Cell Compartment Research Articles

Initial regional cytoarchitectonic differences in dorsal and orbitobasal human developing frontal cortex revealed by spatial transcriptomics.

Early development of the human fetal cerebral cortex involves a set of precisely coordinated molecular processes that remains rather underexplored. Previous studies indicate that the laminar identity and the molecular specification of cortical neurons driven by genetic programming, as well as associated histogenetic events begin during early fetal development. Our recent study discovered unique regional cytoarchitectonic features in the developing human frontal lobe, including migratory waves of postmitotic neurons in the dorsal frontal cortex and the "double plate" feature in orbitobasal cortex (Kopić et al. in Cells 12:231, 2023). Notably, neurons of these two cytoarchitectonic features typically express deep projection neuron (DPN) markers (TBR1, TLE4, SOX5). This paper aims to conduct an in-depth investigation of these cytoarchitectonic features at the transcriptomic level, whilst preserving spatial information. Here, we employed NanoString GeoMx™ Digital Spatial Profiler (DSP) technology to examine gene expression differences in the transient cortical compartments of the dorsal and ventral regions of the developing frontal lobe, focusing specifically on 15 post-conceptional weeks (PCW), that is a critical period for subplate formation. We identified multiple differentially expressed genes between the transient cellular compartments of the dorsal and orbitobasal regions of the developing human frontal cortex. These new findings additionally confirm that regional patterning and specification of the prospectivehigher-order association prefrontal cortex emerges early in fetal development, contributing to the highly organized cortical architecture of the human brain.

Lipids: Emerging Players of Microglial Biology.

Lipids are small molecule immunomodulators that play critical roles in maintaining cellular health and function. Microglia, the resident immune cells of the central nervous system, regulate lipid metabolism both in the extracellular environment and within intracellular compartments through various mechanisms. For instance, glycerophospholipids and fatty acids interact with protein receptors on the microglial surface, such as the Triggering Receptor Expressed on Myeloid Cells 2, influencing cellular functions like phagocytosis and migration. Moreover, cholesterol is essential not only for microglial survivalbut,along with other lipids such as fatty acids, is crucial for the formation, function, and accumulation of lipid droplets,which modulate microglial activity in inflammatory diseases. Other lipids, including acylcarnitines and ceramides, participate in various signaling pathways within microglia. Despite the complexity of the microglial lipidome, only a few studies have investigated the effects of specific lipid classes on microglial biology. In this review, we focus on major lipid classes and their roles in modulating microglial function. We also discuss novel analytical techniques for characterizing the microglial lipidome and highlight gaps in current knowledge, suggesting new directions for future research on microglial lipid biology.

Longevity of antibody responses is associated with distinct antigen-specific B cell subsets early after infection

IntroductionUpon infection, T cell-driven B cell responses in GC reactions induce memory B cells and antibody-secreting cells that secrete protective antibodies. How formation of specifically long-lived plasma cells is regulated via the interplay between specific B and CD4+ T cells is not well understood. Generally, antibody levels decline over time after clearance of the primary infection.MethodIn this study, convalescent individuals with stable RBD antibody levels (n=14, “sustainers”) were compared with donors (n=13) with the greatest antibody decline from a cohort of 132. To investigate the role of the cellular immune compartment in the maintenance of antibody levels, SARS-CoV-2-specific responses at 4 to 6 weeks post-mild COVID-19 infection were characterized using deep immune profiling.ResultsBoth groups had similar frequencies of total SARS-CoV-2-specific B and CD4+ T cells. Sustainers had fewer Spike-specific IgG+ memory B cells early after infection and increased neutralizing capacity of RBD antibodies over time, unlike the declining group. However, declining IgG titers correlated with lower frequency of Spike-specific CD4+ T cells.ConclusionThese data suggest that “sustainers” have unique dynamics of GC reactions, yield different outputs of terminally differentiating cells, and improve the quality of protective antibodies over time. This study helps identify factors controlling formation of long-lived PC and sustained antibody responses.

Enzymatic Reaction Network-driven Polymerization-induced Transient Coacervation.

A living cell has a highly complex microenvironment whereas numerous enzyme-driven processes are active at once. These procedures are incredibly accurate and efficient, although comparable control has not yet been established in vitro. Here, we design an enzymatic reaction network (ERN) that combines antagonistic and orthogonal enzymatic networks to produce adjustable dynamics of ATP-fueled transient coacervation. Using horseradish peroxidase (HRP)-mediated Biocatalytic Atom Transfer Radical Polymerization (BioATRP), we synthesized poly(dimethylaminoethyl methacrylate), which subsequently formed coacervates with ATP. We rationally explored enzymatic control over coacervation and dissolution, using orthogonal and antagonistic enzyme pairs viz., alkaline phosphatase, Creatine phosphokinase, hexokinase, esterase, and urease. ATP-fuelled coacervates also demonstrate the enzymatic catalysis to prove its potential to be exploited as a cellular microreactor. Additionally, we developed ERN-polymerization-induced transient coacervation (ERN-PIC), with complete control over the system, polymerization, coacervation, and dissolution. Notably, the coacervation process itself determines functional properties, as seen in selective cargo uptake. The strategy offers cutting-edge biomimetic applications, and insights into cellular compartmentalization by bridging the gap between synthetic and biological systems. The development of temporally programmed coacervation is promising for the spatial arrangement of multienzyme cascades, and offers novel ideas on the architecture of artificial cells.

Antioxidant and Ultrastructural Alterations in Wheat During Drought-Induced Leaf Senescence

Wheat is one of the most important crops to ensure food production globally. Understanding the mechanism of leaf senescence in wheat plays a crucial role in improving its productivity and resilience under various stress scenarios. In this study, we investigated biochemical, functional, and ultrastructural changes during leaf senescence in wheat genotypes with contrasting drought tolerance. For this, key parameters such as chlorophyll and total protein content, membrane stability, malondialdehyde level, and the activity of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, benzidine peroxidase, and catalase) were comparatively analyzed during both natural and drought-induced senescence. Additionally, the expression of superoxide dismutase isoform genes functioning in different cellular compartments was studied, alongside ultrastructural changes in flag leaves. The experiments involved genotypes of bread wheat (Triticum aestivum L.) and durum (Triticum durum Desf.) wheat. The plants were grown in controlled environment chambers under control and drought conditions using a completely randomized design. After the booting stage, irrigation was discontinued for drought-treated plants. Flag leaves were sampled at 7, 14, 21, 28, and 35 days after anthesis. Drought-tolerant genotypes exhibited slower chlorophyll degradation, lower lipid peroxidation, enhanced membrane stability, and stronger antioxidant responses, allowing them to maintain cellular function longer, whereas sensitive genotypes showed accelerated leaf senescence. Transcript levels of FeSOD increased significantly post-flowering but declined as senescence progressed, while MnSOD expression exhibited a rise towards the later stages of ontogenesis across all studied genotypes. Ultrastructural analysis revealed progressive damage to chloroplast membranes, thylakoid structures, and mesophyll cell walls under stress conditions, particularly in sensitive genotypes. These findings contribute to a deeper understanding of the physiological and molecular responses of wheat to drought stress, offering potential targets for improving crop performance in water-limited environments.

The Impact of HIV on B Cell Compartment and Its Implications for COVID-19 Vaccinations in People with HIV

HIV causes intense polyclonal activation of B cells, resulting in increased numbers of spontaneously antibody-secreting cells in the circulation and hypergammaglobulinemia. It is accompanied by significant perturbations in various B cell subsets, such as increased frequencies of immature/transitional B cells, activated memory B cells, atypical memory B cells, short-lived plasmablasts and regulatory B cells, as well as by decreased frequencies of resting memory and resting naïve B cells. Furthermore, both memory and antigen-inexperienced naïve B cells show exhausted and immune-senescent phenotypes. HIV also drives the expansion and functional impairment of CD4+ T follicular helper cells, which provide help to B cells, crucial for the generation of germinal center reactions and production of long-lived plasma and memory B cells. By suppressing viral replication, anti-retroviral therapy reverses the virus-induced perturbations and functional defects, albeit inadequately. Due to HIV’s lingering impact on B cells, immune senescence and residual chronic inflammation, people with HIV (PWH), especially immune non-responders, are immunocompromised and mount suboptimal antibody responses to vaccination for SARS-CoV-2. Here, we review how functionally and phenotypically distinct B cell subsets are induced in response to a vaccine and an infection and how HIV infection and anti-retroviral therapy (ART) impact them. We also review the role played by HIV-induced defects and perturbations in B cells in the induction of humoral immune responses to currently used anti-SARS-CoV-2 vaccines in PWH on ART. We also outline different strategies that could potentially enhance the vaccine-induced antibody responses in PWH. The review will provide guidance and impetus for further research to improve the immunogenicity of these vaccines in this human population.

Treatment of high risk myelodysplastic syndrome.

The myelodysplastic syndrome (MDS) is considered to be a heterogeneous myeloid malignancy with a common origin in the hematopoietic stem cell compartment, generally divided into lower and higher risk. While treatment goal for lower risk MDS (LR-MDS) is to decrease transfusion burden and transformation into acute leukemia major aim for high risk MDS is to prolong survival and ultimately cure. While novel agents such as luspatercept or imetelstat have recently been approved as new treatment options for LR-MDS, hypomethylating agents (HMA) remain currently the only approved non-transplant option for HR-MDS and is the standard of care for non-transplant-eligible patients. Combinations with other drugs as first-line treatment has to date not proven more efficacious than monotherapy in HR-MDS, and outcome after HMA failure is poor. The only potential cure and standard of care for eligible patients is allogeneic stem cell transplantation (HSCT) and even if the number of transplanted - especially older - MDS patients increased over time due to a better management and donor availability the majority of MDS patients will not be eligible for this curative approach. Current challenges encompass to decrease the relapse risk, the main cause of HSCT failure. This review will summarize current knowledge of options of transplant- and non-transplant treatment approaches for these patients and demonstrate the unmet clinical need for more effective therapies.

Left Ventricular Hypertrophy in Aortic Stenosis: Early Cell and Matrix Regression 2 Months Post-Aortic Valve Replacement.

In aortic stenosis, the myocardium responds with left ventricular hypertrophy, which is characterized by increased left ventricular mass due to cellular hypertrophy and extracellular matrix expansion. Following aortic valve replacement (AVR), left ventricular hypertrophy regression occurs, but early cellular and extracellular dynamics are unknown. Patients with severe symptomatic aortic stenosis undergoing surgical or transcatheter AVR were prospectively recruited. Pre- and early post-AVR cardiac magnetic resonance imaging assessed left ventricular remodeling, global longitudinal strain, and T1 mapping to determine extracellular volume fraction and volume of cellular and extracellular compartments. In all, 39 patients (aged 71.4±9.8 years, male 79%, aortic valve peak velocity 4.4±0.5 m/s) underwent cardiac magnetic resonance before and at median 7.7 weeks post-AVR. Left ventricular mass index reduced significantly by 15.4% (P<0.001*), primarily driven by cellular compartment regression (18.7%, P<0.001*), with a smaller reduction in the extracellular compartment (7.2%, P<0.001*). This unbalanced regression led to an apparent increase in extracellular volume fraction (27.4±3.1% to 30.2±2.8%; P<0.001*). Although there was no significant change in global longitudinal strain post-AVR, an increase in extracellular volume fraction was associated with worsening of global longitudinal strain (Pearson r=0.41, P=0.01). Mode of intervention (transcatheter versus surgical) did not influence the above myocardial parameters post-AVR (all P>0.05). The asterisk in P values indicates a statistical significance of <0.05. Within 8 weeks of AVR for aortic stenosis, substantial left ventricular hypertrophy regression occurs involving both cellular and extracellular compartments, demonstrating the early myocardial adaptability to afterload relief. Cellular compartment regression is greater than extracellular regression, leading to an apparent increase in extracellular volume fraction. Mode of intervention did not affect degree of reverse remodeling, indicating that both are effective at resulting beneficial changes post-AVR. URL: https://www.isrctn.com; Unique identifier: NCT04627987.

Engineering a Silk Protein-Mediated Customizable Compartment for Modular Metabolic Synthesis.

Microbial cell factories provide a nontoxic, economical way for the synthesis of various chemicals and drugs, garnering significant attention from researchers. However, excessive dispersion of enzymes and accumulation of intermediate metabolites in the production process will weaken the reaction efficiency of the pathway enzyme. In this study, a cellular compartment was constructed to isolate the enzyme reaction space and optimize the modular metabolic synthesis. First, a special spider silk protein was designed and constructed to form protein condensates in microbial cells, and its synthetic microcompartment effects were investigated. Second, the interaction of short peptide pairs or direct fusion based on the silk protein was used to recruit a variety of enzymes to improve the efficiency of enzyme catalysis. Third, the 2'-fucosyllactose (2'-FL) de novo synthesis pathway and its modular optimization were carried out to verify the mode. Finally, a synthetic compartment was introduced into the pathway to directly aggregate the 2'-FL synthesis pathway, thus obtaining synthetic-compartment-mediated multienzyme aggregates. The experimental results showed that the titer of 2'-FL was significantly improved compared with those of wild-type and modular-optimized free enzymes. The utilization of this cell microcompartment offers a novel avenue for the aggregation of diverse enzymes, thereby offering an innovative approach for enhancing the efficiency of the microbial modular metabolic pathway.

Structure of yeast RAVE bound to a partial V1 complex

Vacuolar-type ATPases (V-ATPases) are membrane-embedded proton pumps that acidify intracellular compartments in almost all eukaryotic cells. Homologous with ATP synthases, these multisubunit enzymes consist of a soluble catalytic V1 subcomplex and a membrane-embedded proton-translocating VO subcomplex. The V1 and VO subcomplexes can undergo reversible dissociation to regulate proton pumping, with reassociation of V1 and VO requiring the protein complex known as RAVE (regulator of the ATPase of vacuoles and endosomes). In the yeast Saccharomyces cerevisiae, RAVE consists of subunits Rav1p, Rav2p, and Skp1p. We used electron cryomicroscopy (cryo-EM) to determine a structure of yeast RAVE bound to V1. In the structure, RAVE is an L-shaped complex with Rav2p pointing toward the membrane and Skp1p distant from both the membrane and V1. Only Rav1p interacts with V1, binding to a region of subunit A not found in the corresponding ATP synthase subunit. When bound to RAVE, V1 is in a rotational state suitable for binding the free VO complex, but in the structure, it is partially disrupted, missing five of its 16 subunits. Other than these missing subunits and the conformation of the inhibitory subunit H, the V1 complex with RAVE appears poised for reassembly with VO.

The ABC transporter Opp imports reduced glutathione, while Gsi imports glutathione disulfide in Escherichia coli

Glutathione is the major thiol-based antioxidant in a wide variety of biological systems, ranging from bacteria to eukaryotes. As a redox couple, consisting of reduced glutathione (GSH) and its oxidized form, glutathione disulfide (GSSG), it is crucial for the maintenance of the cellular redox balance. Glutathione transport out of and into cellular compartments and the extracellular space is a determinant of the thiol-disulfide redox state of the organelles and bodily fluids in question, but is currently not well understood. Here we use the genetically-encoded, glutathione-measuring redox probe Grx1-roGFP2 to comprehensively elucidate the import of extracellular glutathione into the cytoplasm of the model organism Escherichia coli. The elimination of only two ATP-Binding Cassette (ABC) transporter systems, Gsi and Opp, completely abrogates glutathione import into E. coli’s cytoplasm, both in its reduced and oxidized form. The lack of only one of them, Gsi, completely prevents import of GSSG, while the lack of the other, Opp, substantially retards the uptake of reduced glutathione (GSH).

Monosaccharide coating modulate the intracellular trafficking of gold nanoparticles in dendritic cells

Dendritic cells (DCs) have emerged as a promising target for drug delivery and immune modulation due to their pivotal role in initiating the adaptive immune response. Gold nanoparticles (AuNPs) have garnered interest as a platform for targeted drug delivery due to their biocompatibility, low toxicity and precise control over size, morphology and surface functionalization. Our investigation aimed to elucidate the intracellular uptake and trafficking of AuNPs coated with different combinations of monosaccharides (mannose, galactose, and fucose) in DCs. We used 30 unique polymer-tethered monosaccharide combinations to coat 16 nm diameter spherical gold nanoparticles and investigated their effect on DCs phenotype, uptake, and intracellular trafficking.DCs internalised AuNPs coated with 100% fucose, 100% mannose, 90% mannose + 10% galactose, and 80% mannose + 20% galactose with highest efficiency. Flow cytometry analysis indicated that 100% fucose-coated AuNPs showed increased lysosomal and endosomal contents compared to other conditions and uncoated AuNPs. Imaging flow cytometry further demonstrated that 100% fucose-coated AuNPs had enhanced co-localisation with lysosomes, while 100% mannose-coated AuNPs exhibited higher co-localisation with endosomes.Furthermore, our data showed that the uptake of carbohydrate-coated AuNPs predominantly occurred through receptor-mediated endocytosis, as evidenced by a marked reduction of uptake upon treatment of DCs with methyl-β-cyclodextrins, known to disrupt receptor-mediated endocytosis. These findings highlight the utility of carbohydrate coatings to enable more targeted delivery of nanoparticles and their payload to distinct intracellular compartments in immune cells with potential applications in drug delivery and immunotherapy.

Determination of diffusion coefficients through gels with non-negligible finite-volume effects in the compartments of the diffusion cell

Diffusion cells are used to measure diffusion coefficients (DM) in gels. These measurements are of interest to understand and predict the availability of nutritive or toxic chemical species in waters, soils and sediments. When the diffusive flux from the donor to the acceptor compartment is constant (steady-state regime), DM is determined from the slope of the linear plot of the acceptor concentration vs time. However, at long enough times, there is a non-negligible concentration depletion in the donor compartment concomitant to a concentration increase in the acceptor compartment. Accordingly, the accumulation plot bends downwards preventing a linear fitting. This is the case of metals whose solubility (especially depending on pH values) limits the concentration in the donor compartment and the time required to reach concentrations above the limit of quantification in the acceptor compartment implies a non-negligible decrease of the concentration in the donor compartment.In this work, a simple linear regression is shown to provide the diffusion coefficient values from experiments exhibiting finite-volume effects. This expression is validated against rigorous numerical simulation as well as reported values in the literature. Diffusion coefficients of Zn, Ni and Pb in agarose cross-linked polyacrylamide (APA) gels (used in Diffusive Gradients in Thin-Film devices, DGT) are determined under finite-volume effects. The resulting values agree with those obtained under the standard linear regime.

Polymeric Vehicles for Nucleic Acid Delivery: Enhancing the Therapeutic Efficacy and Cellular Uptake.

Therapeutic gene delivery may be facilitated by the use of polymeric carriers. When combined with nucleic acids to form nanoparticles or polyplexes, a variety of polymers may shield the cargo from in vivo breakdown and clearance while also making it easier for it to enter intracellular compartments. Polymer synthesis design choices result in a wide variety of compounds and vehicle compositions. Depending on the application, these characteristics may be changed to provide enhanced endosomal escape, longer-lasting distribution, or stronger connection with nucleic acid cargo and cells. Here, we outline current methods for delivering genes in preclinical and clinical settings using polymers. Significant therapeutic outcomes have previously been attained using genetic material- delivering polymer vehicles in both in-vitro and animal models. When combined with nucleic acids to form nanoparticles or polyplexes, a variety of polymers may shield the cargo from in vivo breakdown and clearance while also making it easier for it to enter intracellular compartments. Many innovative diagnoses for nucleic acids have been investigated and put through clinical assessment in the past 20 years. Polymer-based carriers have additional delivery issues due to their changes in method and place of biological action, as well as variances in biophysical characteristics. We cover recent custom polymeric carrier architectures that were tuned for nucleic acid payloads such genomemodifying nucleic acids, siRNA, microRNA, and plasmid DNA. In conclusion, the development of polymeric carriers for gene delivery holds promise for therapeutic applications. Through careful design and optimization, these carriers can overcome various challenges associated with nucleic acid delivery, offering new avenues for treating a wide range of diseases.

Isolation, Purification, and Comprehensive Flow Cytometry Assessment of Lung Stromal Cells.

Stromal cells are non-hematopoietic cells that consist of endothelial cells and various mesenchymal cell populations. The composition of the stromal cell compartment is diverse in different organs. Numerous recent studies demonstrated that the lung environment contains heterogeneous mesenchymal stromal cell populations with distinctive genomic signatures and location preferences. Besides their role in supporting organ structure and remodeling tissue, mesenchymal stromal cells fulfill critical immune functions. These stromal cells show alterations during lung fibrosis and infectious disorders like COVID-19 or flu infection. To date, their identification and isolation were challenging, and most information about their heterogeneity was derived from scRNAseq data. In this protocol, we describe an isolation, comprehensive flow cytometry assessment, and purification strategy for murine lung stromal cells. The described method is optimized for minimizing cell death while keeping a high level of cell purity. This protocol can be also used for ex-vivo analysis of these cells in downstream functional assays. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of stromal cells from murine lung tissue Basic Protocol 2: Flow cytometry assessment of lung stromal populations Basic Protocol 3: Purification of lung fibroblastic stromal cells Alternate Protocol: Positive selection of fibroblastic stromal cells.

Molecular principles of the assembly and construction of a carboxysome shell.

Intracellular compartmentalization enhances biological reactions, crucial for cellular function and survival. An example is the carboxysome, a bacterial microcompartment for CO2 fixation. The carboxysome uses a polyhedral protein shell made of hexamers, pentamers, and trimers to encapsulate Rubisco, increasing CO2 levels near Rubisco to enhance carboxylation. Despite their role in the global carbon cycle, the molecular mechanisms behind carboxysome shell assembly remain unclear. Here, we present a structural characterization of α-carboxysome shells generated from recombinant systems, which contain all shell proteins and the scaffolding protein CsoS2. Atomic-resolution cryo-electron microscopy of the shell assemblies, with a maximal size of 54 nm, unveil diverse assembly interfaces between shell proteins, detailed interactions of CsoS2 with shell proteins to drive shell assembly, and the formation of heterohexamers and heteropentamers by different shell protein paralogs, facilitating the assembly of larger empty shells. Our findings provide mechanistic insights into the construction principles of α-carboxysome shells and the role of CsoS2 in governing α-carboxysome assembly and functionality.

Interaction of molecular mechanisms of plant-derived metabolites in Type 2 diabetes mellitus: A network pharmacology, docking and molecular dynamics approach on AKT1 kinase

BackgroundT2DM is a common metabolic disease with enormous effects on health worldwide; moreover, the use of phytochemicals as therapeutic compounds has drawn increasing attention. Therefore, the objective of this study was to assess the effectiveness of these phytochemicals in combating diabetes through a comprehensive evaluation of their interactions with biological networks through network pharmacology, molecular docking, and molecular dynamics simulations. ObjectivesThe first goal of this study was to search and screen potential phytochemicals for binding with key proteins involved in T2DM, with special emphasis on AKT1 kinase, an integral component of the insulin signaling pathway. MethodsNetwork pharmacology analysis was carried out, and the interaction network of targets associated with T2DM was generated using KEGG, STRING and Cytoscape 3.9.1 software's. To determine the specific metabolic processes, cellular compartments, and molecular functions involved in T2DM, we performed Gene Ontology and KEGG analyses. An initial and short molecular docking study was conducted to analyze the binding modes, while the molecular dynamics simulations provided insights into the binding energy and stability of phytochemicals at target sites, with emphasis on rutin engaged with AKT1. ResultsIn total, 10 hub genes were proposed to be involved in T2DM and can be considered candidate therapeutic targets, namely MTOR, CASP3, CCND1, TNF, MMP9, ALB, MDM2, AKT1, and HSP90AA1. Rutin was found to have the highest binding score for AKT1 in docking studies, while MD simulations identified the structural stability and persistence of the compound's activity at the target enzyme loci. ConclusionsThis study identified rutin and flavonoids as potential anti-diabetes phytochemicals. Based on these observations, an opportunity for other in vitro experiments and additional in vivo studies to confirm these buildings as multi-target drugs in T2DM patients is provided.

Identification of novel interacting partners of the TRIM29 E3-ubiquitin ligase in immortalized cells of normal prostate basal epithelium

BACKGROUND: Based on the most recent findings, the E3 ubiquitin ligase TRIM29 participates in numerous cellular processes and holds significance in both the formation of normal basal epithelium and prostate cancer. Exploring these molecular mechanisms could be pivotal in developing novel cancer therapies. AIM: Identification of protein partners of TRIM29 and its truncated forms, followed by functional analysis of these proteins. Identification of the key molecular processes involving TRIM29. METHODS: Cell cultures from normal basal epithelium of the prostate with overexpression of the chimeric protein TRIM29-FLAG or its truncated forms lacking the B-Box domain or the Coiled-Coil domain were conducted. Protein partners of TRIM29 and its truncated forms were identified through protein immunoprecipitation followed by proteomic (HPLC-MS/MS) analysis. The findings were validated through Western blot analysis and immunocytochemistry. RESULTS: TRIM29 interacts with 288 proteins in normal prostate basal epithelium. Deletion of the B-Box has minimal impact on TRIM29's protein interactions, while deletion of the Coiled-Coil domain significantly reduces its protein partners and disrupts its dimerization. Our research indicates that TRIM29 is present in both the nucleus and cytoplasm, and deletion of functional domains does not affect its localization but alters binding to compartment-specific proteins. TRIM29 binds to cytoskeletal proteins, cellular stress response proteins, and RNA-binding proteins. Furthermore, TRIM29 enhances cell resistance to genotoxic agents and influences RNA splicing. CONCLUSION: Proteomic analysis reveals that in normal prostate basal epithelium, the E3-ubiquitin ligase TRIM29 binds to a broad spectrum of proteins with distinct functions in various cell compartments. Our findings align with other studies demonstrating involvement of TRIM29 in cytoskeletal rearrangement, cellular response to viral infections, and DNA damage. Additionally, our research demonstrates the interaction of TRIM29 with RNA-binding proteins and its potential role in regulating RNA splicing, particularly through the Coiled-Coil domain.

The rapid degradation of translated upstream regions points to an inefficient translation initiation process.

Large-scale experimental analyses find ever more abundant evidence of translation from start codons upstream of the canonical start site. This translation either generates entirely new proteins (from novel upstream open reading frames) or produces isoforms with extended N-terminals when the novel start codon is in frame Most extended N-terminals are likely to just add a disordered region to the canonical protein isoform, but some may also block the recognition of the signal peptide causing the isoform to accumulate in the incorrect cellular compartment. This analysis finds evidence that upstream translations that would interfere with signal peptides are detected in expected quantities in ribosome profiling experiments, but that the equivalent N-terminally extended protein isoforms are significantly reduced in multiple proteomics experiments. This suggests that these isoforms are likely to be degraded shortly after translation by the ubiquitination pathway, thus preventing the build up of potentially harmful proteins with hydrophobic regions in the cytoplasm. In addition, this is further evidence that most of the transcripts translated from upstream start sites are the result of an inefficient translation initiation process. This has implications for the annotation of proteins given the huge numbers of upstream translations that are being detected in large-scale experiments.

Quantitative determination of the spatial distribution of components in single cells with CellDetail

The distribution of biomolecules within cells changes upon aging and diseases. To quantitatively determine the spatial distribution of components inside cells, we built the user-friendly open-source 3D-cell-image analysis platform Cell Detection and Analysis of Intensity Lounge (CellDetail). The algorithm within CellDetail is based on the concept of the dipole moment. CellDetail provides quantitative values for the distribution of the polarity proteins Cdc42 and Tubulin in young and aged hematopoietic stem cells (HSCs). Septin proteins form networks within cells that are critical for cell compartmentalization. We uncover a reduced level of organization of the Septin network within aged HSCs and within senescent human fibroblasts. Changes in the Septin network structure might therefore be a common feature of aging. The level of organization of the network of Septin proteins in aged HSCs can be restored to a youthful level by pharmacological attenuation of the activity of the small RhoGTPase Cdc42.