The glioma pathogenesis-related 1 (GLIPR1) family consists of three genes [GLIPR1, GLIPR1-like 1 (GLIPR1L1), and GLIPR1-like 2 (GLIPR1L2)] and forms a distinct subgroup within the cysteine-rich secretory protein (CRISP), antigen 5, and pathogenesis-related 1 (CAP) superfamily. CAP superfamily proteins are found in phyla ranging from plants to humans and, based largely on expression and limited functional studies, are hypothesized to have roles in carcinogenesis, immunity, cell adhesion, and male fertility. Specifically data from a number of systems suggests that sequences within the C-terminal CAP domain of CAP proteins have the ability to promote cell-cell adhesion. Herein we cloned mouse Glipr1l1 and have shown it has a testis-enriched expression profile. GLIPR1L1 is posttranslationally modified by N-linked glycosylation during spermatogenesis and ultimately becomes localized to the connecting piece of elongated spermatids and sperm. After sperm capacitation, however, GLIPR1L1 is also localized to the anterior regions of the sperm head. Zona pellucida binding assays indicate that GLIPR1L1 has a role in the binding of sperm to the zona pellucida surrounding the oocyte. These data suggest that, along with other members of the CAP superfamily and several other proteins, GLIPR1L1 is involved in the binding of sperm to the oocyte complex. Collectively these data further strengthen the role of CAP domain-containing proteins in cellular adhesion and propose a mechanism whereby CAP proteins show overlapping functional significance during fertilization.
Acute myeloid leukaemia (AML) is an aggressive blood cancer, fatal if not responsive to treatment. The standard induction treatment for AML, for the past three decades, involves a (7+3) chemotherapy regimen where a continuous infusion of cytosine arabinoside (ara-c) is given for 7 days in combination with an anthracycline, usually daunorubicin for 3 days [1 ]. Primary treatment failure occurs in 20-40% of adult cases, with higher rates of chemoresistance observed in elderly patients [2-4]. The activation of the phosphoinositide 3-kinase (PI3K) pathway is a feature of many cancers including acute myeloid leukaemia (AML). In fact, constitutive activation of the P13K target AKT is seen in the majority (50%-80%) of AML cases and associated with poor prognosis [5]. The basis for activated AKT in AML remains poorly understood. Unlike in solid tumours [6, 7], activating mutations of AKT or PIK3CA have not been reported in AML [8-10]. The P13-K/Akt pathway is regulated by a triad of lipid phosphatases, known functionally as inositol polyphosphatase (INPP) enzymes, such as PTEN (3-phosphatase), SHIP1 (5-phosphatases) and INPP48 (4-phosphatases). Studies have found mutations or loss of PTEN to be absent in AML whereas mutations in SH/P-1 to account for only extremely rare cases (11-13]. There are a number of other INPP enzymes whose expression and function have not been explored in AML. We, therefore, sought to identify the presence and consequences of pathological expression of INPPs in AML. We utilized the Sequenom® MassARRAY platform to quantitatively screen the gene expression profile of key human INPP enzymes in primary AML and normal bone marrow (BM) samples. Contrary to our expectation, we identified significantly increased expression of the 4-phosphatase INPP48 in AML compared to normal BM (p=0.02). Overexpression of INPP4B in AML bone marrow (2::50% blasts positive) was confirmed in 12% of AML cases at diagnosis (n=205). INPP48 overexpression was associated with an inferior response to chemotherapy leading to a significantly shorter leukaemia-free survival (LFS) (median 6.2 vs 11.8 months; p=0.01)and overall survival (OS) (median 11.5 vs 26.6 months; p<0.01) outcomes. Multivariate analysis revealed that high INPP4B was an independent predictor of poor OS outcome for patients with AML at diagnosis (hazard ratio [HR] 2.2), along with adverse risk karyotypes (HR 1.7) and increased age (HR 2.1 ). Ectopic overexpression of INPP48 in human leukaemic cell lines conferred resistance to standard cytotoxic drugs used to treat AML, including ara-C and anthracyclines using in vitro cell death and colony-forming assays. INPP4B overexpression also led to impaired clearance of bone marrow blasts by cytarabine in vivo, as well as significantly reduced OS in human xenograft models of AML (p<0.0001 ). Although INPP48 phosphatase function was proven to be catalytically active in primary AML samples, expression of a phosphatase inactive mutant (INPP4B C842A) did not abrogate chemoresistance in vitro or in vivo. In contrast, siRNA-mediated knockdown of endogenously overexpressed INPP4B sensitized AML cell lines and primary cells to araC in vitro. Although, INPP4B has previously been reported to be a putative tumour suppressor in epithelial cancers [14-16], these findings infer 1) the presence of a novel phosphataseindependent function for INPP4B and 2) an unsuspected role for INPP48 overexpression in diminishing chemotherapy responses leading to poor survival outcome in AML. The reduction in chemoresistance associated with INPP48 inhibition highlights a potential role for this protein as a therapeutic target for future development in AML.
The CAP superfamily is defined by two signature sequence motifs. Using these motifs 1281 CAP proteins are identified in 481 species. Within the human and mouse there are 31 and 33 CAP proteins respectively, contained within four distinct protein groups: (1) the human glioma pathogenesis-related 1 (GLIPR1) proteins; (2) the Golgi-associated pathogenesis-related 1 (GAPR-1) proteins; (3) the peptidase inhibitor 15 (Pi15) proteins and (4) the cysteine-rich secretory proteins (CRISPs). All four members of the CRISP sub-group are expressed in the male reproductive tract. While screening a testis expression library we identified a novel sequence with homology to the CAP protein superfamily. The sequence showed greatest similarity to the GLIPR1 family of proteins and was called GLIPR1 like-1 (GLIPR1L1). We showed by Northern blot and EST analysis that GLIPR1L11 expression was testis-specific. Following generation of a specific GLIPR1L1 antisera (T32) a developmental Western blot of testis proteins showed a T32 immunoreactive band at the expected size of 25 kDa at day 14 post-partum. From day 22, coinciding with the appearance of round spermatids, a T32 immunoreactive band at 37 kDa was observed which increased in intensity in the adult testis. The 25 kDa form was absent in epididymal sperm. Interestingly however, within the epididymis a proportion of the 37 kDa form was additionally modified to produce a 51 kDa immunoreactive band. Addition of ~12 kDa units is suggestive of post-translational modification by sumoylation. We confirmed successive sumoylation of GLIPR1L1 using dual fluorescence labeling Western blots and isoform-specific SUMO antibodies. GLIPR1L1 is the first protein shown to be directly sumoylated during within sperm and during epididymal maturation. Using immunofluorescence on testicular sections, epididymal mouse sperm and in vitro capacitated mouse sperm we showed that GLIPR1L1 is initially localized to the redundant nuclear envelop (RNE) where it is subsequently redistributed following capacitation to the apical aspect of the sperm head. This redistribution is consistent with a role for GLIPR1L1 in interaction with the oocyte and a role in fertilization and is also consistent with the reports of CAP function in sperm egg interaction. We have characterized a novel testis-specific sperm protein GLIPR1L1, have shown that sumoylation is an active mechanism during sperm maturation and that GLIPR1L1 undergoes an impressive redistribution to the peri-acrosomal region consistent with a role in fertilization.
Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.
Background information . CRISP2 (cysteine‐rich secretory protein 2) is a sperm acrosome and tail protein with the ability to regulate Ca 2+ flow through ryanodine receptors. Based on these properties, CRISP2 has a potential role in fertilization through the regulation of ion signalling in the acrosome reaction and sperm motility. The purpose of the present study was to determine the expression, subcellular localization and the role in spermatogenesis of a novel CRISP2‐binding partner, which we have designated SHTAP (sperm head and tail associated protein). Results . Using yeast two‐hybrid screens of an adult testis expression library, we identified SHTAP as a novel mouse CRISP2‐binding partner. Sequence analysis of all Shtap cDNA clones revealed that the mouse Shtap gene is embedded within a gene encoding the unrelated protein NSUN4 (NOL1/NOP2/Sun domain family member 4). Five orthologues of the Shtap gene have been annotated in public databases. SHTAP and its orthologues showed no significant sequence similarity to any known protein or functional motifs, including NSUN4. Using an SHTAP antiserum, multiple SHTAP isoforms (∼20–87 kDa) were detected in the testis, sperm, and various somatic tissues. Interestingly, only the ∼26 kDa isoform of SHTAP was able to interact with CRISP2. Furthermore, yeast two‐hybrid assays showed that both the CAP (CRISP/antigen 5/pathogenesis related‐1) and CRISP domains of CRISP2 were required for maximal binding to SHTAP. SHTAP protein was localized to the peri‐acrosomal region of round spermatids, and the head and tail of the elongated spermatids and sperm tail where it co‐localized with CRISP2. During sperm capacitation, SHTAP and the SHTAP—CRISP2 complex appeared to be redistributed within the head. Conclusions . The present study is the first report of the identification, annotation and expression analysis of the mouse Shtap gene. The redistribution observed during sperm capacitation raises the possibility that SHTAP and the SHTAP—CRISP2 complex play a role in the attainment of sperm functional competence.
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