Inositol Polyphosphate 5-phosphatase Research Articles

Base editing correction of OCRL in Lowe syndrome: ABE-mediated functional rescue in patient-derived fibroblasts.

Lowe syndrome, a rare X-linked multisystem disorder presenting with major abnormalities in the eyes, kidneys, and central nervous system, is caused by mutations in OCRL gene (NG_008638.1). Encoding an inositol polyphosphate 5-phosphatase, OCRL catalyzes the hydrolysis of PI(4,5)P2 into PI4P. There are no effective targeted treatments for Lowe syndrome. Here, we demonstrate a novel gene therapy for Lowe syndrome in patient fibroblasts using an adenine base editor (ABE) that can efficiently correct pathogenic point mutations. We show that ABE8e-NG-based correction of a disease-causing mutation in a Lowe patient-derived fibroblast line containing R844X mutation in OCRL gene, restores OCRL expression at mRNA and protein levels. It also restores cellular abnormalities that are hallmarks of OCRL dysfunction, including defects in ciliogenesis, microtubule anchoring, α-actinin distribution, and F-actin network. The study indicates that ABE-mediated gene therapy is a feasible treatment for Lowe syndrome, laying the foundation for therapeutic application of ABE in the currently incurable disease.

Plausible role of INPP4A dysregulation in idiopathic pulmonary fibrosis.

INPP4A has been shown to be involved in the regulation of cell proliferation and apoptosis of multiple cell types including fibroblasts. Previous reports from our group have demonstrated the role of inositol polyphosphate 4-phosphatase Type I A (INPP4A) in these functions. Though existing evidences suggest a critical role for INPP4A in the maintenance of lung homeostasis, its role in chronic lung diseases is relatively under explored. In the current study, we made an attempt to understand the regulation of INPP4A in idiopathic pulmonary fibrosis (IPF). Through integration of relevant INPP4A gene expression data from public repositories with our results from invitro experiments and mouse models, we show that INPP4A is altered in IPF. Interestingly, the direction of the change is dependent both on the disease stage and the region of the lung used. INPP4A was found to be upregulated when analyzed in lung sample representative of the whole lung, but was downregulated in the fibrotic regions of the lung. Similarly, INPP4A was found to be high, compared to controls, only in the early stage of the disease. Though the observed increase in INPP4A was found to be negatively correlated to physiological indices, FVC, and DLCO, of lung function, treatment with anti-INPP4A antibody worsened the condition in bleomycin treated mice. These contrasting results taken together are suggestive of a nuanced regulation of INPP4A in IPF which is dependent on the disease stage, cellular state and extent of fibrosis in the lung region being analyzed.

Knockdown of INPP5K compromises the differentiation of N2A cells.

Inositol polyphosphate 5-phosphatase K (INPP5K), also known as SKIP (skeletal muscle and kidney-enriched inositol phosphatase), is a cytoplasmic enzyme with 5-phosphatase activity toward phosphoinositides (PIs). Mutations in INPP5K are associated with autosomal recessive congenital muscular dystrophy with cataracts and intellectual disability (MDCCAID). Notably, muscular dystrophy is characterized by the hypoglycosylation of dystroglycan. Thus, far, the underlying mechanisms are only partially understood. In this study, we show that INPP5K expression increases during brain development. Knockdown of INPP5K in the neuroblastoma-derived cell line N2A impaired their neuronal-like differentiation and interfered with protein glycosylation.

INPP4B ensures that ILC1s and NK cells set up a productive home office.

In this issue of JEM, Peng et al. (https://doi.org/10.1084/jem.20230124) identify inositol polyphosphate 4-phosphatase type II (encoded by Inpp4b) as an important enzyme for tissue-resident ILC1 and NK cell survival, signal transduction, and anti-tumor immunity.

Identifying Qingkailing ingredients-dependent mesenchymal-epithelial transition factor-axiation "π" structuring module with angiogenesis and neurogenesis effects.

To explore the functional role of the drug-dependent mesenchymal-epithelial transition (Met)-axiation "π" structural module of neurogenesis after processing by three components of Qingkailing injection in neurogenesis and angiogenesis in cerebral ischemia. We used a Glutathione S-transferase (GST)-pull down assay, isothermal titration calorimetry assay, and other related methods to identify the relationships among Met, inositol polyphosphate phosphatase like 1 (Inppl1), and death associated protein kinase 3 (Dapk3) in this allosteric module. The biological effects of the modules of neurons generation composed of Met, Inppl1, and Dapk3 were measured through Western blot, apoptosis analysis, and double immunofluorescence labeling. The GST-pull down assay revealed that proline-serine-threonine rich domain of Met binds to the Src homology domain of Inppl1 to form a protein-protein complex; Dapk3 with a C-terminal domain interacts weakly with the protein kinase C domain of Met in the intracellular region. Thus, we obtained a "π" structuring module considered a neural regeneration module. The biological effects of angiogenesis and neurogenesis modules composed of Met, Inppl1, and Dapk3 were also verified. The study suggested that understanding the functional modules that contribute to pharmaceutics might provide novel signatures that can be used as endpoints to define disease processes under stroke or cerebral ischemia conditions.

Role of INPP4B in the proliferation, migration, invasion, and survival of human endometrial cancer cells.

Inositol polyphosphate 4-phosphatase type II (INPP4B) has been identified as a tumor repressor in several human cancers while its role in endometrial cancer has not been investigated yet. Therefore, the current study was designed to determine whether INPP4B participates in the progression of endometrial cancer by utilizing clinical data and experimental determination. We first include six chemotherapy-treated patients with recurrent and metastatic endometrioid carcinoma to determine the relationship between INPP4B mutation and relative tumor burden. By using siRNA-mediated gene silencing and vector-mediated gene overexpression, we further determined the effect of manipulating INPP4B expression on the proliferation, invasion, and survival of endometrial cancer cells. Furthermore, the repressing effect of INPP4B together with its role in chemotherapy was further validated by xenograft tumor-bearing mice models. Western blot analysis was used to explore further downstream signaling modulated by INPP4B expression manipulation. Two of the patients were found to have INPP4B mutations and the mutation frequency of INPP4B increased during the progression of chemotherapy resistance. Endometrial cancer cells with silenced INPP4B expression were found to have promoted tumor cell proliferation, invasion, and survival. Endometrial cancer cells overexpressing INPP4B were found to have decreased tumor cell proliferation, invasion, and survival. An in vivo study using six xenograft tumor-bearing mice in each group revealed that INPP4B overexpression could suppress tumor progression and enhance chemosensitivity. Furthermore, INPP4B overexpression was found to modulate the activation of Wnt3a signaling. The current study suggested that INPP4B could be a suppressor in endometrial cancer progression and might be a target for endometrial cancer treatment. Also, INPP4B might serve as a predictor of chemosensitivity determination.

MARCKS and PI(4,5)P2 reciprocally regulate actin-based dendritic spine morphology.

Myristoylated, alanine-rich C-kinase substrate (MARCKS) is an F-actin and phospholipid binding protein implicated in numerous cellular activities, including the regulation of morphology in neuronal dendrites and dendritic spines. MARCKS contains a lysine-rich effector domain that mediates its binding to plasma membrane phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) in a manner controlled by PKC and calcium/calmodulin. In neurons, manipulations of MARCKS concentration and membrane targeting strongly affect the numbers, shapes, and F-actin properties of dendritic spines, but the mechanisms remain unclear. Here, we tested the hypothesis that the effects of MARCKS on dendritic spine morphology are due to its capacity to regulate the availability of plasma membrane PI(4,5)P2. We observed that the concentration of free PI(4,5)P2 on the dendritic plasma membrane was inversely proportional to the concentration of MARCKS. Endogenous PI(4,5)P2 levels were increased or decreased, respectively, by acutely overexpressing either phosphatidylinositol-4-phosphate 5-kinase (PIP5K) or inositol polyphosphate 5-phosphatase (5ptase). PIP5K, like MARCKS depletion, induced severe spine shrinkage; 5ptase, like constitutively membrane-bound MARCKS, induced aberrant spine elongation. These phenotypes involved changes in actin properties driven by the F-actin severing protein cofilin. Collectively, these findings support a model in which neuronal activity regulates actin-dependent spine morphology through antagonistic interactions of MARCKS and PI(4,5)P2.

Structure-Activity Studies on Bis-Sulfonamide SHIP1 Activators.

The SH2-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) enzyme opposes the activity of PI3K and therefore is of interest in the treatment of inflammatory disorders. Recent results also indicate that SHIP1 promotes phagolysosomal degradation of lipids by microglia, suggesting that the enzyme may be a target for the treatment of Alzheimer's disease. Therefore, small molecules that increase SHIP1 activity may have benefits in these areas. Recently we discovered a bis-sulfonamide that increases the enzymatic activity of SHIP1. A series of similar SHIP1 activators have been synthesized and evaluated to determine structure-activity relationships and improve in vivo stability. Some new analogs have now been found with improved potency. In addition, both the thiophene and the thiomorpholine in the parent structure can be replaced by groups without a low valent sulfur atom, which provides a way to access activators that are less prone to oxidative degradation.

SAT053 Development Of A Novel Peptide-based Insulin Sensitizer Through Disruption Of Inositol Polyphosphate 5-Phosphatase K (SKIP) - p21-activated Kinase 1 (Pak1) Interactions

Abstract Disclosure: A.M. Kimbrough: None. J. Dev Gaddameedi: None. A.M. Lipchik: None. Type 2 diabetes (T2D) is a prominent, chronic metabolic disease that affects approximately 462 million people worldwide. The driving force behind T2D is insulin resistance (IR), defined as an impaired biological response to insulin, and leads to a multitude of more severe conditions including heart disease, stroke, and neuropathy. Most therapies fail to improve insulin sensitivity, and instead drive physiological compensation. It is imperative to develop new therapies to correct the underlying pathology by targeting mechanisms of IR. Inositol polyphosphate 5-phosphatase K (INPP5K/SKIP) is a potential target to treat IR. SKIP resides in the endoplasmic reticulum (ER) in resting cells, and translocates to the plasma membrane (PM) following growth factor stimulation. SKIP is recruited to the PM through binding to p21 activated kinase 1 (Pak1) and inhibits growth factor signaling through dephosphorylation of phosphoinositol-(3,4,5)-triphosphate. In obesity and IR, SKIP is overexpressed leading to PM localization and suppression of insulin signaling. Here, we developed a novel insulin sensitizer utilizing a peptide derived from Pak1 to inhibit SKIP-Pak1 interactions and prevent PM localization. We found that the Pak1 derived peptide displayed alpha helical properties and thermostabilized SKIP. In controlled in vitro assays, the Pak1 peptide was successfully taken up in target tissues. Additionally, treatment with the Pak1 peptides was sufficient to restore insulin sensitivity in skeletal muscle, adipose, and liver models of IR. Together these results demonstrate the use of a peptide-based insulin sensitizer as a novel therapeutic for T2D that requires further investigation. Presentation: Saturday, June 17, 2023

SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late-onset Alzheimer's disease.

The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. 3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-contaning inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays.A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health.SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic.The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.

Multiple Inositol Polyphosphate Phosphatase Compartmentalization Separates Inositol Phosphate Metabolism from Inositol Lipid Signaling.

Multiple inositol polyphosphate phosphatase (MINPP1) is an enigmatic enzyme that is responsible for the metabolism of inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6 pentakisphosphate (Ins(1,3,4,5,6)P5 in mammalian cells, despite being restricted to the confines of the ER. The reason for this compartmentalization is unclear. In our previous studies in the insulin-secreting HIT cell line, we expressed MINPP1 in the cytosol to artificially reduce the concentration of these higher inositol phosphates. Undocumented at the time, we noted cytosolic MINPP1 expression reduced cell growth. We were struck by the similarities in substrate preference between a number of different enzymes that are able to metabolize both inositol phosphates and lipids, notably IPMK and PTEN. MINPP1 was first characterized as a phosphatase that could remove the 3-phosphate from inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). This molecule shares strong structural homology with the major product of the growth-promoting Phosphatidyl 3-kinase (PI3K), phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and PTEN can degrade both this lipid and Ins(1,3,4,5)P4. Because of this similar substrate preference, we postulated that the cytosolic version of MINPP1 (cyt-MINPP1) may not only attack inositol polyphosphates but also PtdIns(3,4,5)P3, a key signal in mitogenesis. Our experiments show that expression of cyt-MINPP1 in HIT cells lowers the concentration of PtdIns(3,4,5)P3. We conclude this reflects a direct effect of MINPP1 upon the lipid because cyt-MINPP1 actively dephosphorylates synthetic, di(C4:0)PtdIns(3,4,5)P3 in vitro. These data illustrate the importance of MINPP1's confinement to the ER whereby important aspects of inositol phosphate metabolism and inositol lipid signaling can be separately regulated and give one important clarification for MINPP1's ER seclusion.

Duplicated zebrafish (Danio rerio) inositol phosphatases inpp5ka and inpp5kb diverged in expression pattern and function

One hurdle in the development of zebrafish models of human disease is the presence of multiple zebrafish orthologs resulting from whole genome duplication in teleosts. Mutations in inositol polyphosphate 5-phosphatase K (INPP5K) lead to a syndrome characterized by variable presentation of intellectual disability, brain abnormalities, cataracts, muscle disease, and short stature. INPP5K is a phosphatase acting at position 5 of phosphoinositides to control their homeostasis and is involved in insulin signaling, cytoskeletal regulation, and protein trafficking. Previously, our group and others have replicated the human phenotypes in zebrafish knockdown models by targeting both INPP5K orthologs inpp5ka and inpp5kb. Here, we show that inpp5ka is the more closely related orthologue to human INPP5K. While both inpp5ka and inpp5kb mRNA expression levels follow a similar trend in the developing head, eyes, and tail, inpp5ka is much more abundantly expressed in these tissues than inpp5kb. In situ hybridization revealed a similar trend, also showing unique localization of inpp5kb in the pineal gland and retina indicating different transcriptional regulation. We also found that inpp5kb has lost its catalytic activity against its preferred substrate, PtdIns(4,5)P2. Since most human mutations are missense changes disrupting phosphatase activity, we propose that loss of inpp5ka alone can be targeted to recapitulate the human presentation. In addition, we show that the function of inpp5kb has diverged from inpp5ka and may play a novel role in the zebrafish.

Tumor Suppressor Role of INPP4B in Chemoresistant Retinoblastoma.

The chemotherapy of retinoblastoma (RB), a malignant ocular childhood disease, is often limited by the development of resistance against commonly used drugs. We identified inositol polyphosphate 4-phosphatase type II (INPP4B) as a differentially regulated gene in etoposide-resistant RB cell lines, potentially involved in the development of RB resistances. INPP4B is controversially discussed as a tumor suppressor and an oncogenic driver in various cancers, but its role in retinoblastoma in general and chemoresistant RB in particular is yet unknown. In the study presented, we investigated the expression of INPP4B in RB cell lines and patients and analyzed the effect of INPP4B overexpression on etoposide resistant RB cell growth in vitro and in vivo. INPP4B mRNA levels were significantly downregulated in RB cells lines compared to the healthy human retina, with even lower expression levels in etoposide-resistant compared to the sensitive cell lines. Besides, a significant increase in INPP4B expression was observed in chemotherapy-treated RB tumor patient samples compared to untreated tumors. INPP4B overexpression in etoposide-resistant RB cells resulted in a significant reduction in cell viability with reduced growth, proliferation, anchorage-independent growth, and in ovo tumor formation. Caspase-3/7-mediated apoptosis was concomitantly increased, suggesting a tumor suppressive role of INPP4B in chemoresistant RB cells. No changes in AKT signaling were discernible, but p-SGK3 levels increased following INPP4B overexpression, indicating a potential regulation of SGK3 signaling in etoposide-resistant RB cells. RNAseq analysis of INPP4B overexpressing, etoposide-resistant RB cell lines revealed differentially regulated genes involved in cancer progression, mirroring observed in vitro and in vivo effects of INPP4B overexpression and strengthening INPP4B's importance for cell growth control and tumorigenicity.

MiR-155 regulates M2 polarization of hepatitis B virus-infected tumour-associated macrophages which in turn regulates the malignant progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) initiated by hepatitis B virus (HBV) infection is a complicated process. MiR-155 can alter the immune microenvironment to affect the host's anti-infective ability. This study investigated the mechanism by which miR-155 affects tumour-associated macrophage (TAM) polarization at a molecular level, thus affecting the malignant progression of HBV+ HCC. MiR-155 and TAM-related cytokine expression were analysed by qRT-PCR. The distribution of TAMs was detected by immunohistochemistry. The effect of the aberrant miR-155 expression on macrophage polarization was examined by flow cytometry. The targeted relationship was verified by dual-luciferase assay, and the protein level of src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) was detected by western blot. The proliferation of HCC cells was examined by CCK-8 and colony formation assays. Invasion and migration of HCC cells were detected by transwell assay. In HBV+ HCC tissues, miR-155 was significantly highly expressed and the number of CD206-positive TAM (CD206+ TAM) and CD68-positive TAM (CD68+ TAM) were higher than those in HBV- HCC tissues. In addition, miR-155 overexpression significantly promoted M2-type macrophage polarization, whilst miR-155 silencing expression significantly promoted M1-type macrophage polarization. Besides, the miR-155/SHIP1 axis accelerated HCC cell invasion, proliferation and migration by inducing M2-type macrophage polarization. MiR-155 accelerates HCC cell proliferation, migration and invasion by targeting SHIP1 expression and inducing macrophage M2 polarization. This finding provides new insights into the development of novel therapeutic strategies for combatting HBV+ HCC and a new reference for exploring anti-tumour immunotherapy.

Transcriptome Sequencing of the Diatom Asterionellopsis thurstonii and In Silico Identification of Enzymes Potentially Involved in the Synthesis of Bioactive Molecules

Microalgae produce a plethora of primary and secondary metabolites with possible applications in several market sectors, including cosmetics, human nutrition, aquaculture, biodiesel production and treatment/prevention of human diseases. Diatoms, in particular, are the most diversified microalgal group, many species of which are known to have anti-cancer, anti-oxidant, anti-diabetes, anti-inflammatory and immunomodulatory properties. Compounds responsible for these activities are often still unknown. The aim of this study was to de novo sequence the full transcriptome of two strains of the diatom Asterionellopsis thurstonii, sampled from two different locations and cultured in both control and phosphate starvation conditions. We used an RNA-sequencing approach to in silico identify transcripts potentially involved in the synthesis/degradation of compounds with anti-cancer and immunomodulatory properties. We identified transcript coding for L-asparaginase I, polyketide cyclase/dehydrase, bifunctional polyketide phosphatase/kinase, 1-deoxy-D-xylulose-5-phosphate synthase (fragment), inositol polyphosphate 5-phosphatase INPP5B/F, catechol O-Methyltransferase, digalactosyldiacylglycerol synthase (DGD1), 1,2-diacylglycerol-3-beta-galactosyltransferase and glycerolphosphodiester phosphodiesterase. Differential expression analysis also allowed to identify in which culturing condition these enzymes are more expressed. Overall, these data give new insights on the annotation of diatom genes, enzymatic pathways involved in the generation of bioactive molecules and possible exploitation of Asterionellopsis thurstonii.

Loss of INPP5K attenuates IP3-induced Ca2+ responses in the glioblastoma cell line U-251 MG cells

INPP5K (inositol polyphosphate 5-phosphatase K) is an endoplasmic reticulum (ER)-resident enzyme that acts as a phosphoinositide (PI) 5-phosphatase, capable of dephosphorylating various PIs including PI 4,5-bisphosphate (PI(4,5)P2), a key phosphoinositide found in the plasma membrane. Given its ER localization and substrate specificity, INPP5K may play a role in ER-plasma membrane contact sites. Furthermore, PI(4,5)P2 serves as a substrate for phospholipase C, an enzyme activated downstream of extracellular agonists acting on Gq-coupled receptors or tyrosine-kinase receptors, leading to IP3 production and subsequent release of Ca2+ from the ER, the primary intracellular Ca2+ storage organelle. In this study, we investigated the impact of INPP5K on ER Ca2+ dynamics using a previously established INPP5K-knockdown U-251 MG glioblastoma cell model. We here describe that loss of INPP5K impairs agonist-induced, IP3 receptor (IP3R)-mediated Ca2+ mobilization in intact cells, while the ER Ca2+ content and store-operated Ca2+ influx remain unaffected. To further elucidate the underlying mechanisms, we examined Ca2+ release in permeabilized cells stimulated with exogenous IP3. Interestingly, the absence of INPP5K also disrupted IP3-induced Ca2+ release events. These results suggest that INPP5K may directly influence IP3R activity through mechanisms yet to be resolved. The findings from this study point towards role of INPP5K in modulating ER calcium dynamics, particularly in relation to IP3-mediated signaling pathways. However, further work is needed to establish the general nature of our findings and to unravel the exact molecular mechanisms underlying the interplay between INNP5K function and Ca2+ signaling.

Proteomics of Plasma from Patients with Sickle Cell Disease with and without Chronic Pain: A Pilot Biomarker Discovery Study

Background Acute, severe, episodic nociceptive pain, or vaso-occlusive crises (VOCs) have been the hallmark of sickle cell disease (SCD), and responsible for >90% of health care encounters for this population. The concept that SCD patients experience a period of "normalcy" without any pain between these episodes of varying frequency has been challenged by several studies in the past two decades. It has been shown that ~30% of adults with SCD have chronic pain, defined as pain >50% of time for at least 6 months (Smith et al, 2008). The evolution of chronic pain is age dependent, and likely involves several different mechanisms. Studies in transgenic mouse models have shown that one mechanism involves neurogenic inflammation, triggered by mast cell activation (Vincent et al, 2013), through mediators such as tryptase and substance P. In a previous study (Albo et al, 2021) it was shown that patients with SCD and chronic pain had higher plasma levels of Tryptase and substance P, consistent with mast cell activation and neurogenic inflammation. Additionally, nerve growth factor (NGS), which has been incriminated in the pathophysiology of several chronic pain syndromes, was also found to be significantly elevated in the plasma of SCD patients with chronic pain. In this study, we conducted a pilot proteomics study of plasma samples from patients with and without chronic pain collected during the Albo et al study, and stored at -80 C. Methods IRB approval was obtained, and informed consent was obtained from 72 patients enrolled in the study, whose plasma were stored. Discovery proteomics was conducted on 20 plasma samples from two groups (10 with chronic pain, 10 without chronic pain) through semiquantitative LC/MS/MS proteomics on the Thermo Fisher Orbitrap Mass Spectrometer. Samples were subjected to affinity depletion of major proteins to discern out lower concentration proteins. Filters were then utilized to remove excess proteins using two rules; a protein that only showed up in one patient and proteins that were not present in at least 5 patients. Differential expression was then used to yield proteins significantly different between the chronic pain and non-chronic pain groups Results Table 1 shows the demographics, hydroxyurea therapy, opioid use, and pain scores of the 20 subjects included in the pilot study. Mass spectrometry of 20 samples revealed 5219 individual proteins. The filters cut down the protein count to 417, and differential expression yielded 16 proteins that were significantly different between the two groups. These included alpha-1 antitrypsin, Hepatocyte growth factor receptor, creatine kinase M-type, serine/threonine protein kinase SMG1, F-box only protein, Alstrom syndrome protein, beta transforming growth factor, serine/threonine protein mTOR, apolipoprotein C-1, keratin gylceraldehyde 3 phosphate dehydrogenase, Nesprin 1, retinol binding protein 3, Protein Wnt 16, multiple inositol polyphosphate phosphatase, immunoglobulin J-chain. Figure 1 demonstrates a heat map of these proteins down regulated and upregulated expression. Conclusion This pilot study has yielded 16 differentially expressed proteins in the plasma of SCD patients with and without chronic pain. Validation of these findings in a larger cohort are under way. It is hoped that if confirmed, these findings will lead to the identification of biomarkers for chronic pain in SCD. In addition to the potential utility of these biomarkers in differential diagnosis of pain syndromes in SCD, targets for therapeutic intervention could also be identified. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

INPP4B inhibits glioma cell proliferation and immune escape via inhibition of the PI3K/AKT signaling pathway.

INPP4B (Inositol polyphosphate 4-phosphatase type II) has been regarded as a suppressor of several human tumors, but its biological function, expression, and clinical significance in glioma tissues and cell lines are unclear. Notably, whether INPP4B participates in immune escape of glioma deserves urgent attention. Here, we confirmed that INPP4B expression is often downregulated in low- and high-grade human glioma tissues, in tissues from an orthotopic mouse model of brain glioma and in glioma cells. We found that INPP4B overexpression restrained the proliferation, migration, apoptosis resistance, PD-L1 expression, and T cell suppression by glioma cells, whereas INPP4B silencing had the opposite effects. Moreover, we showed that INPP4B inhibited glioma cell proliferation, migration, and PD-L1 expression by downregulating PI3K/AKT signaling. Collectively, these data support that INPP4B may inhibit glioma progression, and particularly, glioma’s immune escape. Thus, INPP4B may constitute a valuable target for glioma treatment.

Computational Evaluation of Designed Phosphatase from Conserved Sequence Scratch for Diverse Substrate Specificity

The ability to design efficient enzymes for a broad class of different reactions would be of tremendous practical interest in both science and industry. Computer-assisted designing is a novel approach to generating industrial enzymes for biotechnological applications. Objectives: The main aim of this study was to design an enzyme construct with diverse substrate-binding specificity based on the evolutionary conservation of archaeal vanadium-dependent phosphatases. Materials and methods: A rational 3D structural model of enzyme construct was developed from conserved sequence scratch encompassing a vanadium-binding site and functional domain. Substrate-binding specificity of the designed enzyme was computed with different myo-inositol polyphosphate analogous by a molecular docking program. Results: A designed enzyme has shown more substrate-binding specificity with 1D-myo-inositol 3, 4, 5, 6-tetrakisphosphate. Its catalytic function closely resembled myo-inositol polyphosphate-5-phosphatase and multiple inositol polyphosphate phosphatases. Moreover, the enzyme construct was energetically stable with a low degree of conformational changes upon substrate-binding. Conclusion: Substrate specificity and catalytic competence of designed enzymes were computationally evaluated for further biotechnological applications.

Characterisation of a soil MINPP phytase with remarkable long-term stability and activity from Acinetobacter sp.

Phylogenetic analysis, homology modelling and biochemical methods have been employed to characterize a phytase from a Gram-negative soil bacterium. Acinetobacter sp. AC1-2 phytase belongs to clade 2 of the histidine (acid) phytases, to the Multiple Inositol Polyphosphate Phosphatase (MINPP) subclass. The enzyme was extraordinarily stable in solution both at room temperature and 4°C, retaining near 100% activity over 755 days. It showed a broad pH activity profile from 2-8.5 with maxima at 3, 4.5-5 and 6. The enzyme showed Michaelis-Menten kinetics and substrate inhibition (Vmax, Km, and Ki, 228 U/mg, 0.65 mM and 2.23 mM, respectively). Homology modelling using the crystal structure of a homologous MINPP from a human gut commensal bacterium indicated the presence of a potentially stabilising polypeptide loop (a U-loop) straddling the active site. By employ of the enantiospecificity of Arabidopsis inositol tris/tetrakisphosphate kinase 1 for inositol pentakisphosphates, we show AC1-2 MINPP to possess D6-phytase activity, which allowed modelling of active site specificity pockets for InsP6 substrate. While phytase gene transcription was unaltered in rich media, it was repressed in minimal media with phytic acid and orthophosphate as phosphate sources. The results of this study reveal AC1-2 MINPP to possess desirable attributes relevant to biotechnological use.