Abstract
Inositol polyphosphates (InsPs) play key signaling roles in diverse cellular functions, including calcium homeostasis, cell survival and death. Multiple inositol polyphosphate phosphatase 1 (Minpp1) affects the cellular levels of InsPs and cell functions. The Minpp1 is an endoplasmic reticulum (ER) resident but localizes away from its cytosolic InsPs substrates. The current study examines the heterogeneity of Minpp1 and the potential physiologic impact of Minpp1 isoforms, distinct motifs, subcellular distribution, and enzymatic potential. The NCBI database was used to analyze the proteome diversity of Minpp1 using bioinformatics tools. The analysis revealed that translation of three different Minpp1 variants resulted in three isoforms of Minpp1 of varying molecular weights. A link between the minpp1 variant-2 gene and ER-stress, using real-time PCR, suggests a functional similarity between minpp1 variant-1 and variant-2. A detailed study on motifs revealed Minpp1 isoform-2 is the only other isoform, besides isoform-1, that carries a phosphatase motif for InsPs hydrolysis but no ER-retention signal. The confocal microscopy revealed that the Minpp1 isoform-1 predominantly localized near the nucleus with a GRP-78 ER marker, while Minpp1 isoform-2 was scattered more towards the cell periphery where it co-localizes with the plasma membrane-destined multivesicular bodies biomarker CD63. MCF-7 cells were used to establish that Minpp1 isoform-2 is secreted into exosomes. Brefeldin A treatment resulted in overexpression of the exosome-associated Minpp1 isoform-2, suggesting its secretion via an unconventional route involving endocytic-generated vesicles and a link to ER stress. Results further demonstrated that the exosome-associated Minpp1 isoform-2 was enzymatically active. Overall, the data support the possibility that an extracellular form of enzymatically active Minpp1 isoform-2 mitigates any anti-proliferative actions of extracellular InsPs, thereby also impacting the makeup of the tumor microenvironment.
Highlights
Inositol polyphosphates (InsPs) are naturally occurring compounds widely distributed from unicellular to multicellular organisms
This study examined the heterogeneity in Multiple inositol polyphosphate phosphatase 1 (Minpp1) and demonstrated an extra-endoplasmic reticulum (ER) Minpp1 isoform-2 secreted in exosomes
Computational studies previously published from this lab predicted four different isoforms of Minpp1 [22] in humans
Summary
Inositol polyphosphates (InsPs) are naturally occurring compounds widely distributed from unicellular to multicellular organisms. Higher organisms are well recognized for their critical signaling role in maintaining cellular calcium homeostasis [1]. Among the InsPs, Inositol (1,2,3,4,5,6) hexakisphosphate (InsP6), known as phytic acid, is the most abundant in nature, regulates critical cell biological processes in both plants and animals [2–6]. InsP6, a substrate, produces highly energetic pyrophosphates (PP-InsPs: InsP7 and InsP8), implicated in various cellular processes such as apoptosis [7], vesicular trafficking [8, 9], ribosome biogenesis [10], aging [11] and energy metabolism [12]. InsP6 and its derivatives have been reported as anti-proliferative agents that counteract tumorigenic processes reverting a cell to normalcy [13, 14]. The homeostasis of InsPs is maintained by a complex metabolic network of InsPs regulated by InsPs specific kinases and phosphatases [15, 16]
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