Spliceosome component PHD finger 5A is essential for early B lymphopoiesis.

The spliceosome, a multi-megadalton ribonucleoprotein complex, is essential for pre-mRNA splicing in the nucleus and ensuring genomic stability. Its precise and dynamic assembly is pivotal for its function. Spliceosome malfunctions can lead to developmental abnormalities and potentially contribute to tumorigenesis. The specific role of the spliceosome in B cell development is poorly understood. Here, we reveal that the spliceosomal U2 snRNP component PHD finger protein 5A (Phf5a) is vital for early B cell development. Loss of Phf5a results in pronounced defects in B cell development, causing an arrest at the transition from pre-pro-B to early pro-B cell stage in the bone marrow of mutant mice. Phf5a-deficient B cells exhibit impaired immunoglobulin heavy (IgH) chain expression due to defective V-to-DJ gene rearrangement. Mechanistically, our findings suggest that Phf5a facilitates IgH gene rearrangement by regulating the activity of recombination-activating gene endonuclease and influencing chromatin interactions at the Igh locus.

Identification of a novel mitochondria-localized LKB1 variant required for the regulation of the oxidative stress response.

The tumor suppressor Liver Kinase B1 (LKB1) is a multifunctional serine/threonine protein kinase that regulates cell metabolism, polarity, and growth and is associated with Peutz-Jeghers Syndrome and cancer predisposition. The LKB1 gene comprises 10 exons and 9 introns. Three spliced LKB1 variants have been documented, and they reside mainly in the cytoplasm, although two possess a nuclear-localization sequence (NLS) and are able to shuttle into the nucleus. Here, we report the identification of a fourth and novel LKB1 isoform that is, interestingly, targeted to the mitochondria. We show that this mitochondria-localized LKB1 (mLKB1) is generated from alternative splicing in the 5' region of the transcript and translated from an alternative initiation codon encoded by a previously unknown exon 1b (131 bp) hidden within the long intron 1 of LKB1 gene. We found by replacing the N-terminal NLS of the canonical LKB1 isoform, the N-terminus of the alternatively spliced mLKB1 variant encodes a mitochondrial transit peptide that allows it to localize to the mitochondria. We further demonstrate that mLKB1 colocalizes histologically with mitochondria-resident ATP Synthase and NAD-dependent deacetylase sirtuin-3, mitochondrial (SIRT3) and that its expression is rapidly and transiently upregulated by oxidative stress. We conclude that this novel LKB1 isoform, mLKB1, plays a critical role in regulating mitochondrial metabolic activity and oxidative stress response.

Mettl14-Mediated m6A Modification Is Essential for Germinal Center B Cell Response.

The germinal center (GC) response is essential for generating memory B and long-lived Ab-secreting plasma cells during the T cell-dependent immune response. In the GC, signals via the BCR and CD40 collaboratively promote the proliferation and positive selection of GC B cells expressing BCRs with high affinities for specific Ags. Although a complex gene transcriptional regulatory network is known to control the GC response, it remains elusive how the positive selection of GC B cells is modulated posttranscriptionally. In this study, we show that methyltransferase like 14 (Mettl14)-mediated methylation of adenosines at the position N 6 of mRNA (N 6-methyladenosine [m6A]) is essential for the GC B cell response in mice. Ablation of Mettl14 in B cells leads to compromised GC B cell proliferation and a defective Ab response. Interestingly, we unravel that Mettl14-mediated m6A regulates the expression of genes critical for positive selection and cell cycle regulation of GC B cells in a Ythdf2-dependent but Myc-independent manner. Furthermore, our study reveals that Mettl14-mediated m6A modification promotes mRNA decay of negative immune regulators, such as Lax1 and Tipe2, to upregulate genes requisite for GC B cell positive selection and proliferation. Thus, our findings suggest that Mettl14-mediated m6A modification plays an essential role in the GC B cell response.

RNA-Editing Enzyme ADAR1 p150 Isoform Is Critical for Germinal Center B Cell Response.

Adenosine deaminase acting on RNA (ADAR)1 is the principal enzyme for adenosine-to-inosine editing, an RNA modification-avoiding cytosolic nucleic acid sensor's activation triggered by endogenous dsRNAs. Two ADAR1 isoforms exist in mammals, a longer IFN-inducible and mainly cytoplasm-localized p150 isoform and a shorter constitutively expressed and primarily nucleus-localized p110 isoform. Studies of ADAR1 mutant mice have demonstrated that ADAR1 is essential for multiple physiological processes, including embryonic development, innate immune response, and B and T lymphocyte development. However, it remained unknown whether ADAR1 plays a role in the humoral immune response. In this study, we conditionally delete Adar1 in activated B cells and show that ADAR1-deficient mice have a defective T cell-dependent Ab response and diminished germinal center (GC) B cells. Using various double mutant mice concurrently deficient in ADAR1 and different downstream dsRNA sensors, we demonstrate that ADAR1 regulates the GC response by preventing hyperactivation of the melanoma differentiation-associated protein 5 (MDA5) but not the protein kinase R or RNase L pathway. We also show that p150 is exclusively responsible for ADAR1's function in the GC response, and the p110 isoform cannot substitute for the p150's role, even when p110 is constitutively expressed in the cytoplasm. We further demonstrated that the dsRNA-binding but not the RNA-editing activity is required for ADAR1's function in the GC response. Thus, our data suggest that the ADAR1 p150 isoform plays a crucial role in regulating the GC B cell response.

Brain microglia activation and peripheral adaptive immunity in Parkinson's disease: a multimodal PET study.

BACKGROUND: Abnormal activation of immune system is an important pathogenesis of Parkinson's disease, but the relationship between peripheral inflammation, central microglia activation and dopaminergic degeneration remains unclear. OBJECTIVES: To evaluate the brain regional microglia activation and its relationship with clinical severity, dopaminergic presynaptic function, and peripheral inflammatory biomarkers related to adaptive immunity. METHODS: In this case-control study, we recruited 23 healthy participants and 24 participants with early-stage Parkinson's disease. 18F-PBR06 PET/MR for microglia activation, 18F-FP-DTBZ for dopaminergic denervation, total account of T cells and subpopulations of T helper (Th1/Th2/Th17) cells, and the levels of serum inflammatory cytokines were assessed. Sanger sequencing was used to exclude the mix-affinity binders of 18F-PBR06-PET. RESULTS: Compared to healthy controls, patients with Parkinson's disease had an increased 18F-PBR06-PET standardized uptake value ratio (SUVR) in the putamen, particularly in the ipsilateral side of the motor onset. 18F-PBR06-PET SUVR was positively associated with 18F-FP-DTBZ-PET SUVR in the brainstem and not associated with disease severity measured by Hoehn and Yahr stage, MDS-UPDRS III scores. Patients with Parkinson's disease had elevated frequencies of Th1 cells and serum levels of IL10 and IL17A as compared to healthy controls. No significant association between peripheral inflammation markers and microglia activation in the brain of PD was observed. CONCLUSION: Parkinson's disease is associated with early putaminal microglial activation and peripheral phenotypic Th1 bias. Peripheral adaptive immunity might be involved in microglia activation in the process of neurodegeneration in PD indirectly, which may be a potential biomarker for the early detection and the target for immunomodulating therapy.

Conditional disruption of AMP kinase in dopaminergic neurons promotes Parkinson's disease-associated phenotypes in vivo.

Emerging studies implicate energy dysregulation as an underlying trigger for Parkinson's disease (PD), suggesting that a better understanding of the molecular pathways governing energy homeostasis could help elucidate therapeutic targets for the disease. A critical cellular energy regulator is AMP kinase (AMPK), which we have previously shown to be protective in PD models. However, precisely how AMPK function impacts on dopaminergic neuronal survival and disease pathogenesis remains elusive. Here, we showed that Drosophila deficient in AMPK function exhibits PD-like features, including dopaminergic neuronal loss and climbing impairment that progress with age. We also created a tissue-specific AMPK-knockout mouse model where the catalytic subunits of AMPK are ablated in nigral dopaminergic neurons. Using this model, we demonstrated that loss of AMPK function promotes dopaminergic neurodegeneration and associated locomotor aberrations. Accompanying this is an apparent reduction in the number of mitochondria in the surviving AMPK-deficient nigral dopaminergic neurons, suggesting that an impairment in mitochondrial biogenesis may underlie the observed PD-associated phenotypes. Importantly, the loss of AMPK function enhances the susceptibility of nigral dopaminergic neurons in these mice to 6-hydroxydopamine-induced toxicity. Notably, we also found that AMPK activation is reduced in post-mortem PD brain samples. Taken together, these findings highlight the importance of neuronal energy homeostasis by AMPK in PD and position AMPK pathway as an attractive target for future therapeutic exploitation.

Faim knockout leads to gliosis and late-onset neurodegeneration of photoreceptors in the mouse retina.

Fas Apoptotic Inhibitory Molecule protein (FAIM) is a death receptor antagonist and an apoptosis regulator. It encodes two isoforms, namely FAIM-S (short) and FAIM-L (long), both with significant neuronal functions. FAIM-S, which is ubiquitously expressed, is involved in neurite outgrowth. In contrast, FAIM-L is expressed only in neurons and it protects them from cell death. Interestingly, FAIM-L is downregulated in patients and mouse models of Alzheimer's disease before the onset of neurodegeneration, and Faim transcript levels are decreased in mouse models of retinal degeneration. However, few studies have addressed the role of FAIM in the central nervous system, yet alone the retina. The retina is a highly specialized tissue, and its degeneration has proved to precede pathological mechanisms of neurodegenerative diseases. Here we describe that Faim depletion in mice damages the retina persistently and leads to late-onset photoreceptor death in older mice. Immunohistochemical analyses showed that Faim knockout (Faim-/- ) mice present ubiquitinated aggregates throughout the retina from early ages. Moreover, retinal cells released stress signals that can signal to Müller cells, as shown by immunofluorescence and qRT-PCR. Müller cells monitor retinal homeostasis and trigger a gliotic response in Faim-/- mice that becomes pathogenic when sustained. In this regard, we observed pronounced vascular leakage at later ages, which may be caused by persistent inflammation. These results suggest that FAIM is an important player in the maintenance of retinal homeostasis, and they support the premise that FAIM is a plausible early marker for late photoreceptor and neuronal degeneration.

A 1.7-Mb chromosomal inversion downstream of a PpOFP1 gene is responsible for flat fruit shape in peach.

Flat peaches have become popular worldwide due to their novelty and convenience. The peach flat fruit trait is genetically controlled by a single gene at the S locus, but its genetic basis remains unclear. Here, we report a 1.7-Mb chromosomal inversion downstream of a candidate gene encoding OVATE Family Protein, designated PpOFP1, as the causal mutation for the peach flat fruit trait. Genotyping of 727 peach cultivars revealed an occurrence of this large inversion in flat peaches, but absent in round peaches. Ectopic overexpression of PpOFP1 resulted in oval-shaped leaves and shortened siliques in Arabidopsis, suggesting its role in repressing cell elongation. Transcriptional activation of PpOFP1 by the chromosomal inversion may repress vertical elongation in flat-shaped fruits at early stages of development, resulting in the flat fruit shape. Moreover, PpOFP1 can interact with fruit elongation activator PpTRM17, suggesting a regulatory network controlling fruit shape in peach. Additionally, screening of peach wild relatives revealed an exclusive presence of the chromosomal inversion in P. ferganensis, supporting that this species is the ancestor of the domesticated peach. This study provides new insights into mechanisms underlying fruit shape evolution and molecular tools for genetic improvement of fruit shape trait in peach breeding programmes.

Two amino acid changes in the R3 repeat cause functional divergence of two clustered MYB10 genes in peach.

R2R3-MYB genes play a pivotal role in regulating anthocyanin accumulation. Here, we report two tandemly duplicated R2R3-MYB genes in peach, PpMYB10.1 and PpMYB10.2, with the latter showing lower ability to induce anthocyanin accumulation than the former. Site-directed mutation assay revealed two amino acid changes in the R3 repeat, Arg/Lys66 and Gly/Arg93, responsible for functional divergence between these two PpMYB10 genes. Anthocyanin-promoting activity of PpMYB10.2 was significantly increased by a single amino acid replacement of Arg93 with Gly93. However, either the Gly93 → Arg93 or Arg66 → Lys66 substitutions alone showed little impact on anthocyanin-promoting activity of PpMYB10.1, but simultaneous substitutions caused a significant decrease. Reciprocal substitution of Arg/Gly93 could significantly alter binding affinity to PpbHLH3, while the Arg66 → Lys66 substitution is predicted to affect the folding of the MYB DNA-binding domain, instead of PpbHLH3-binding affinity. Overall, the change of anthocyanin-promoting activity was accompanied with that of bHLH-binding affinity, suggesting that DNA-binding affinity of R2R3-MYBs depends on their bHLH partners. Our study is helpful for understanding of functional evolution of R2R3-MYBs and their interaction with DNA targets.

Blebbistatin modulates prostatic cell growth and contrapctility through myosin II signaling.

To investigate the effect of blebbistatin (BLEB, a selective myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions. BLEB was incubated with cell lines of BPH-1 and WPMY-1, and intraprostatically injected into rats. Cell growth was determined by flow cytometry, and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM) myosin isoform (SM1/2, SM-A/B, and LC17a/b) expression was determined via competitive reverse transcriptase PCR. SM myosin heavy chain (MHC), non-muscle (NM) MHC isoforms (NMMHC-A and NMMHC-B), and proteins related to cell apoptosis were further analyzed via Western blotting. Masson's trichrome staining was applied to tissue sections. BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western blotting confirmed the effects of BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with down-regulation of Bcl-2 and up-regulation of BAX and cleaved caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally, BLEB decreased SM cell number and SM MHC expression, along with attenuated phenylephrine-induced contraction and altered prostate SMM isoform composition with up-regulation of SM-B and down-regulation of LC17a, favoring a faster contraction. Our novel data demonstrate BLEB regulated myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM isoform composition.

Adaptor protein DOK3 promotes plasma cell differentiation by regulating the expression of programmed cell death 1 ligands.

The adaptor Downstream-of-Kinase (DOK) 3 functions as a negative regulator and attenuates B-cell receptor-mediated calcium signaling. Although DOK3 is dispensable for early B-cell development, its role in plasma cell (PC) differentiation is unknown. Here, we show that Dok3(-/-) mice have increased populations of T follicular-helper (Tfh) and germinal center (GC) B cells upon immunization with a T-cell-dependent antigen. However, interestingly, they generate significantly fewer PCs. Bone marrow reconstitution experiments show that the PC defect is B-cell intrinsic and due to the inability of Dok3(-/-) B cells to sustain programmed cell death 1 (PD-1) ligand 1 (PDL1) and up-regulate PD-1 ligand 2 (PDL2) expressions that are critical for PC differentiation. Overexpression of PDL2 rectifies the PC differentiation defect in Dok3(-/-) B cells. We further demonstrate that calcium signaling suppresses the transcription of PD-1 ligands. Abrogation of calcium signaling in B cells by deleting BTK or PLCγ2 or inhibiting calcineurin with cyclosporine A leads to increased expression of PD-1 ligands. Thus, our study reveals DOK3 as a nonredundant regulator of PC differentiation by up-regulating PD-1 ligand expression through the attenuation of calcium signaling.

Loss of miR-182 affects B-cell extrafollicular antibody response.

MicroRNAs have been shown to play a role in B-cell differentiation and activation. Here, we found miR-182 to be highly induced in activated B cells. However, mice lacking miR-182 have normal B-cell and T-cell development. Interestingly, mutant mice exhibited a defective antibody response at early time-points in the immunization regimen when challenged with a T-cell-dependent antigen. Germinal centres were formed but the generation of extrafollicular plasma cells was defective in the spleens of immunized miR-182-deficient mice. Mutant mice were also not able to respond to a T-cell-independent type 2 antigen, which typically elicited an extrafollicular B-cell response. Taken together, the data indicated that miR-182 plays a critical role in driving extrafollicular B-cell antibody responses.

DOK3 is required for IFN-ß production by enabling TRAF3/TBK1 complex formation and IRF3 activation.

The downstream of kinase (DOK) family of adaptors is generally involved in the negative regulation of signaling pathways. DOK1, 2, and 3 were shown to attenuate TLR4 signaling by inhibiting Ras-ERK activation. In this study, we elucidated a novel role for DOK3 in IFN-ß production. Macrophages lacking DOK3 were impaired in IFN-ß synthesis upon influenza virus infection or polyinosinic-polyribocytidylic acid stimulation. In the absence of DOK3, the transcription factor IFN regulatory factor 3 was not phosphorylated and could not translocate to the nucleus to activate ifn-ß gene expression. Interestingly, polyinosinic-polyribocytidylic acid-induced formation of the upstream TNFR-associated factor (TRAF) 3/TANK-binding kinase (TBK) 1 complex was compromised in dok3(-/-) macrophages. DOK3 was shown to bind TBK1 and was required for its activation. Furthermore, we demonstrated that overexpression of DOK3 and TBK1 could significantly enhance ifn-ß promoter activity. DOK3 was also shown to bind TRAF3, and the binding of TRAF3 and TBK1 to DOK3 required the tyrosine-rich C-terminal domain of DOK3. We further revealed that DOK3 was phosphorylated by Bruton's tyrosine kinase. Hence, DOK3 plays a critical and positive role in TLR3 signaling by enabling TRAF3/TBK1 complex formation and facilitating TBK1 and IFN regulatory factor 3 activation and the induction of IFN-ß production.

Deficiency in TNFRSF13B (TACI) expands T-follicular helper and germinal center B cells via increased ICOS-ligand expression but impairs plasma cell survival.

Mutations in TNFRSF13B, better known as transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), contribute to common variable immunodeficiency and autoimmunity in humans. How TACI regulates these two opposing conditions is unclear, however. TACI binds the cytokines BAFF and APRIL, and previous studies using gene KO mice indicated that loss of TACI affected only T-cell-independent antibody responses. Here we demonstrate that Taci(-/-) mice have expanded populations of T follicular helper (T(fh)) and germinal center (GC) B cells in their spleens when immunized with T-cell-dependent antigen. The increased numbers of T(fh) and GC B cells in Taci(-/-) mice are largely a result of up-regulation of inducible costimulator (ICOS) ligand on TACI-deficient B cells, given that ablation of one copy of the Icosl allele restores normal levels of T(fh) and GC B cells in Taci(-/-) mice. Interestingly, despite the presence of increased T(fh) and antigen-specific B cells, immunized Taci(-/-) mice demonstrate defective antigen-specific antibody responses resulting from significantly reduced numbers of antibody-secreting cells (ASCs). This effect is attributed to the failure to down-regulate the proapoptotic molecule BIM in Taci(-/-) plasma cells. Ablation of BIM could rescue ASC formation in Taci(-/-) mice, suggesting that TACI is more important for the survival of plasma cells than for the differentiation of these cells. Thus, our data reveal dual roles for TACI in B-cell terminal differentiation. On one hand, TACI modulates ICOS ligand expression and thereby limits the size of T(fh) and GC B-cell compartments and prevents autoimmunity. On the other hand, it regulates the survival of ASCs and plays an important role in humoral immunity.

Bruton's tyrosine kinase phosphorylates Toll-like receptor 3 to initiate antiviral response.

Toll-like receptor 3 (TLR3) mediates antiviral response by recognizing double-stranded RNA. Its cytoplasmic domain is tyrosine phosphorylated upon ligand binding and initiates downstream signaling via the adapter TIR-containing adaptor inducing interferon-ß (TRIF). However, the kinase responsible for TLR3 phosphorylation remains unknown. We show here that Bruton's tyrosine kinase (BTK)-deficient macrophages failed to secrete inflammatory cytokines and IFN-ß upon TLR3 stimulation and were impaired in clearing intracellular dengue virus infection. Mutant mice were also less susceptible to d-galactosamine/p(I:C)-induced sepsis. In the absence of BTK, TLR3-induced phosphoinositide 3-kinase (PI3K), AKT and MAPK signaling and activation of NFκB, IRF3, and AP-1 transcription factors were all defective. We demonstrate that BTK directly phosphorylates TLR3 and in particular the critical Tyr759 residue. BTK point mutations that abrogate or led to constitutive kinase activity have opposite effects on TLR3 phosphorylation. Loss of BTK also compromises the formation of the downstream TRIF/receptor-interacting protein 1 (RIP1)/TBK1 complex. Thus, BTK plays a critical role in initiating TLR3 signaling.

The RNase III enzyme Dicer is essential for germinal center B-cell formation.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression and are important for pre-B and follicular B lymphopoiesis as demonstrated, respectively, by mb-1-Cre- and cd19-Cre-mediated deletion of Dicer, the RNase III enzyme critical for generating mature miRNAs. To explore the role of miRNAs in B-cell terminal differentiation, we use Aicda-Cre to specifically delete Dicer in activated B cells where activation-induced cytidine deaminase is highly expressed. We demonstrate that mutant mice fail to produce high-affinity class-switched antibodies and generate memory B and long-lived plasma cells on immunization with a T cell-dependent antigen. More importantly, germinal center (GC) B-cell formation is drastically compromised in the absence of Dicer, as a result of defects in cell proliferation and survival. Dicer-deficient GC B cells express higher levels of cell cycle inhibitor genes and proapoptotic protein Bim. Ablation of Bim could partially rescue the defect in GC B-cell formation in Dicer-deficient mice. Taken together, our data suggest that Dicer and probably miRNAs are critical for GC B-cell formation during B-cell terminal differentiation.

High-precision rotation angle measurement method based on monocular vision.

To accurately measure the attitude angles (pitch, roll, and yaw) of a rigid object that rotates in a space, we propose a high-precision rotation angle measurement method based on monocular vision. This method combines camera self-calibration, multiview geometry, and 3D measurement. This monocular vision measuring system consists of an area scan CCD, a prime lens, and a spots array target, which are fixed on the measured object. We can calculate the rotation angle according to the rebuilt rotating spots array target by using this monocular vision measuring system. The measurement precision of rotation angle can reach 1 arc sec in this paper's experiments. This method has high measurement precision and good stability. Therefore we can widely use this method in machinery manufacturing, engineering measurement, aerospace, and the military.

Tumor-associated NK cells drive MDSC-mediated tumor immune tolerance through the IL-6/STAT3 axis.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell-to-myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell-derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m-knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell-mediated mechanism that drives the development of MDSCs during tumor immune escape.

Identification of HEXIM1 as a positive regulator of p53.

Hexamethylene bisacetamide-inducible protein 1 (HEXIM1) is best known as the inhibitor of positive transcription elongation factor b (P-TEFb), which regulates the transcription elongation of RNA polymerase II and controls 60-70% of mRNA synthesis. Our previous studies show that HEXIM1 interacts with two key p53 regulators, nucleophosmin and human double minute-2 protein (HDM2), implying a possible connection between HEXIM1 and the p53 signaling pathway. Here we report the interaction between p53 and HEXIM1 in breast cancer, acute myeloid leukemia, and colorectal carcinoma cells. The C-terminal regions of p53 and HEXIM1 are required for the protein-protein interaction. Overexpression of HEXIM1 prevents the ubiquitination of p53 by HDM2 and enhances the protein stability of p53, resulting in up-regulation of p53 target genes, such as Puma and p21. Induction of p53 can be achieved by several means, such as UV radiation and treatment with anti-cancer agents (including doxorubicin, etoposide, roscovitine, flavopiridol, and nutlin-3). Under all the conditions examined, elevated protein levels of p53 are found to associate with the increased p53-HEXIM1 interaction. In addition, knockdown of HEXIM1 significantly inhibits the induction of p53 and releases the cell cycle arrest caused by p53. Finally, the transcription of the p53 target genes is regulated by HEXIM1 in a p53-dependent fashion. Our results not only identify HEXIM1 as a positive regulator of p53, but also propose a novel molecular mechanism of p53 activation caused by the anti-cancer drugs and compounds.

Alpha-synuclein promotes early neurite outgrowth in cultured primary neurons.

We previously showed that alpha-synuclein (α-Syn), a protein implicated in the pathogenesis of several neurodegenerative diseases, is a microtubule-associated protein (MAP), facilitating the polymerization of tubulin into microtubules. Therefore, we hypothesized that α-Syn might promote neurite outgrowth, a process that requires microtubule assembly. To test this hypothesis, recombinant human wild type (WT) and mutant (A30P and A53T) α-Syn proteins were added to cultured primary rat cortical neurons, and their effects on early neurite outgrowth were observed. The WT and mutant α-Syn proteins entered the neurons after 1-4 h of incubation. However, a significant increase in neurite outgrowth was observed only in neurons treated with WT α-Syn. MES23.5 dopaminergic neuronal cells overexpressing WT α-Syn also exhibited enhanced neurite outgrowth, indicating that the ability of α-Syn to promote neurite outgrowth was not due to a direct action on the cell membrane or by the membrane translocation process. Co-immunoprecipitation demonstrated that the recombinant human α-Syn was bound to tubulin. In addition, the α-Syn-treated neurons displayed increased levels of polymerized tubulin. Because α-Syn's MAP functionality is mediated by specific domains, we generated N-terminal (a.a. 1-65), non-amyloid-ß (non-Aß) component (NAC) (a.a. 61-95) and C-terminal (a.a. 96-140) fragments and added them to the primary neurons. After 1-4 h of incubation, the various α-Syn fragments had entered the neurons. However, only the NAC and C-terminal fragments, which have been previously shown to mediate MAP functionality, promoted neurite outgrowth. These results suggest that α-Syn promotes neurite outgrowth by facilitating the polymerization of tubulin into microtubules.