Interactome Analysis Identifies the Role of BZW2 in Promoting Endoplasmic Reticulum-Mitochondria Contact and Mitochondrial Metabolism.

Understanding the molecular functions of less-studied proteins is an important task of life science research. Despite reports of basic leucine zipper and W2 domain-containing protein 2 (BZW2) promoting cancer progression first emerging in 2017, little is known about its molecular function. Using a quantitative proteomic approach to identify its interacting proteins, we found that BZW2 interacts with both endoplasmic reticulum (ER) and mitochondrial proteins. We thus hypothesized that BZW2 localizes to and promotes the formation of ER-mitochondria contact sites and that such localization would promote calcium transport from ER to the mitochondria and promote ATP production. Indeed, we found that BZW2 localized to ER-mitochondria contact sites and that BZW2 knockdown decreased ER-mitochondria contact, mitochondrial calcium levels, and ATP production. These findings provide key insights into molecular functions of BZW2, the potential role of BZW2 in cancer progression, and highlight the utility of interactome data in understanding the function of less-studied proteins.

Control of asparagine homeostasis in Bacillus subtilis: identification of promiscuous amino acid importers and exporters.

The Gram-positive model bacterium B. subtilis is able to import all proteinogenic amino acids from the environment as well as to synthesize them. However, the players involved in the acquisition of asparagine have not yet been identified for this bacterium. In this work, we used d-asparagine as a toxic analog of l-asparagine to identify asparagine transporters. This revealed that d- but not l-asparagine is taken up by the malate/lactate antiporter MleN. Specific strains that are sensitive to the presence of l-asparagine due to the lack of the second messenger cyclic di-AMP or due to the intracellular accumulation of this amino acid were used to isolate and characterize suppressor mutants that were resistant to the presence of otherwise growth-inhibiting concentrations of l-asparagine. These screens identified the broad-spectrum amino acid importers AimA and BcaP as responsible for the acquisition of l-asparagine. The amino acid exporter AzlCD allows detoxification of l-asparagine in addition to 4-azaleucine and histidine. This work supports the idea that amino acids are often transported by promiscuous importers and exporters. However, our work also shows that even stereo-enantiomeric amino acids do not necessarily use the same transport systems.IMPORTANCETransport of amino acid is a poorly studied function in many bacteria, including the model organism Bacillus subtilis. The identification of transporters is hampered by the redundancy of transport systems for most amino acids as well as by the poor specificity of the transporters. Here, we apply several strategies to use the growth-inhibitive effect of many amino acids under defined conditions to isolate suppressor mutants that exhibit either reduced uptake or enhanced export of asparagine, resulting in the identification of uptake and export systems for l-asparagine. The approaches used here may be useful for the identification of transporters for other amino acids both in B. subtilis and in other bacteria.

NDC1 promotes hepatocellular carcinoma tumorigenesis by targeting BCAP31 to activate PI3K/AKT signaling.

Hepatocellular carcinoma (HCC) is among the world's worst malignancies. Nuclear division cycle 1 (NDC1) is an essential membrane-integral nucleoporin, found in this study to be significantly increased in primary HCC. A multivariate analysis revealed that higher NDC1 expression was linked to worse outcome in HCC patients. Mouse xenograft tumors overexpressing NDC1 grew rapidly, and HCC cells overexpressing NDC1 showed enhanced proliferation, invasion, and migration in vitro. In contrast, knocking down NDC1 had the opposite effects in vitro. Furthermore, co-immunoprecipitation and liquid chromatograph mass spectrometer analyses revealed that NDC1 activated PI3K/AKT signaling by interacting with BCAP31. In summary, NDC1 and BCAP31 cooperate to promote the PI3K/AKT pathway, which is essential for HCC carcinogenesis. This suggests that NDC1 is predictive of prognosis in HCC.

BCAP31 is involved in modulating colorectal cancer cell proliferation via the Emerin/ß-catenin axis.

Understanding the mechanisms of colorectal cancer (CRC) progression is critical for developing innovative treatment strategies. As an endoplasmic reticulum-located protein, B cell receptor-associated protein 31 (BCAP31) has been identified to be highly expressed in multiple cancers. However, its function and molecular mechanism in CRC remain not fully understood. In the present study, BCAP31 expression and its correlation with the clinical stage were analyzed based on TCGA database. We demonstrated that loss of BCAP31 suppressed CRC cell proliferation in vitro and tumor growth in vivo. Mechanistically, we demonstrated that Emerin was an interaction partner and downstream molecule of BCAP31. Knockdown of BCAP31 promoted the nuclear envelope localization of Emerin, leading to a reduction of ß-catenin accumulation in the nucleus, which resulted in downregulation of Wnt/ß-catenin downstream target genes, including c-Myc, cyclin D1, Survivin, and Mcl-1. Moreover, downregulation of Emerin partially restored the BCAP31 depletion-mediated ß-catenin protein level and tumor suppressive effects in CRC cells.Our data highlights the pivotal role of BCAP31 depletion in inhibiting cell proliferation in CRC cells, and mechanistically via Emerin/ß-catenin signaling, which may serve as a promising target for CRC treatment.

Molecular Characterization of a B Cell Adaptor for Phosphoinositide 3-Kinase Homolog in Lamprey (Lampetra japonica) and Its Function in the Immune Response.

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys.

De novo mutation and skewed X-inactivation in girl with BCAP31-related syndrome.

Full genome analysis of a young girl with deafness, dystonia, central hypomyelination, refractory seizure, and fluctuating liver function impairment revealed a heterozygous, de novo variant in the BCAP31 gene on chromosome Xq28 (NM_001256447.2:c.92G>A), mutations of which caused the X-linked recessive severe neurologic disorder deafness, dystonia, and cerebral hypomyelination. Reverse transcription-polymerase chain reaction of the patient's white blood cells showed the absence of wild-type BCAP31 messenger RNA (mRNA) but the presence of two novel BCAP31 mRNAs. The major alternatively spliced mRNA is due to Exon 2 skipping and the utilization of a new initiation site in Exon 3 that leads to a frameshift and truncated transcript while the minor novel mRNA has a 110 nucleotide insertion to Exon 2. Phasing studies showed that the de novo variant arose in the paternal X chromosome. X chromosome inactivation assay was done and confirmed that the patient's maternal X chromosome was preferentially inactivated, providing evidence that the mutated BCAP31 gene was the one predominantly expressed. According to the American College of Medical Genetics and Genomics guideline, this variant is deemed "pathogenic" (PS2, PS3, PM2, PP3, and PP4) and deleterious. This is the first reported female patient in BCAP31-related syndrome resulted from skewed X-inactivation and a de novo mutation in the active X chromosome.

Schimke XLID syndrome results from a deletion in BCAP31.

A family with three affected males and a second family with a single affected male with intellectual disability, microcephaly, ophthalmoplegia, deafness, and Involuntary limb movements were reported by Schimke and Associates in 1984. The affected males with Schimke X-linked intellectual disability (XLID) syndrome (OMIM# 312840) had a similar facial appearance with deep-set eyes, downslanting palpebral fissures, hypotelorism, narrow nose and alae nasi, cupped ears and spacing of the teeth. Two mothers had mild hearing loss but no other manifestations of the disorder. The authors considered the disorder to be distinctive and likely X-linked. Whole genome sequencing in the single affected male available and the three carrier females from one of the families with Schimke XLID syndrome identified a 2 bp deletion in the BCAP31 gene. During the past decade, pathogenic alterations of the BCAP31 gene have been associated with deafness, dystonia, and central hypomyelination, an XLID condition given the eponym DDCH syndrome. A comparison of clinical findings in Schimke XLID syndrome and DDCH syndrome shows them to be the same clinical entity. The BCAP31 protein functions in endoplasmic reticulum-associated degradation to promote ubiquitination and destruction of misfolded proteins.

Immunohistochemical expression of BCAP 31 in chronic plaque psoriasis.

Psoriasis is a common chronic skin inflammatory disease characterized by an exaggerated proliferation of keratinocytes. B-cell receptor-associated protein 31 (BCAP 31) plays critical roles in induction of proliferation and apoptosis. The current study aimed at evaluation of the immunohistochemical localization of BCAP 31 in psoriatic skin compared to normal skin in addition of correlating BCAP31 expression with the clinical and pathological parameters of psoriasis. The present study was carried out on skin biopsies from 30 psoriatic patients and 10 normal skin (control group). BCAP31 was not expressed in normal skin either epidermis or dermis, while it was expressed in epidermis of 15 psoriatic cases and in dermis of 13 cases with a significant difference between the two groups (p < .05). Strong epidermal BCAP 31 expression was associated with marked parakeratosis (p = .025). There was a significant co-parallel epidermal and dermal expression of BCAP31 in psoriasis (p < .05). The role of BCAP 31 is not only confined to its expression by affected keratinocytes but extended to its localization to dermal lymphocytes where they were correlated with each other. The up- regulation of BCAP 31 in psoriatic lesion compared to normal skin may suggest its use as a target therapy for treatment of psoriasis that necessitates further studies to clarify.

Structure of the Toll/Interleukin-1 Receptor (TIR) Domain of the B-cell Adaptor That Links Phosphoinositide Metabolism with the Negative Regulation of the Toll-like Receptor (TLR) Signalosome.

Ligand binding to Toll-like receptors (TLRs) results in dimerization of their cytosolic Toll/interleukin-1 receptor (TIR) domains and recruitment of post-receptor signal transducers into a complex signalosome. TLR activation leads to the production of transcription factors and pro-inflammatory molecules and the activation of phosphoinositide 3-kinases (PI3K) in a process that requires the multimodular B-cell adaptor for phosphoinositide 3-kinase (BCAP). BCAP has a sequence previously proposed as a "cryptic" TIR domain. Here, we present the structure of the N-terminal region of human BCAP and show that it possesses a canonical TIR fold. Dimeric BCAP associates with the TIR domains of TLR2/4 and MAL/TIRAP, suggesting that it is recruited to the TLR signalosome by multitypic TIR-TIR interactions. BCAP also interacts with the p85 subunit of PI3K and phospholipase Cγ, enzymes that deplete plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), and these interactions provide a molecular explanation for BCAP-mediated down-regulation of inflammatory signaling.

BCAP31-associated encephalopathy and complex movement disorder mimicking mitochondrial encephalopathy.

BCAP31, encoded by BCAP31, is involved in the export of transmembrane proteins from the endoplasmic reticulum. Pathogenic variants in BCAP31 results in global developmental delay, dystonia, deafness and dysmorphic features in males, called deafness, dystonia, and cerebral hypomyelination (DDCH) syndrome. We report a new patient with BCAP3-associated encephalopathy, DDCH syndrome, sensorineural hearing loss, generalized dystonia, and choreoathetosis. This 3.5-year-old boy had microcephaly and failure to thrive within the first 3 months of life. His brain MRI showed bilateral increased signal intensity in globus pallidus at age 3 months raising the suspicion of mitochondrial encephalopathy. His muscle biopsy revealed pleomorphic subsarcolemmal mitochondria collection in electron microscopy. Respiratory chain enzyme activities were normal in muscle. He was enrolled to a whole exome sequencing research study, which identified a hemizygous likely pathogenic truncating variant (c.533_536dup; p.Ser180AlafsX6) in BCAP31, inherited from his mother, who had sensorineural hearing loss and normal cognitive functions. We report a new patient with BCAP31-associated encephalopathy, DDCH syndrome, mimicking mitochondrial encephalopathy. We also report a heterozygous mother who has bilateral sensorineural hearing loss. This patient's clinical features, muscle histopathology, brain MRI features, and family history were suggestive of mitochondrial encephalopathy. Whole exome sequencing research study confirmed the diagnosis of BCAP31-associated encephalopathy, DDCH syndrome.

pH-sensitive pHluorins as a molecular sensor for in situ monitoring of enzyme-catalyzed prodrug activation.

This work examines the feasibility of using a pH-sensitive fluorescent protein as a molecular reporter for enzyme-catalyzed prodrug activation reaction. Specifically, a ratiometric pHluorins was examined for detection of the activity of horseradish peroxidase (HRP) for the activation of indole-3-acetic acid. The pHluorins and HRP were conjugated chemically, forming a biocatalyst with a self-reporting function. Results showed that the characteristic fluorescence intensity ratio of the conjugate shifted from 1.47 to 1.40 corresponding to the progress of the prodrug activation reaction. The effectiveness of applying the conjugate for inhibition of the growth of Bcap-37 cells was also demonstrated simultaneously with reaction monitoring. The results reveal a very promising approach to realizing in situ monitoring of enzyme activities based on pH shifting for enzyme-based prodrug therapy applications.

Distal Xq28 microdeletions: clarification of the spectrum of contiguous gene deletions involving ABCD1, BCAP31, and SLC6A8 with a new case and review of the literature.

The contiguous ABCD1/DXS1375E (BCAP31) deletion syndrome (CADDS) is a rare X-linked contiguous gene deletion syndrome with a severe clinical phenotype that includes marked delays, significant growth failure, liver dysfunction, and early death. The X-linked creatine transporter deficiency is a considerably more common and a cause of X-linked intellectual disability; however, multi-exon deletions of the creatine transporter are rare. We report the fifth case of CADDS, who also has a deletion of the X-linked creatine transporter. We also review reported cases of deletions in this region in order to clarify the clinical spectrum of contiguous microdeletions in this region.

Whole exome sequencing reveals a dual diagnosis of BCAP31-related syndrome and glutaric aciduria III.

Background: Biochemical testing is a common first-tier approach in the setting of genetic evaluation of patients with unexplained developmental delay. However, results can be unclear, and a plan for second-tier analysis must be determined based on the patient's biochemical results and clinical presentation - in many cases, triggering a diagnostic odyssey. Case presentation: A male patient from the United States presenting with unexplained developmental delay, microcephaly, hypotonia, and feeding difficulties was referred for clinical genetic evaluation at age 8 months. Biochemical testing revealed an isolated marked elevation of glutaric acid on urine organic acid profile, without elevations of related metabolites. Further testing included GCDH sequencing, a neurometabolic gene panel, chromosomal microarray, Prader Willi/Angelman testing, and lysosomal disease enzyme panel, all of which were non-diagnostic. The patient had persistent developmental delay and hypotonia, dystonia, sensorineural hearing loss, and abnormal brain myelination on magnetic resonance imaging. Whole exome sequencing (WES) was performed and revealed a dual diagnosis of glutaric aciduria III (GA III) and BCAP31-related disorder, an X-linked intellectual disability syndrome, caused by a novel pathogenic variant. Conclusions: GA III has historically been considered clinically benign, with few reported cases. This patient's presenting symptoms were similar to those commonly seen in GA I and GA II, however the biochemical abnormalities were not consistent with these disorders, prompting additional molecular and biochemical testing. Ultimately, WES confirmed a diagnosis of BCAP31-related syndrome, a rare neurological disorder, which explained the patient's presenting symptoms. WES also identified a secondary diagnosis of GA III. We present a patient with two rare genetic conditions, highlighting the importance of deep phenotyping and the utility of WES in the setting of a patient with dual genetic diagnoses.

Presence of phosphodiesterase type 5 in the spinal cord and its involvement in bladder outflow obstruction related bladder overactivity.

PURPOSE: Phosphodiesterase type 5 inhibitors were recently introduced as a new treatment option for men with lower urinary tract symptoms. Safety and clinical effectiveness are well documented but the mode of action is still unclear. We determined and compared the expression of phosphodiesterase type 5 in the spinal cord of normal (sham operated) rats and rats with partial urethral obstruction induced bladder overactivity. We also assessed the urodynamic effects of intravenously and intrathecally administered sildenafil in the rats to determine whether phosphodiesterase type 5 inhibitors exert effects on the sacral spinal cord. MATERIALS AND METHODS: A total of 65 male Sprague-Dawley rats were used for molecular/morphological and functional experiments. Bladder overactivity was induced via surgical partial urethral obstruction in 39 of 65 rats. Spinal phosphodiesterase type 5 expression was assessed by histology and polymerase chain reaction. The effects of sildenafil administered intravenously or intrathecally were studied urodynamically. RESULTS: Phosphodiesterase type 5 was expressed in various regions of the lumbosacral spinal cord, including the sacral regions of micturition control. Expression was similar in normal rats and rats with partial urethral obstruction/bladder overactivity. In normal rats intravenous and intrathecal sildenafil had no urodynamic effect. When administered intravenously and intrathecally to rats with partial urethral obstruction/bladder overactivity, sildenafil decreased micturition frequency and bladder pressure. Doses tested intrathecally had no effect when given intravenously. CONCLUSIONS: Phosphodiesterase type 5 is expressed in the rat spinal cord. Intravenous sildenafil may exert part of its urodynamic effect in rats with partial urethral obstruction/bladder overactivity via an effect on the sacral spinal cord.

Interstitial lung disease and pancreatic exocrine insufficiency in CADDS: Phenotypic expansion and literature review.

Contiguous ABCD1/ DXS1357E deletion syndrome (CADDS) is a rare deletion syndrome involving two contiguous genes on Xq28, ABCD1 and BCAP31 (formerly known as DXS1357E). Only nine individuals with this diagnosis have been reported in the medical literature to date. Intragenic loss-of-function variants in BCAP31 cause the deafness, dystonia, and cerebral hypomyelination syndrome (DDCH). Isolated pathogenic intragenic variants in ABCD1 are associated with the most common peroxisomal disorder, X-linked adrenoleukodystrophy (X-ALD), a single transporter deficiency, which in its more severe cerebral form is characterised by childhood-onset neurodegeneration and high levels of very-long-chain fatty acids (VLCFA). While increased VLCFA levels also feature in CADDS, the few patients described to date all presented as neonates with a severe phenotype. Here we report a tenth individual with CADDS, a male infant with dysmorphic facial features who was diagnosed through ultra-rapid whole genome sequencing (WGS) in the setting of persistent cholestatic liver disease, sensorineural hearing loss, hypotonia and growth failure and developmental delay. Biochemical studies showed elevated VLCFA and mildly reduced plasmalogens. He died at 7 months having developed pancreatic exocrine deficiency and interstitial lung disease, two features we propose to be possible extensions to the CADDS phenotype. We also review the genetic, phenotypic, and biochemical features in previously reported individuals with CADDS.

Possible mitochondrial dysfunction in a patient with deafness, dystonia, and cerebral hypomyelination (DDCH) due to BCAP31 Mutation.

BACKGROUND: Deafness, dystonia, and cerebral hypomyelination (DDCH) is an X-linked disorder due to hemizygous mutations of BCAP31. METHODS: We report an 8-year-old boy with DDCH who possibly accompanied mitochondrial dysfunction. Clinical evaluation, respiratory chain enzyme assay, and whole exome sequencing analysis were performed. RESULTS: Mitochondrial dysfunction was suspected by respiratory chain enzyme assay on his cultured skin fibroblasts which showed significantly decreased complex I enzyme activity. Whole exome sequencing analysis revealed a recurrent BCAP31 mutation (c.97C>T:p.Gln33*) which confirmed the diagnosis of DDCH for the patient. CONCLUSION: We speculate that mitochondrial dysfunction may be a feature in patients with DDCH.

Profiling of Circulating microRNAs in Prostate Cancer Reveals Diagnostic Biomarker Potential.

Early detection of prostate cancer (PC) is paramount as localized disease is generally curable, while metastatic PC is generally incurable. There is a need for improved, minimally invasive biomarkers as current diagnostic tools are inaccurate, leading to extensive overtreatment while still missing some clinically significant cancers. Consequently, we profiled the expression levels of 92 selected microRNAs by RT-qPCR in plasma samples from 753 patients, representing multiple stages of PC and non-cancer controls. First, we compared plasma miRNA levels in patients with benign prostatic hyperplasia (BPH) or localized prostate cancer (LPC), versus advanced prostate cancer (APC). We identified several dysregulated microRNAs with a large overlap of 59 up/down-regulated microRNAs between BPH versus APC and LPC versus APC. Besides identifying several novel PC-associated dysregulated microRNAs in plasma, we confirmed the previously reported upregulation of miR-375 and downregulation of miR-146a-5p. Next, by randomly splitting our dataset into a training and test set, we identified and successfully validated a novel four microRNA diagnostic ratio model, termed bCaP (miR-375*miR-33a-5p/miR-16-5p*miR-409-3p). Combined in a model with prostate specific antigen (PSA), digital rectal examination status, and age, bCaP predicted the outcomes of transrectal ultrasound (TRUS)-guided biopsies (negative vs. positive) with greater accuracy than PSA alone (Training: area under the curve (AUC), model = 0.84; AUC, PSA = 0.63. Test set: AUC, model = 0.67; AUC, PSA = 0.56). It may be possible in the future to use this simple and minimally invasive bCaP test in combination with existing clinical parameters for a more accurate selection of patients for prostate biopsy.

MicroRNA-362-3p Inhibits Migration and Invasion via Targeting BCAP31 in Cervical Cancer.

Cervical cancer (CC) is the most common malignant tumor in gynecology, and metastasis is an important cause of patient death. MiRNAs (microRNAs) have been found to play key roles in cervical cancer metastasis, but the effect of miR-362-3p in CC is unclear. This study aimed to investigate the role of miR-362-3p in cervical cancer migration and invasion. We compared the expression levels of miR-362-3p in cervical cancer tissues and adjacent normal cervical tissues. In CC tissues, miR-362-3p expression was significantly down-regulated, which is related to the cancer stage and patient survival. MiR-362-3p can effectively inhibit the migration and invasion of cervical cancer cells. The dual-luciferase reporter assay results showed that BCAP31 (B cell receptor associated protein 31) is a direct target protein of miR-362-3p. The results of the immunohistochemical examination of clinical tissue samples showed that BCAP31 was abnormally highly expressed in cervical cancer, which was positively correlated with the clinical stage. BCAP31 knockdown exerted similar effects as miR-362-3p overexpression. Further GSEA analysis showed that BCAP31 may participate in multiple biological processes, such as protein transport, metabolism, and organelle organization. Our results suggest that miR-362-3p inhibits migration and invasion via directly targeting BCAP31 in cervical cancer, and restoring miR-362-3p levels may be a new treatment strategy for cervical cancer in the future.

Case Study: The Recurrent Fusion RNA DUS4L-BCAP29 in Noncancer Human Tissues and Cells.

Traditional gene fusions are involved in the development of various neoplasias. DUS4L-BCAP29, a chimeric fusion RNA, has been reported to be a cancer-related fusion in prostate and gastric cancers. This chimeric RNA is believed to play a tumorigenic role. Here, we showed that the DUS4L-BCAP29 fusion transcript exists in a variety of normal tissues. It is also present in noncancerous epithelial and fibroblast cell lines. Quantitatively, the fusion transcript has a similar expression level in noncancerous gastric and prostate cell lines and tissues to its expression in cancerous cell lines and tissues. Previously, a loss-of-function approach was used to report a probable functionality for this fusion. However, this approach is not sufficient to prove such functionality. Alternatively, a gain-of-function approach showed that overexpression of DUS4L-BCAP29 promotes cell growth and motility, even in noncancerous cell lines. Finally, we provide further evidence that the fusion transcript is a product of cis-splicing between adjacent genes. In summary, we believe that in contrast to traditional gene fusions, DUS4L-BCAP29 cannot be used as a cancer biomarker. Instead, it is a fusion transcript that exists in normal physiology and its progrowth effect is not unique to cancer situations.