Cyto-IL-15 synergizes with the STING agonist ADU-S100 to eliminate prostate tumors and confer durable immunity in mouse models.

Introduction: Prostate cancer is one of the most commonly diagnosed malignancies in men with high mortality rates. Despite the recent therapeutic advances, such as immunotherapies, survival of patients with advance disease remains significantly low. Blockade of immune checkpoints has led to low response rates in these patients probably due to the immunosuppressive microenvironment and low mutation burden of prostate tumors. Combination of multiple immunotherapeutic regimes has also been unsatisfactory due to augmented adverse effects. To activate multiple immune-stimulatory pathways in the hostile prostate cancer microenvironment, we used a combination of cytotopically modified interleukin-15 (cyto-IL-15) with the stimulator of interferon genes (STING) agonist, ADU-S100. Methods: To determine whether this combination regime could lead to both local and systemic anti-tumor effects, intratumoral administration of these agents was used in murine models of prostate cancer. Tumor growth and mouse survival were monitored, and ex vivo analyses, and RNA sequencing were performed on the tumors. Results: Intratumorally injected ADU-S100 and cyto-IL-15 synergized to eliminate tumors in 58-67% of mice with unilateral tumors and promoted abscopal immunity in 50% of mice with bilateral tumors treated only at one side. Moreover, this combination regime offered immunoprotection against tumor rechallenge in 83% of cured mice. The efficacy of the combination treatment was associated with a strong innate and adaptive immune activation and induction of apoptotic and necrotic cell death. Cytokines, including type I and II interferons, and cytokine signalling pathways were activated, NK and T cell mediated cytotoxicity was increased, and B cells were activated both locally and systemically. While ADU-S100 led to an ulcerative pathology at the injection site, no other adverse effects were observed. Discussion: Localised administration of a STING agonist together with cyto-IL-15 can confer significant systemic benefits and long-lasting immunity against prostate tumors while reducing immune related toxicities.

Production and Purification of Phosphatidylinositol Mannosides from Mycobacterium smegmatis Biomass.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, is regarded as the most successful pathogen of humankind and a major threat to global health. The mycobacterial cell wall is vital for cell growth, virulence, and resistance to antibiotics, and thus constitutes a unique target for drug development. To characterize the enzymes catalyzing the synthesis of the cell wall components, considerable amounts of substrates are required. Since many mycobacterial cell wall lipids, particularly phosphatidylinositol mannosides (PIMs), are not commercially available, isolation from cell biomass is the most straightforward way to obtain these compounds. In this study, we optimized a protocol to extract and purify PIM species, in particular Ac1 PIM2 and Ac1 PIM4 , which can be further used for the identification and characterization of target enzymes. PIMs were extracted from Mycobacterium smegmatis mc2 155 ΔPimE using organic solvents, and purified through three consecutive chromatography steps. Thin-layer chromatography (TLC) was used in-between purification steps to evaluate the success of lipid separation, and nuclear magnetic resonance (NMR) was used for product quantification and to assess purity. Typically, from a 60 g batch of M. smegmatis biomass we were able to isolate approximately 9 mg of Ac1 PIM2 and 1.8 mg of Ac1 PIM4 . This is the first time the purification of phosphatidylinositol tetramannoside has been reported. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Growth of M. smegmatis mc2 155 ∆PimE Basic Protocol 2: Extraction of lipids from M. smegmatis mc2 155 ∆PimE Basic Protocol 3: Treatment of the lipid extract for isolation of phospholipids Basic Protocol 4: Isolation of phosphatidylinositol mannosides Basic Protocol 5: Quantification of phosphatidylinositol mannosides.

Cytotopic (Cyto-) IL-15 as a New Immunotherapy for Prostate Cancer: Recombinant Production in Escherichia coli and Purification.

Interleukin-15 (IL-15) is a cytokine previously suggested as a potential immunotherapy for cancer treatment. IL-15 can effectively reduce tumor growth in many preclinical tumor models including prostate cancer. This is due to its ability to expand and activate immune cells, such as CD8+ T cells and natural killer cells. To increase the potency of IL-15, we have engineered a protein variant that can be modified to localize and be retained in tissues where it is administered. However, the production of recombinant IL-15, the purity, and correct refolding of the final protein is not always ideal. In the current study, we aimed to optimize the methodology for production and purification of a modified recombinant human IL-15 and investigate the efficacy of the produced protein in the treatment of prostate tumors. Human IL-15 with its polypeptide sequence modified at the C-terminus to enable thiol conjugation with membrane localizing peptides, was produced in E. coli and purified using mild denaturing conditions (2M urea) from a washing step or from solubilization of inclusion bodies. The purified protein from the wash fraction was conjugated to a myristoylated peptide to form a membrane-localizing IL-15 (cyto-IL-15). The efficacy of cyto-IL-15 was investigated in subcutaneous TRAMP-C2 prostate tumors in mice and compared with cyto-IL-15 derived from protein purified from inclusion bodies (cyto-IL-15 Gen). When mild denaturing conditions were used for purification, the largest amount of IL-15 was collected from the wash fraction and a smaller amount from inclusion bodies. The protein from the wash fraction was mainly present as a monomer, whereas the one from inclusion bodies formed homodimers and higher complexes. After cytotopic modification, the purified IL-showed great efficacy in delaying prostate tumor growth (∼50%) and increased mice survival by ∼1.8-fold compared with vehicle. This study demonstrates an alternative, inexpensive and efficient method to produce and purify a modified version of IL-15 using mild denaturing conditions. This IL-15, when cytotopically modified, showed great efficacy as a monotherapy in prostate tumors in mice further highlighting the potential of IL-15 as a cancer immunotherapy.

Functional basis of electron transport within photosynthetic complex I.

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme - the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.

Combination of Interleukin-15 With a STING Agonist, ADU-S100 Analog: A Potential Immunotherapy for Prostate Cancer.

Prostate cancer is the second most commonly diagnosed cancer in men with mortality rates, overtaking those for breast cancer in the last 2 years in the UK. Despite advances in prostate cancer treatments, over 25% of men do not survive over 5 years with advanced disease. Due to the success of immunotherapies in treating other cancers, this treatment modality has been investigated for Prostate cancer, however, the sole FDA approved immunotherapy so far (Provenge™) only extends life by a few months. Therefore, finding immunotherapeutic agents to treat prostate cancer is of major interest. Our group has previously shown that Interleukin-15 (IL-15), unlike other therapeutic cytokines such as IL-2 and IL-12, can stimulate expansion and activity of CD8 T cells and NK cells in vitro when they are exposed to prostate cancer cells, while studies in mice have shown a 50% reduction in tumor size with no apparent toxicity. In this study, we aim to examine potencies of IL-15 in combination with a cyclic dinucleotide (CDN) that activates the Stimulator of Interferon-Gene (STING) receptor. Selected CDNs (also known as STING agonists) have previously been shown to activate both T cells and dendritic cells through STING. We hypothesize that the combination of STING agonists and IL-15 can additively increase NK and T cell activity as they act to increase type I interferons (IFNs) through STING activation and IFN-γ through IL-15. In prostate cancer-lymphocyte co-cultures we now show that combination of IL-15 and the STING agonist ADU-S100 analog induces a marked killing of cancer cells above that seen with IL-15 or ADU-S100 alone. We show that this is related to a potent activation of NK cells resulting in increased perforin and CD69 expression, and up to a 13-fold increase in IFNγ secretion in the co-cultures. NK cells are responsible for killing of the cancer cells, as shown by a lack of cytotoxicity in NK depleted lymphocyte-tumor cell co-cultures, or in co-cultures of B and T cells with tumor cells. In summary, we propose that the combination of IL-15 and the sting agonist ADU-S100 analog may be potently effective in treatment of prostate cancer.

Prostate cancer cells enhance interleukin-15-mediated expansion of NK cells.

OBJECTIVES: To identify cytokines that can activate and expand NK cells in the presence of prostate cancer cells in order to determine whether these agents may be useful in future intra-tumoural administration in pre-clinical and clinical prostate cancer trials. MATERIALS AND METHODS: Lymphocytes isolated from normal donor blood were set up in co-cultures with either cancer or non-cancerous prostate cell lines, together with each of the cytokines interleukin (IL)-2, IL-12, IL-15, interferon (IFN)-γ or IL-21 for a period of 7 days. Then, expansion of NK cells, NKT cells and CD8 T cells was measured by flow cytometry and compared with the expansion of the same cells in the absence of prostate cells. The cytotoxic activity of NK cells, as measured by perforin and tumour cell killing, was also assessed. NK cell receptors and their corresponding ligands on prostate tumour cells were analysed to determine whether any of these were modulated by co-culture. The role of the tumour-secreted heat shock proteins HSP90 and HSP70 in the expansion of NK cells in the co-cultures was also investigated because of their effects on NK and CD8 T-cell activation. RESULTS: We showed that, among a panel of cytokines known to cause NK cell activation and expansion, only IL-15 could actively induce expansion of NK, NKT and CD8 T cells in the presence of prostate cancer cell lines. Furthermore, the expansion of NK cells was far greater (up to 50% greater) in the presence of the cancer cells (LNCaP, PC3) than when lymphocytes were incubated alone. In contrast, non-cancerous cell lines (PNT2 and WPMY-1) did not exert any expansion of NK cells. The cytolytic activity of the NK cells, as measured by perforin, CD107a and killing of tumour cells, was also greatest in co-cultures with IL-15. Examination of NK cell receptors shows that NKG2D is upregulated to a greater degree in the presence of prostate cancer cells, compared with the upregulation with IL-15 in lymphocytes alone. However, blocking of NKG2D does not inhibit the enhanced expansion of NK cells in the presence of tumour cells. CONCLUSIONS: Among a panel of NK cell-activating cytokines, IL-15 was the only cytokine that could stimulate expansion of NK cells in the presence of prostate cancer cells; therefore IL-15 may be a good candidate for novel future intra-tumoural therapy of the disease.

Combined transcriptomics-metabolomics profiling of the heat shock response in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a remarkable archaeon able to grow at temperatures around 100 °C. To gain insight into how this model hyperthermophile copes with heat stress, we compared transcriptomic and metabolomic data of cells subjected to a temperature shift from 90 °C to 97 °C. In this study, we used RNA-sequencing to characterize the global variation in gene expression levels, while nuclear magnetic resonance (NMR) and targeted ion exchange liquid chromatography-mass spectrometry (LC-MS) were used to determine changes in metabolite levels. Of the 552 differentially expressed genes in response to heat shock conditions, 257 were upregulated and 295 were downregulated. In particular, there was a significant downregulation of genes for synthesis and transport of amino acids. At the metabolite level, 37 compounds were quantified. The level of di-myo-inositol phosphate, a canonical heat stress solute among marine hyperthermophiles, increased considerably (5.4-fold) at elevated temperature. Also, the levels of mannosylglycerate, UDP-N-acetylglucosamine (UDPGlcNac) and UDP-N-acetylgalactosamine were enhanced. The increase in the pool of UDPGlcNac was concurrent with an increase in the transcript levels of the respective biosynthetic genes. This work provides the first metabolomic analysis of the heat shock response of a hyperthermophile.

X-ray structure of a CDP-alcohol phosphatidyltransferase membrane enzyme and insights into its catalytic mechanism.

Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed.

Mannosylglycerate and di-myo-inositol phosphate have interchangeable roles during adaptation of Pyrococcus furiosus to heat stress.

Marine hyperthermophiles accumulate small organic compounds, known as compatible solutes, in response to supraoptimal temperatures or salinities. Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at temperatures near 100°C. This organism accumulates mannosylglycerate (MG) and di-myo-inositol phosphate (DIP) in response to osmotic and heat stress, respectively. It has been assumed that MG and DIP are involved in cell protection; however, firm evidence for the roles of these solutes in stress adaptation is still missing, largely due to the lack of genetic tools to produce suitable mutants of hyperthermophiles. Recently, such tools were developed for P. furiosus, making this organism an ideal target for that purpose. In this work, genes coding for the synthases in the biosynthetic pathways of MG and DIP were deleted by double-crossover homologous recombination. The growth profiles and solute patterns of the two mutants and the parent strain were investigated under optimal growth conditions and also at supraoptimal temperatures and NaCl concentrations. DIP was a suitable replacement for MG during heat stress, but substitution of MG for DIP and aspartate led to less efficient growth under conditions of osmotic stress. The results suggest that the cascade of molecular events leading to MG synthesis is tuned for osmotic adjustment, while the machinery for induction of DIP synthesis responds to either stress agent. MG protects cells against heat as effectively as DIP, despite the finding that the amount of DIP consistently increases in response to heat stress in the nine (hyper)thermophiles examined thus far.

Three-dimensional structure of mannosyl-3-phosphoglycerate phosphatase from Thermus thermophilus HB27: a new member of the haloalcanoic acid dehalogenase superfamily.

Mannosyl-3-phosphoglycerate phosphatase (MpgP) is a key mediator in the physiological response to thermal and osmotic stresses, catalyzing the hydrolysis of mannosyl-3-phosphoglycerate (MPG) to the final product, α-mannosylglycerate. MpgP is a metal-dependent haloalcanoic acid dehalogenase-like (HAD-like) phosphatase, preserving the catalytic motifs I-IV of the HAD core domain, and classified as a Cof-type MPGP (HAD-IIB-MPGP family; SCOP [117505]) on the basis of its C2B cap insertion module. Herein, the crystallographic structures of Thermus thermophilus HB27 MpgP in its apo form and in complex with substrates, substrate analogues, and inhibitors are reported. Two distinct enzyme conformations, open and closed, are catalytically relevant. Apo-MpgP is primarily found in the open state, while holo-MpgP, in complex with the reaction products, is found in the closed state. Enzyme activation entails a structural rearrangement of motifs I and IV with concomitant binding of the cocatalytic Mg(2+) ion. The closure motion of the C2B domain is subsequently triggered by the anchoring of the phosphoryl group to the cocatalytic metal center, and by Arg167 fixing the mannosyl moiety inside the catalytic pocket. The results led to the proposal that in T. thermophilus HB27 MpgP the phosphoryl transfer employs a concerted D(N)S(N) mechanism with assistance of proton transfer from the general acid Asp8, forming a short-lived PO(3)(-) intermediate that is attacked by a nucleophilic water molecule. These results provide new insights into a possible continuum of phosphoryl transfer mechanisms, ranging between those purely associative and dissociative, as well as a picture of the main mechanistic aspects of phosphoryl monoester transfer catalysis, common to other members of the HAD superfamily.

Crystallization and preliminary X-ray analysis of mannosyl-3-phosphoglycerate phosphatase from Thermus thermophilus HB27.

Mannosylglycerate (MG) is primarily known as an osmolyte and is widely distributed among (hyper)thermophilic marine microorganisms. The synthesis of MG via mannosyl-3-phosphoglycerate synthase (MpgS) and mannosyl-3-phosphoglycerate phosphatase (MpgP), the so-called two-step pathway, is the most prevalent route among these organisms. The phosphorylated intermediate mannosyl-3-phosphoglycerate is synthesized by the first enzyme and is subsequently dephosphorylated by the second. The structure of MpgS from the thermophilic bacterium Thermus thermophilus HB27 has recently been solved and characterized. Here, the cloning, expression, purification, crystallization and preliminary crystallographic analysis of MpgP from T. thermophilus HB27 are reported. Size-exclusion chromatography assays suggested a dimeric assembly in solution for MpgP at pH 6.3 and together with differential scanning fluorimetry data showed that high ionic strength and charge compensation were required to produce a highly pure and soluble protein sample for crystallographic studies. The crystals obtained belonged to the monoclinic space group P2(1), with unit-cell parameters a=39.52, b=70.68, c=95.42 Å, ß=92.95°. Diffraction data were measured to 1.9 Šresolution. Matthews coefficient calculations suggested the presence of two MpgP monomers in the asymmetric unit and the calculation of a self-rotation Patterson map indicated that the two monomers could be related by a noncrystallographic twofold rotation axis, forming a dimer.

Structural analysis of Thermus thermophilus HB27 mannosyl-3-phosphoglycerate synthase provides evidence for a second catalytic metal ion and new insight into the retaining mechanism of glycosyltransferases.

Mannosyl-3-phosphoglycerate synthase is a glycosyltransferase involved in the two-step synthetic pathway of mannosylglycerate, a compatible solute that accumulates in response to salt and/or heat stresses in many microorganisms thriving in hot environments. The three-dimensional structure of mannosyl-3-phosphoglycerate synthase from Thermus thermophilus HB27 in its binary complex form, with GDP-alpha-D-mannose and Mg(2+), shows a second metal binding site, about 6 A away from the mannose moiety. Kinetic and mutagenesis studies have shown that this metal site plays a role in catalysis. Additionally, Asp(167) in the DXD motif is found within van der Waals contact distance of the C1' atom in the mannopyranose ring, suggesting its action as a catalytic nucleophile, either in the formation of a glycosyl-enzyme intermediate according to the double-displacement S(N)2 reaction mechanism or in the stabilization of the oxocarbenium ion-like intermediate according to the D(N)*A(Nss) (S(N)i-like) reaction mechanism. We propose that either mechanism may occur in retaining glycosyltransferases with a GT-A fold, and, based on the gathered structural information, we identified an extended structural signature toward a common scaffold between the inverting and retaining glycosyltransferases.