Single Cell Spatial Analysis Reveals the Topology of Immunomodulatory Purinergic Signaling in Glioblastoma

Glioblastoma develops an immunosuppressive microenvironment that fosters tumorigenesis and resistance to current therapeutic strategies. Here we use multiplexed tissue imaging and single-cell RNA-sequencing to characterize the composition, spatial organization, and clinical significance of extracellular purinergic signaling in glioblastoma. We show that glioblastoma exhibit strong expression of CD39 and CD73 ectoenzymes, correlating with increased adenosine levels. Microglia are the predominant source of CD39, while CD73 is principally expressed by tumor cells, particularly in tumors with amplification of EGFR and astrocyte-like differentiation. Spatially-resolved single-cell analyses demonstrate strong spatial correlation between tumor CD73 and microglial CD39, and that their spatial proximity is associated with poor clinical outcomes. Together, this data reveals that tumor CD73 expression correlates with tumor genotype, lineage differentiation, and functional states, and that core purine regulatory enzymes expressed by neoplastic and tumor-associated myeloid cells interact to promote a distinctive adenosine-rich signaling niche and immunosuppressive microenvironment potentially amenable to therapeutic targeting.

Synthetic Biology Approaches to Hydrocarbon Biosensors: A Review

Monooxygenases are a class of enzymes that facilitate the bacterial degradation of alkanes and alkenes. The regulatory components associated with monooxygenases are nature’s own hydrocarbon sensors, and once functionally characterised, these components can be used to create rapid, inexpensive and sensitive biosensors for use in applications such as bioremediation and metabolic engineering. Many bacterial monooxygenases have been identified, yet the regulation of only a few of these have been investigated in detail. A wealth of genetic and functional diversity of regulatory enzymes and promoter elements still remains unexplored and unexploited, both in published genome sequences and in yet-to-be-cultured bacteria. In this review we examine in detail the current state of research on monooxygenase gene regulation, and on the development of transcription-factor-based microbial biosensors for detection of alkanes and alkenes. A new framework for the systematic characterisation of the underlying genetic components and for further development of biosensors is presented, and we identify focus areas that should be targeted to enable progression of more biosensor candidates to commercialisation and deployment in industry and in the environment.

Physiological Ovarian Aging Is Associated with Altered Expression of Post-Translational Modifications in Mice

Post-translational modifications (PTMs) have been confirmed to be involved in multiple female reproductive events, but their role in physiological ovarian aging is far from elucidated. In this study, mice aged 3, 12 or 17 months (3M, 12M, 17M) were selected as physiological ovarian aging models. The expression of female reproductive function-related genes, the global profiles of PTMs, and the level of histone modifications and related regulatory enzymes were examined during physiological ovarian aging in the mice by quantitative real-time PCR and western blot, respectively. The results showed that the global protein expression of Kbhb (lysineβ-hydroxybutyryllysine), Khib (lysine 2-hydroxyisobutyryllysine), Kglu (lysineglutaryllysine), Kmal (lysinemalonyllysine), Ksucc (lysinesuccinyllysine), Kcr (lysinecrotonyllysine), Kbu (lysinebutyryllysine), Kpr (lysinepropionyllysine), SUMO1 (SUMO1 modification), ub (ubiquitination), P-Typ (phosphorylation), and 3-nitro-Tyr (nitro-tyrosine) increased significantly as mice aged. Moreover, the modification level of Kme2 (lysinedi-methyllysine) and Kac (lysineacetyllysine) was the highest in the 3M mice and the lowest in 12M mice. In addition, only trimethylation of histone lysine was up-regulated progressively and significantly with increasing age (p < 0.001), H4 ubiquitination was obviously higher in the 12M and 17M mice than 3M (p < 0.001), whereas the modification of Kpr (lysinepropionylation) and O-GlcNA in 17M was significantly decreased compared with the level in 3M mice (p < 0.05, p < 0.01). Furthermore, the expression levels of the TIP60, P300, PRDM9, KMT5B, and KMT5C genes encoding PTM regulators were up-regulated in 17M compared to 3M female mice (p < 0.05). These findings indicate that altered related regulatory enzymes and PTMs are associated with physiological ovarian aging in mice, which is expected to provide useful insights for the delay of ovarian aging and the diagnosis and treatment of female infertility.

Therapeutic Potential of Dietary Polyphenols

The chapter summarizes available research on polyphenols and the potential for polyphenol based therapeutics. Polyphenols have the potential to be used in a multi-target fashion therapeutically. The majority of the polyphenol benefits appear to share positive effects across multiple disease states including inflammatory diseases, diseases of metabolic dysregulation and cancer. The reviewed literature includes human, animal and cell culture based studies. Selected mechanisms within each disease state are highlighted including interleukin inflammatory markers, NF-κB, acetyl-CoA concentration regulation of metabolism, and p-glycoprotein multidrug efflux pump associated with cancer treatment failures. Reviewed studies discuss polyphenols inhibiting transcription factors that control expression on inflammatory factors as well as activating other transcription factors that increase expression of enzymes protective of oxidative damage. Levels of metabolic regulatory enzymes are also affected positively by polyphenol addition through epigenetic modifications. Epigenetic modifications affecting cancer development and progression appear positively affected by polyphenol treatment. Additionally, oxidative damage protection of normal cells can be achieved by polyphenol treatment thus limiting chemotherapeutic damage. Upon review of the available literature, a strong case for the potential use of polyphenols in therapeutic situations stands out. Potential risks included are that the purity and specific concentrations required to achieve therapeutic benefits without potential side effects need to be examined prior to the adoption of therapeutics.

Lysine Fatty Acylation: Regulatory Enzymes, Research Tools, and Biological Function

Post-translational acylation of lysine side chains is a common mechanism of protein regulation. Modification by long-chain fatty acyl groups is an understudied form of lysine acylation that has gained increasing attention recently due to the characterization of enzymes that catalyze the addition and removal this modification. In this review we summarize what has been learned about lysine fatty acylation in the approximately 30 years since its initial discovery. We report on what is known about the enzymes that regulate lysine fatty acylation and their physiological functions, including tumorigenesis and bacterial pathogenesis. We also cover the effect of lysine fatty acylation on reported substrates. Generally, lysine fatty acylation increases the affinity of proteins for specific cellular membranes, but the physiological outcome depends greatly on the molecular context. Finally, we will go over the experimental tools that have been used to study lysine fatty acylation. While much has been learned about lysine fatty acylation since its initial discovery, the full scope of its biological function has yet to be realized.

Comparative study on the influence of silicon and selenium to mitigate arsenic induced stress by modulating TCA cycle, GABA and polyamine synthesis in rice seedlings

Arsenic contamination of groundwater is a major concern for its use as drinking water and crop irrigation in many regions of the world. Arsenic is absorbed by rice plants from arsenic contaminated water during irrigation, hampers growth and agricultural productivity. The aim of the study was to mitigate the activity of TCA cycle, synthesis of γ-aminobutyric acid (GABA) and polyamines (PAs) in rice (Oryza sativa L. cv. MTU-1010) seedlings under arsenate (As-V) stress [25 µM, 50 µM and 75 µM] by silicon (Si) [2 mM] and selenium (Se) [5 µM] amendments, and to investigate which chemical was more potential to combat this threat. As(V) application decreased the activities of tested respiratory enzymes while the levels of organic acids (OAs) were increased in the test seedlings. Co-application of Si and As(V) increased the activities of respiratory enzymes, consequently further increased accumulation of OAs that were more than Se with As(V) application in the test seedlings. GABA accumulation along with the activities of its regulatory enzymes were enhanced under As(V) stress. During joint application of Si and As(V) and Se and As(V) said parameters were decreased showing defensive role of these chemicals to resist As(V) toxicity in rice but amendment of Si was more potential than Se amendment resulted reduction of stress induced damage in the test seedlings. PAs trigger tolerance mechanism against stress in plants. PAs viz., Putrescine, spermidine and spermine were synthesized more during Si and Se amendments in As(V) contaminated rice seedlings to combat the effect of stress. Si amendment substantially modulated the toxic effects caused by As(V) over Se amendment in As(V) challenged test seedlings. Thus in future application Si enriched fertilizer will be beneficial than application of Se enriched fertilizer to grow rice plants with normal vigor in arsenic contaminated soil.

Kinase-anchoring proteins in ciliary signal transduction

Historically, the diffusion of chemical signals through the cell was thought to occur within a cytoplasmic soup bounded by the plasma membrane. This theory was predicated on the notion that all regulatory enzymes are soluble and moved with a Brownian motion. Although enzyme compartmentalization was initially rebuffed by biochemists as a ‘last refuge of a scoundrel', signal relay through macromolecular complexes is now accepted as a fundamental tenet of the burgeoning field of spatial biology. A-Kinase anchoring proteins (AKAPs) are prototypic enzyme-organizing elements that position clusters of regulatory proteins at defined subcellular locations. In parallel, the primary cilium has gained recognition as a subcellular mechanosensory organelle that amplifies second messenger signals pertaining to metazoan development. This article highlights advances in our understanding of AKAP signaling within the primary cilium and how defective ciliary function contributes to an increasing number of diseases known as ciliopathies.

Intestinal-epithelial LSD1 controls goblet cell maturation and effector responses required for gut immunity to bacterial and helminth infection

Infectious and inflammatory diseases in the intestine remain a serious threat for patients world-wide. Reprogramming of the intestinal epithelium towards a protective effector state is important to manage inflammation and immunity and can be therapeutically targeted. The role of epigenetic regulatory enzymes within these processes is not yet defined. Here, we use a mouse model that has an intestinal-epithelial specific deletion of the histone demethylase Lsd1 (cKO mice), which maintains the epithelium in a fixed reparative state. Challenge of cKO mice with bacteria-induced colitis or a helminth infection model both resulted in increased pathogenesis. Mechanistically, we discovered that LSD1 is important for goblet cell maturation and goblet-cell effector molecules such as RELMß. We propose that this may be in part mediated by directly controlling genes that facilitate cytoskeletal organization, which is important in goblet cell biology. This study therefore identifies intestinal-epithelial epigenetic regulation by LSD1 as a critical element in host protection from infection.

Class I Histone Deacetylases (HDAC1–3) are Histone Lysine Delactylases

Lysine l-lactylation [K(l-la)] is a newly discovered histone mark that can be stimulated under conditions of high glycolysis, such as the Warburg effect. K(l-la) is associated with functions that are different from the widely studied histone acetylation. While K(l-la) can be introduced by the acetyltransferase p300, histone delactylase enzymes remain unknown. Here, we report the systematic evaluation of zinc- and NAD+-dependent HDACs for their ability to cleave ε-N-l-lactyllysine marks. Our screens identified HDACs 1–3 and SIRT1–3 as delactylases in vitro. HDACs 1–3 show robust activity toward not only K(l-la) but also K(d-la) and diverse short-chain acyl modifications. We further confirmed the de-l-lactylase activity of HDACs 1 and 3 in cells. Identification of p300 and HDAC3 as regulatory enzymes suggests that histone lactylation is installed and removed by enzymes as opposed to spontaneous chemical reactivity. Our results therefore represent an important step toward full characterization of this pathway’s regulatory elements.

Kinetochores respond to subtle changes in the stability of microtubule attachments

Proper attachment of spindle microtubules to kinetochores is necessary to satisfy the spindle assembly checkpoint and ensure faithful chromosome segregation. Microtubules detach from kinetochores to correct improperly oriented attachments, and overall kinetochore-microtubule (k-MT) attachment stability is determined in response to regulatory enzymes and the activities of kinetochore-associated microtubule stabilizing and destabilizing proteins. However, it is unknown whether regulatory enzyme activity or kinetochore-associated protein localization respond to subtle changes in k-MT attachment stability. To test for this feedback response, we monitored Aurora B kinase activity and the localization of select kinetochore proteins in metaphase cells following treatments that subtly stabilize or destabilize k-MT attachments using low dose Taxol or UMK57 (an MCAK agonist), respectively. Increasing k-MT stability induced changes in the abundance of some kinetochore proteins. In contrast, reducing k-MT stability induced both increases in Aurora B kinase signaling and changes in the abundance of some kinetochore proteins. Thus, kinetochores dynamically respond to changes in the stability of their attached microtubules. This feedback control contributes to tuning k-MT attachment stability required for efficient error correction to facilitate faithful chromosome segregation.Summary StatementLive cell imaging demonstrates that kinetochore signaling responds to feedback from attached microtubules to tune their stability to ensure faithful chromosome segregation during cell division.