From sensing interactions to controlling the interactions: a novel approach to obtain biological transistors for specific and label-free immunosensing.

Antibody-antigen interactions are shaped by the solution pH level, ionic strength, and electric fields, if present. In biological field-effect transistors (BioFETs), the interactions take place at the sensing area in which the pH level, ionic strength and electric fields are determined by the Poisson-Boltzmann equation and the boundary conditions at the solid-solution interface and the potential applied at the solution electrode. The present study demonstrates how a BioFET solution electrode potential affects the sensing area double layer pH level, ionic strength, and electric fields and in this way shapes the biological interactions at the sensing area. We refer to this as 'active sensing'. To this end, we employed the meta-nano-channel (MNC) BioFET and demonstrate how the solution electrode can determine the antibody-antigen equilibrium constant and allows the control and tuning of the sensing performance in terms of the dynamic range and limit-of-detection. In the current work, we employed this method to demonstrate the specific and label-free sensing of Alpha-Fetoprotein (AFP) molecules from 0.5 µL drops of 1 : 100 diluted serum. AFP was measured during pregnancy as part of the prenatal screening program for fetal anomalies, chromosomal abnormalities, and abnormal placentation. We demonstrate AFP sensing with a limit-of-detection of 10.5 aM and a dynamic range of 6 orders of magnitude in concentration. Extensive control measurements are reported.

The proteasome regulator PSME4 modulates proteasome activity and antigen diversity to abrogate antitumor immunity in NSCLC.

Immunotherapy revolutionized treatment options in cancer, yet the mechanisms underlying resistance in many patients remain poorly understood. Cellular proteasomes have been implicated in modulating antitumor immunity by regulating antigen processing, antigen presentation, inflammatory signaling and immune cell activation. However, whether and how proteasome complex heterogeneity may affect tumor progression and the response to immunotherapy has not been systematically examined. Here, we show that proteasome complex composition varies substantially across cancers and impacts tumor-immune interactions and the tumor microenvironment. Through profiling of the degradation landscape of patient-derived non-small-cell lung carcinoma samples, we find that the proteasome regulator PSME4 is upregulated in tumors, alters proteasome activity, attenuates presented antigenic diversity and associates with lack of response to immunotherapy. Collectively, our approach affords a paradigm by which proteasome composition heterogeneity and function should be examined across cancer types and targeted in the context of precision oncology.

Implantation and Evaluation of Melanoma in the Murine Choroid via Optical Coherence Tomography.

Establishing experimental choroidal melanoma models is challenging in terms of the ability to induce tumors at the correct localization. In addition, difficulties in observing posterior choroidal melanoma in vivo limit tumor location and growth evaluation in real-time. The approach described here optimizes techniques for establishing choroidal melanoma in mice via a multi-step sub-choroidal B16LS9 cell injection procedure. To enable precision in injecting into the small dimensions of the mouse uvea, the complete procedure is performed under a microscope. First, a conjunctival peritomy is formed in the dorsal-temporal area of the eye. Then, a tract into the sub-choroidal space is created by inserting a needle through the exposed sclera. This is followed by the insertion of a blunt needle into the tract and the injection of melanoma cells into the choroid. Immediately after injection, noninvasive optical coherence tomography (OCT) imaging is utilized to determine tumor location and progress. Retinal detachment is evaluated as a predictor of tumor site and size. The presented method enables the reproducible induction of choroid-localized melanoma in mice and the live imaging of tumor growth evaluation. As such, it provides a valuable tool for studying intraocular tumors.

Oxidative stress facilitates exogenous mitochondria internalization and survival in retinal ganglion precursor-like cells.

Ocular cells are highly dependent on mitochondrial function due to their high demand of energy supply and their constant exposure to oxidative stress. Indeed, mitochondrial dysfunction is highly implicated in various acute, chronic, and genetic disorders of the visual system. It has recently been shown that mitochondrial transplantation (MitoPlant) temporarily protects retinal ganglion cells (RGCs) from cell death during ocular ischemia. Here, we characterized MitoPlant dynamics in retinal ganglion precursor-like cells, in steady state and under oxidative stress. We developed a new method for detection of transplanted mitochondria using qPCR, based on a difference in the mtDNA sequence of C57BL/6 and BALB/c mouse strains. Using this approach, we show internalization of exogenous mitochondria already three hours after transplantation, and a decline in mitochondrial content after twenty four hours. Interestingly, exposure of target cells to moderate oxidative stress prior to MitoPlant dramatically enhanced mitochondrial uptake and extended the survival of mitochondria in recipient cells by more than three fold. Understanding the factors that regulate the exogenous mitochondrial uptake and their survival may promote the application of MitoPlant for treatment of chronic and genetic mitochondrial diseases.

Maintaining Golgi Homeostasis: A Balancing Act of Two Proteolytic Pathways.

The Golgi apparatus is a central hub for cellular protein trafficking and signaling. Golgi structure and function is tightly coupled and undergoes dynamic changes in health and disease. A crucial requirement for maintaining Golgi homeostasis is the ability of the Golgi to target aberrant, misfolded, or otherwise unwanted proteins to degradation. Recent studies have revealed that the Golgi apparatus may degrade such proteins through autophagy, retrograde trafficking to the ER for ER-associated degradation (ERAD), and locally, through Golgi apparatus-related degradation (GARD). Here, we review recent discoveries in these mechanisms, highlighting the role of the Golgi in maintaining cellular homeostasis.

Histone degradation by the proteasome regulates chromatin and cellular plasticity.

Histones constitute the primary protein building blocks of the chromatin and play key roles in the dynamic control of chromatin compaction and epigenetic regulation. Histones are regulated by intricate mechanisms that alter their functionality and stability, thereby expanding the regulation of chromatin-transacting processes. As such, histone degradation is tightly regulated to provide spatiotemporal control of cellular histone abundance. While several mechanisms have been implicated in controlling histone stability, here, we discuss proteasome-dependent degradation of histones and the protein modifications that are associated with it. We then highlight specific cellular and physiological states that are associated with altered histone degradation by cellular proteasomes.

SUMOylation of linker histone H1 drives chromatin condensation and restriction of embryonic cell fate identity.

The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.

Gatekeepers of the Gut: The Roles of Proteasomes at the Gastrointestinal Barrier.

The gut epithelial barrier provides the first line of defense protecting the internal milieu from the environment. To circumvent the exposure to constant challenges such as pathogenic infections and commensal bacteria, epithelial and immune cells at the gut barrier require rapid and efficient means to dynamically sense and respond to stimuli. Numerous studies have highlighted the importance of proteolysis in maintaining homeostasis and adapting to the dynamic changes of the conditions in the gut environment. Primarily, proteolytic activities that are involved in immune regulation and inflammation have been examined in the context of the lysosome and inflammasome activation. Yet, the key to cellular and tissue proteostasis is the ubiquitin-proteasome system, which tightly regulates fundamental aspects of inflammatory signaling and protein quality control to provide rapid responses and protect from the accumulation of proteotoxic damage. In this review, we discuss proteasome-dependent regulation of the gut and highlight the pathophysiological consequences of the disarray of proteasomal control in the gut, in the context of aberrant inflammatory disorders and tumorigenesis.

Altered Protein Abundance and Localization Inferred from Sites of Alternative Modification by Ubiquitin and SUMO.

Protein modification by ubiquitin or SUMO can alter the function, stability or activity of target proteins. Previous studies have identified thousands of substrates that were modified by ubiquitin or SUMO on the same lysine residue. However, it remains unclear whether such overlap could result from a mere higher solvent accessibility, whether proteins containing those sites are associated with specific functional traits, and whether selectively perturbing their modification by ubiquitin or SUMO could result in different phenotypic outcomes. Here, we mapped reported lysine modification sites across the human proteome and found an enrichment of sites reported to be modified by both ubiquitin and SUMO. Our analysis uncovered thousands of proteins containing such sites, which we term Sites of Alternative Modification (SAMs). Among more than 36,000 sites reported to be modified by SUMO, 51.8% have also been reported to be modified by ubiquitin. SAM-containing proteins are associated with diverse biological functions including cell cycle, DNA damage, and transcriptional regulation. As such, our analysis highlights numerous proteins and pathways as putative targets for further elucidating the crosstalk between ubiquitin and SUMO. Comparing the biological and biochemical properties of SAMs versus other non-overlapping modification sites revealed that these sites were associated with altered cellular localization or abundance of their host proteins. Lastly, using S. cerevisiae as model, we show that mutating the SAM motif in a protein can influence its ubiquitination as well as its localization and abundance.

Metabolic alterations in the tumor microenvironment and their role in oncogenesis.

Metabolic reprogramming is a characteristic feature of both cancer cells and their neighbouring cells in the tumor microenvironment (TME). The latter include stroma fibroblasts and adipocytes, that respectively differentiate to become cancer associated fibroblasts (CAFs) and cancer associated adipocytes (CAAs), and infiltrated immune cells, that collaborate with the stromal cells to provide the tumor a pro-tumorigenic niche. Here we discuss the association between the reprogramming of glucose metabolism in the TME and oncogenic signaling and its reflection in the non-canonical functions of metabolic enzymes. We also discuss the non-canonical actions of oncometabolites and the contribution to oncogenesis of external metabolites that accumulate in the TME as result of crosstalk between the tumor and the TME. Special emphasis is given in this regard to lysophosphatidic acid (LPA) and adenosine, two powerful metabolites, the concentrations of which rise in the TME due to altered metabolism of the tumor and its surrounding cells, allowing their action as external signals.

Golgi organization is regulated by proteasomal degradation.

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies.

Phenotypic Screen Identifies JAK2 as a Major Regulator of FAT10 Expression.

FAT10 is a ubiquitin-like protein suggested to target proteins for proteasomal degradation. It is highly upregulated upon pro-inflammatory cytokines, namely, TNFα, IFNγ, and IL6, and was found to be highly expressed in various epithelial cancers. Evidence suggests that FAT10 is involved in cancer development and may have a pro-tumorigenic role. However, its biological role is still unclear, as well as its biochemical and cellular regulation. To identify pathways underlying FAT10 expression in the context of pro-inflammatory stimulation, which characterizes the cancerous environment, we implemented a phenotypic transcriptional reporter screen with a library of annotated compounds. We identified AZ960, a potent JAK2 inhibitor, which significantly downregulates FAT10 under pro-inflammatory cytokines induction, in an NFκB-independent manner. We validated JAK2 as a major regulator of FAT10 expression via knockdown, and we suggest that the transcriptional effects are mediated through pSTAT1/3/5. Overall, we have elucidated a pathway regulating FAT10 transcription and discovered a tool compound to chemically downregulate FAT10 expression, and to further study its biology.

Revealing the cellular degradome by mass spectrometry analysis of proteasome-cleaved peptides.

Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows for capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells revealed dynamic modulation of the cellular degradome in response to various stimuli, such as proinflammatory signals. Further, we performed analysis of minute amounts of clinical samples by studying cells from the peripheral blood of patients with systemic lupus erythematosus (SLE). We found increased degradation of histones in patient immune cells, thereby suggesting a role of aberrant proteasomal degradation in the pathophysiology of SLE. Thus, MAPP offers a broadly applicable method to facilitate the study of the cellular-degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer.

Post-Translational Modification Profiling-Functional Proteomics for the Analysis of Immune Regulation.

Posttranslational modifications (PTMs) of proteins are an integral part of major cellular regulatory mechanisms dictating protein function, localization, and stability. The capacity to screen PTMs using protein microarrays has advanced our ability to identify their targets and regulatory role. This chapter discusses a unique procedure that combines functional extract-based activity assay with large-scale screening utilities of protein microarrays. This "PTM-profiling" system offers advantages in quantitatively identifying modifications in an unbiased manner in the context of specific cellular conditions. While the possibilities of studying PTMs in different settings are enormous, the immune system presents an attractive model for studying the effects of perturbations in PTMs, and specifically the ubiquitin system, as these were already implicated in both immune function and dysfunction. This chapter discusses the significance of PTM profiling in addressing basic questions in immunology. We describe detailed protocols for the preparation of functional cell extracts from immune cell cultures, following differentiation or induced signals, and screening PTMs on protein arrays, as well as basic guidelines for data analysis and interpretation.

Post-translational modification profiling - A novel tool for mapping the protein modification landscape in cancer.

The ubiquitin system plays an important role in essentially every cellular process, regulating numerous pathways ranging from development, transcription, DNA damage response, cell cycle, and signal transduction. Its best studied role involves removal of faulty proteins or those that are not necessary anymore. Aberrations in the ubiquitin system have been implicated in various pathologies including cancer, where specific mutations in E3 ligases such as Mdm2, pVHL, and BRCA1 have been linked to disease progression, prognosis, and resistance to drugs. Yet, there are hundreds of E3 ligases in the human genome and our knowledge of their target proteins and their dynamic regulation in the cellular environment is largely limited. In addition, fundamental questions related to recognition and specificity in ubiquitin conjugation remain unanswered. It is thus of major importance to characterize the ubiquitin landscape under various cellular conditions, and study how the regulatory network is altered in health and disease. To do so, analytical tools that allow identification of ubiquitin substrates, the conjugation and removal of ubiquitin, and the nature of specific ubiquitin linkages that are formed are needed. In this mini-review, we discuss common proteomic methodologies applied to studying the ubiquitome, and specifically focus on our recently developed post-translational modification (PTM) profiling approach. PTM profiling is a functional assay, amenable to biochemical manipulation, which allows the detection of protein modifications in a high-throughput manner. We discuss in detail the advantages and limitations of this system, focusing primarily on examples for analyzing the ubiquitin system in cancer. Uncovering the intricate signaling dynamics governed by and regulating ubiquitin modifications should clearly evolve into a new paradigm in understanding the molecular basis of malignant transformation and the development of novel therapeutic modalities.

PKD at the crossroads of necrosis and autophagy.

Reactive oxygen species (ROS) that accumulate under oxidative pressure cause severe damage to cellular components, and induce various cellular responses, including apoptosis, programmed necrosis and autophagy, depending on the cellular setting. Various studies have described ROS-induced autophagy, but only a few direct factors that regulate autophagy under oxidative stress are known to date. We have identified DAPK and PKD as such regulators by demonstrating their role in the process of autophagy in general, and specifically during oxidative stress. PKD acts as a downstream effector of DAPk in the regulation of autophagy. Furthermore, PKD functions within the autophagic network as an activator of VPS34, by associating with and phosphorylating VPS34, leading to its activation. Significantly, PKD is recruited to the autophagosomal membranes, placing it within proximity of its autophagic target.

The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase.

The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65ß of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65ß and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation.

The paradox of autophagy and its implication in cancer etiology and therapy.

Autophagy is a cellular self-catabolic process in which cytoplasmic constituents are sequestered in double membrane vesicles that fuse with lysosomes where they are degraded. As this catabolic activity generates energy, autophagy is often induced under nutrient limiting conditions providing a mechanism to maintain cell viability and may be exploited by cancer cells for survival under metabolic stress. However, progressive autophagy can be cytotoxic and autophagy can under certain settings substitute for apoptosis in induction of cell death. Moreover, loss of autophagy is correlated with tumorigenesis and several inducers of autophagy are tumor-suppressor genes. Thus, the relation of autophagy to cancer development is complex and depends on the genetic composition of the cell as well as on the extra-cellular stresses a cell is exposed to. In this review we describe the intricate nature of autophagy and its regulators, particularly those that have been linked to cancer. We discuss the multifaceted relation of autophagy to tumorigenesis and highlight studies supporting a role for autophagy in both tumor-suppression and tumor-progression. Finally, various autophagy-targeting therapeutic strategies for cancer treatment are presented.