

Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To investigate the age-selectivity of cellular pathways, we developed a methodology termed "proteome birthdating" that barcodes proteins based on their time of synthesis. We demonstrate that this approach provides accurate measurements of protein turnover kinetics from a single biological sample encoding multiple labeling time-points. As a first application of the birthdated proteome, we investigated the age distribution of the human ubiquitinome. Our results indicate that the vast majority of ubiquitinated proteins in a cell consist of newly synthesized proteins and that these young proteins constitute the bulk of the degradative flux through the proteasome. Rapidly ubiquitinated nascent proteins are enriched in cytosolic subunits of large protein complexes. Conversely, proteins destined for the secretory pathway and vesicular transport have older ubiquitinated populations. Our data also identify a smaller subset of older ubiquitinated cellular proteins that do not appear to be targeted to the proteasome for rapid degradation. Together, our data provide an age census of the human ubiquitinome and establish proteome birthdating as a robust methodology for investigating the protein age-selectivity of diverse cellular pathways.

A comparative study of tissue protein synthesis rates in an Antarctic, Harpagifer antarcticus and a temperate, Lipophrys pholis teleost.

The affect of temperature on tissue protein synthesis rates has been reported in temperate and tropical, but not Antarctic fishes. Previous studies have generally demonstrated low growth rates in Antarctic fish species in comparison to temperate relatives and elevated levels of protein turnover. This study investigates how low temperatures effect tissue protein synthesis and hence tissue growth in a polar fish species. Groups of Antarctic, Harpagifer antarcticus and temperate, Lipophrys pholis, were acclimated to a range of overlapping water temperatures and protein synthesis was measure in white muscle (WM), liver and gastrointestinal tract (GIT). WM protein synthesis rates increased linearly with temperature in both species (H. antarcticus 0.16-0.23%.d-1, L. pholis, 0.31-0.76%.d-1), while liver (H. antarcticus 0.24-0.27%.d-1, L. pholis, 0.44-1.03%.d-1) and GIT were unaffected by temperature in H. antarcticus but increased non-linearly in L.pholis (H. antarcticus 0.22-0.26%.d-1, L. pholis, 0.40-0.86%.d-1). RNA to protein ratios were unaffected by temperature in H. antarcticus but increased weakly, in L.pholis WM and liver. In L.pholis, RNA translational efficiency increased significantly with temperature in all tissues, but only in liver in H. antarcticus. At the overlapping temperature of 3 °C, protein synthesis (WM 26%, Liver, 39%, GIT, 35%) and RNA translational efficiency (WM 273%, Liver, 271%, GIT, 300%) were significantly lower in H. antarcticus than L.pholis, while RNA to protein ratios were significantly higher (WM 270%, Liver 170%, GIT 186%). Tissue specific effects of temperature are detectable in both species. This study provides the first evidence, that tissue protein synthesis rates are constrained in Antarctic fishes.

Reconsidering the pre-eminence of dietary leucine and plasma leucinemia for predicting the stimulation of postprandial muscle protein synthesis rates.

The regulation of postprandial muscle protein synthesis (MPS) with or without physical activity has been an intensely studied area within nutrition and physiology. The leucine content of dietary protein and the subsequent plasma leucinemia it elicits postingestion is often considered the primary drivers of the postprandial MPS response. This concept, generally known as the leucine "trigger" hypothesis, has also been adopted within more applied aspects of nutrition. Our view is that recent evidence is driving a more nuanced picture of the regulation of postprandial MPS by revealing a compelling dissociation between ingested leucine or plasma leucinemia and the magnitude of the postprandial MPS response. Much of this lack of coherence has arisen as experimental progress has demanded relevant studies move beyond reliance on isolated amino acids and proteins to use increasingly complex protein-rich meals, whole foods, and mixed meals. Our overreliance on the centrality of leucine in this field has been reflected in 2 recent systematic reviews. In this perspective, we propose a re-evaluation of the pre-eminent role of these leucine variables in the stimulation of postprandial MPS. We view the development of a more complex intellectual framework now a priority if we are to see continued progress concerning the mechanistic regulation of postprandial muscle protein turnover, but also consequential from an applied perspective when evaluating the value of novel dietary protein sources.

Effects of improved amino acid balance diet on lysine mammary utilization, whole body protein turnover and muscle protein breakdown on lactating sows.

BACKGROUND: The study objective was to test the hypothesis that low crude protein (CP) diet with crystalline amino acids (CAA) supplementation improves Lys utilization efficiency for milk production and reduces protein turnover and muscle protein breakdown. Eighteen lactating multiparous Yorkshire sows were allotted to 1 of 2 isocaloric diets (10.80 MJ/kg net energy): control (CON; 19.24% CP) and reduced CP with "optimal" AA profile (OPT; 14.00% CP). Sow body weight and backfat were recorded on d 1 and 21 of lactation and piglets were weighed on d 1, 14, 18, and 21 of lactation. Between d 14 and 18, a subset of 9 sows (CON = 4, OPT = 5) was infused with a mixed solution of 3-[methyl-2H3]histidine (bolus injection) and [13C]bicarbonate (priming dose) first, then a constant 2-h [13C]bicarbonate infusion followed by a 6-h primed constant [1-13C]lysine infusion. Serial blood and milk sampling were performed to determine plasma and milk Lys enrichment, Lys oxidation rate, whole body protein turnover, and muscle protein breakdown. RESULTS: Over the 21-d lactation period, compared to CON, sows fed OPT had greater litter growth rate (P < 0.05). Compared to CON, sows fed OPT had greater efficiency of Lys (P < 0.05), Lys mammary flux (P < 0.01) and whole-body protein turnover efficiency (P < 0.05). Compared to CON, sows fed OPT tended to have lower whole body protein breakdown rate (P = 0.069). Muscle protein breakdown rate did not differ between OPT and CON (P = 0.197). CONCLUSION: Feeding an improved AA balance diet increased efficiency of Lys and reduced whole-body protein turnover and protein breakdown. These results imply that the lower maternal N retention observed in lactating sows fed improved AA balance diets in previous studies may be a result of greater partitioning of AA towards milk rather than greater body protein breakdown.

Turnover of EDEM1, an ERAD-enhancing factor, is mediated by multiple degradation routes.

Quality-based protein production and degradation in the endoplasmic reticulum (ER) are essential for eukaryotic cell survival. During protein maturation in the ER, misfolded or unassembled proteins are destined for disposal through a process known as ER-associated degradation (ERAD). EDEM1 is an ERAD-accelerating factor whose gene expression is upregulated by the accumulation of aberrant proteins in the ER, known as ER stress. Although the role of EDEM1 in ERAD has been studied in detail, the turnover of EDEM1 by intracellular degradation machinery, including the proteasome and autophagy, is not well understood. To clarify EDEM1 regulation in the protein level, degradation mechanism of EDEM1 was examined. Our results indicate that both ERAD and autophagy degrade EDEM1 alike misfolded degradation substrates, although each degradation machinery targets EDEM1 in different folded states of proteins. We also found that ERAD factors, including the SEL1L/Hrd1 complex, YOD1, XTP3B, ERdj3, VIMP, BAG6, and JB12, but not OS9, are involved in EDEM1 degradation in a mannose-trimming-dependent and -independent manner. Our results suggest that the ERAD accelerating factor, EDEM1, is turned over by the ERAD itself, similar to ERAD clients.

A proteomics-based survey reveals thrombospondin-4 as a ligand regulated by the mannose receptor in the injured lung.

Receptor-mediated cellular uptake of specific ligands constitutes an important step in the dynamic regulation of individual protein levels in extracellular fluids. With a focus on the inflammatory lung, we here performed a proteomics-based search for novel ligands regulated by the mannose receptor (MR), a macrophage-expressed endocytic receptor. WT and MR-deficient mice were exposed to lipopolysaccharide, after which the protein content in their lung epithelial lining fluid was compared by tandem mass tag-based mass spectrometry. More than 1200 proteins were identified in the epithelial lining fluid using this unbiased approach, but only six showed a statistically different abundance. Among these, an unexpected potential new ligand, thrombospondin-4 (TSP-4), displayed a striking 17-fold increased abundance in the MR-deficient mice. Experiments using exogenous addition of TSP-4 to MR-transfected CHO cells or MR-positive alveolar macrophages confirmed that TSP-4 is a ligand for MR-dependent endocytosis. Similar studies revealed that the molecular interaction with TSP-4 depends on both the lectin activity and the fibronectin type-II domain of MR and that a closely related member of the TSP family, TSP-5, is also efficiently internalized by the receptor. This was unlike the other members of this protein family, including TSPs -1 and -2, which are ligands for a close MR homologue known as urokinase plasminogen activator receptor-associated protein. Our study shows that MR takes part in the regulation of TSP-4, an important inflammatory component in the injured lung, and that two closely related endocytic receptors, expressed on different cell types, undertake the selective endocytosis of distinct members of the TSP family.

Nα-acetyltransferase NAA50 mediates plant immunity independent of the Nα-acetyltransferase A complex.

In humans and plants, 40% of the proteome is co-translationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex is comprised of the catalytic subunit Nα- acetyltransferase 10 (NAA10) and the ribosome-anchoring subunit NAA15. The regulatory subunit Huntingtin Yeast Partner K (HYPK) and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis (Arabidopsis thaliana), only AtHYPK is known to interact with AtNatA. Here we uncover the AtNAA50 interactome and provide evidence for the association of AtNAA50 with NatA at ribosomes. In agreement with the latter, a split-luciferase approach demonstrated close proximity of AtNAA50 and AtNatA in planta. Despite their interaction, AtNatA/HYPK and AtNAA50 exerted different functions in vivo. Unlike NatA/HYPK, AtNAA50 did not modulate drought-tolerance or promote protein stability. Instead, transcriptome and proteome analyses of a novel AtNAA50-depleted mutant (amiNAA50) implied that AtNAA50 negatively regulates plant immunity. Indeed, amiNAA50 plants exhibited enhanced resistance to oomycetes and bacterial pathogens. In contrast to what was observed in NatA-depleted mutants, this resistance was independent of an accumulation of salicylic acid prior to pathogen exposure. Our study dissects the in vivo function of the NatA interactors HYPK and NAA50 and uncovers NatA-independent roles for NAA50 in plants.

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.

Adrenalectomy attenuates hyperalgesia but does not regulate muscle wasting in a female rat model of fibromyalgia.

Although it is well established that fibromyalgia (FM) syndrome is characterized by chronic diffuse musculoskeletal hyperalgesia, very little is known about the effect of this pathology on muscle tissue plasticity. Therefore, the present study aimed to characterize the putative alterations in skeletal muscle mass in female rats subjected to a FM model by inducing chronic diffuse hyperalgesia (CDH) through double injections of acidic saline (pH 4.0) into the left gastrocnemius muscle at 5-day intervals. To determine protein turnover, the total proteolysis, proteolytic system activities and protein synthesis were evaluated in oxidative soleus muscles of pH 7.2 (control) and pH 4.0 groups at 7 days after CDH induction. All animals underwent behavioural analyses of mechanical hyperalgesia, strength and motor performance. Our results demonstrated that, in addition to hyperalgesia, rats injected with acidic saline exhibited skeletal muscle loss, as evidenced by a decrease in the soleus fibre cross-sectional area. This muscle loss was associated with increased proteasomal proteolysis and expression of the atrophy-related gene (muscle RING-finger protein-1), as well as reduced protein synthesis and decreased protein kinase B/S6 pathway activity. Although the plasma corticosterone concentration did not differ between the control and pH 4.0 groups, the removal of the adrenal glands attenuated hyperalgesia, but it did not prevent the increase in muscle protein loss in acidic saline-injected animals. The data suggests that the stress-related hypothalamic-pituitary-adrenal axis is involved in the development of hyperalgesia, but is not responsible for muscle atrophy observed in the FM model induced by intramuscular administration of acidic saline. Although the mechanisms involved in the attenuation of hyperalgesia in rats injected with acidic saline and subjected to adrenalectomy still need to be elucidated, the results found in this study suggest that glucocorticoids may not represent an effective therapeutic approach to alleviate FM symptoms.

Translation is an emerging constraint on protein homeostasis in ageing.

Proteins are molecular machines that provide structure and perform vital transport, signalling and enzymatic roles. Proteins expressed by cells require tight regulation of their concentration, folding, localisation, and modifications; however, this state of protein homeostasis is continuously perturbed by tissue-level stresses. While cells in healthy tissues are able to buffer against these perturbations, for example, by expression of chaperone proteins, protein homeostasis is lost in ageing, and can lead to protein aggregation characteristic of protein folding diseases. Here, we review reports of a progressive disconnect between transcriptomic and proteomic regulation during cellular ageing. We discuss how age-associated changes to cellular responses to specific stressors in the tissue microenvironment are exacerbated by loss of ribosomal proteins, ribosomal pausing, and mistranslation.

In utero pulse injection of isotopic amino acids quantifies protein turnover rates during murine fetal development.

Protein translational control is critical for ensuring that the fetus develops correctly and that necessary organs and tissues are formed and functional. We developed an in utero method to quantify tissue-specific protein dynamics by monitoring amino acid incorporation into the proteome after pulse injection. Fetuses of pregnant mice were injected with isotopically labeled lysine and arginine via the vitelline vein at various embyonic days, and organs and tissues were harvested. By analyzing the nascent proteome, unique signatures of each tissue were identified by hierarchical clustering. In addition, the quantified proteome-wide turnover rates were calculated between 3.81E-5 and 0.424 h-1. We observed similar protein turnover profiles for analyzed organs (e.g., liver vs. brain); however, their distributions of turnover rates vary significantly. The translational kinetic profiles of developing organs displayed differentially expressed protein pathways and synthesis rates, which correlated with known physiological changes during mouse development.

Studying Secretory Protein Synthesis in Nicotiana benthamiana Protoplasts.

Transient gene expression in plant protoplasts facilitates the analysis of hybrid genes in a fast and reproducible manner. The technique is particularly powerful when studying basic conserved biochemical processes including de novo protein synthesis, modification, assembly, transport, and turnover. Unlike individual plants, protoplast suspensions can be divided into almost identical aliquots, allowing the analysis of independent variables with uncertainties restricted to minor pipetting errors/variations. Using the examples of protein secretion and ER retention, we describe the most advanced working practice of routinely preparing, electroporating, and analyzing Nicotiana benthamiana protoplasts. A single batch of electroporation-competent protoplasts permits up to 30 individual transfections. This is ideal to assess the influence of independent variables, such as point mutations, deletions or fusions, or the influence of a co-expressed effector gene in dose-response studies.

Unraveling the interplay between PKA inhibition and Cdk1 activation during oocyte meiotic maturation.

Oocytes are arrested in prophase I. In vertebrates, meiotic resumption is triggered by hormonal stimulation that results in cAMP-dependent protein kinase (PKA) downregulation leading to Cdk1 activation. Yet the pathways connecting PKA to Cdk1 remain unclear. Here, we identify molecular events triggered by PKA downregulation occurring upstream of Cdk1 activation. We describe a two-step regulation controlling cyclin B1 and Mos accumulation, which depends on both translation and stabilization. Cyclin B1 accumulation is triggered by PKA inhibition upstream of Cdk1 activation, while its translation requires Cdk1 activity. Conversely, Mos translation initiates in response to the hormone, but the protein accumulates only downstream of Cdk1. Furthermore, two successive translation waves take place, the first controlled by PKA inhibition and the second by Cdk1 activation. Notably, Arpp19, an essential PKA effector, does not regulate the early PKA-dependent events. This study elucidates how PKA downregulation orchestrates multiple pathways that converge toward Cdk1 activation and induce the oocyte G2/M transition.

Derailed protein turnover in the aging mammalian brain.

Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.

Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.

The functions of proteins depend on their spatial and temporal distributions, which are not directly measured by static protein abundance. Under endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) pathway remediates proteostasis in part by altering the turnover kinetics and spatial distribution of proteins. A global view of these spatiotemporal changes has yet to emerge and it is unknown how they affect different cellular compartments and pathways. Here we describe a mass spectrometry-based proteomics strategy and data analysis pipeline, termed Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently the changes in protein turnover and subcellular distribution in the same experiment. Investigating two common UPR models of thapsigargin and tunicamycin challenge in human AC16 cells, we find that the changes in protein turnover kinetics during UPR varies across subcellular localizations, with overall slowdown but an acceleration in endoplasmic reticulum and Golgi proteins involved in stress response. In parallel, the spatial proteomics component of the experiment revealed an externalization of amino acid transporters and ion channels under UPR, as well as the migration of RNA-binding proteins toward an endosome co-sedimenting compartment. The SPLAT experimental design classifies heavy and light SILAC labeled proteins separately, allowing the observation of differential localization of new and old protein pools and capturing a partition of newly synthesized EGFR and ITGAV to the ER under stress that suggests protein trafficking disruptions. Finally, application of SPLAT toward human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) exposed to the cancer drug carfilzomib, identified a selective disruption of proteostasis in sarcomeric proteins as a potential mechanism of carfilzomib-mediated cardiotoxicity. Taken together, this study provides a global view into the spatiotemporal dynamics of human cardiac cells and demonstrates a method for inferring the coordinations between spatial and temporal proteome regulations in stress and drug response.

Protein turnover in pigs: A review of interacting factors.

Protein turnover defines the balance between two continuous and complex processes of protein metabolism, synthesis and degradation, which determine their deposition in tissues. Although the liver and intestine have been studied extensively for their important roles in protein digestion, absorption and metabolism, the study of protein metabolism has focused mainly on skeletal muscle tissue to understand the basis for its growth. Due to the high adaptability of skeletal muscle, its protein turnover is greatly affected by different internal and external factors, contributing to carcass lean-yield and animal growth. Amino acid (AA) labelling and tracking using isotope tracer methodology, together with the study of myofiber type profiling, signal transduction pathways and gene expression, has allowed the analysis of these mechanisms from different perspectives. Positive stimuli such as increased nutrient availability in the diet (e.g., AA), physical activity, the presence of certain hormones (e.g., testosterone) or a more oxidative myofiber profile in certain muscles or pig genotypes promote increased upregulation of translation and transcription-related genes, activation of mTORC1 signalling mechanisms and increased abundance of satellite cells, allowing for more efficient protein synthesis. However, fasting, animal aging, inactivity and stress, inflammation or sepsis produce the opposite effect. Deepening the understanding of modifying factors and their possible interaction may contribute to the design of optimal strategies to better control tissue growth and nutrient use (i.e., protein and AA), and thus advance the precision feeding strategy.

S-acylation: an orchestrator of the life cycle and function of membrane proteins.

S-acylation is a covalent post-translational modification of proteins with fatty acids, achieved by enzymatic attachment via a labile thioester bond. This modification allows for dynamic control of protein properties and functions in association with cell membranes. This lipid modification regulates a substantial portion of the human proteome and plays an increasingly recognized role throughout the lifespan of affected proteins. Recent technical advancements have propelled the S-acylation field into a 'molecular era', unveiling new insights into its mechanistic intricacies and far-reaching implications. With a striking increase in the number of studies on this modification, new concepts are indeed emerging on the roles of S-acylation in specific cell biology processes and features. After a brief overview of the enzymes involved in S-acylation, this viewpoint focuses on the importance of S-acylation in the homeostasis, function, and coordination of integral membrane proteins. In particular, we put forward the hypotheses that S-acylation is a gatekeeper of membrane protein folding and turnover and a regulator of the formation and dynamics of membrane contact sites.

Proteomic Approach to Investigating Expression, Localization, and Functions of the SOWAHD Gene Protein Product during Granulocytic Differentiation.

Cataloging human proteins and evaluation of their expression, cellular localization, functions, and potential medical significance are important tasks for the global proteomic community. At present, localization and functions of protein products for almost half of protein-coding genes remain unknown or poorly understood. Investigation of organelle proteomes is a promising approach to uncovering localization and functions of human proteins. Nuclear proteome is of particular interest because many nuclear proteins, e.g., transcription factors, regulate functions that determine cell fate. Meta-analysis of the nuclear proteome, or nucleome, of HL-60 cells treated with all-trans-retinoic acid (ATRA) has shown that the functions and localization of a protein product of the SOWAHD gene are poorly understood. Also, there is no comprehensive information on the SOWAHD gene expression at the protein level. In HL-60 cells, the number of mRNA transcripts of the SOWAHD gene was determined as 6.4 ± 0.7 transcripts per million molecules. Using targeted mass spectrometry, the content of the SOWAHD protein was measured as 0.27 to 1.25 fmol/µg total protein. The half-life for the protein product of the SOWAHD gene determined using stable isotope pulse-chase labeling was ~19 h. Proteomic profiling of the nuclear fraction of HL-60 cells showed that the content of the SOWAHD protein increased during the ATRA-induced granulocytic differentiation, reached the peak value at 9 h after ATRA addition, and then decreased. Nuclear location and involvement of the SOWAHD protein in the ATRA-induced granulocytic differentiation have been demonstrated for the first time.