Quantitative description of the phase-separation behavior of the multivalent SLP65-CIN85 complex.

Biomolecular condensates play a major role in cell compartmentalization, besides membrane-enclosed organelles. The multivalent SLP65 and CIN85 proteins are proximal B-cell antigen receptor (BCR) signal effectors and critical for proper immune responses. In association with intracellular vesicles, the two effector proteins form phase separated condensates prior to antigen stimulation, thereby preparing B lymphocytes for rapid and effective activation upon BCR ligation. Within this tripartite system, 6 proline-rich motifs (PRMs) of SLP65 interact promiscuously with 3 SH3 domains of the CIN85 monomer, establishing 18 individual SH3-PRM interactions whose individual dissociation constants we determined. Based on these 18 dissociation constants, we measured the phase-separation properties of the natural SLP65/CIN85 system as well as designer constructs that emphasize the strongest SH3/PRM interactions. By modeling these various SLP65/CIN85 constructs with the program LASSI (LAttice simulation engine for Sticker and Spacer Interactions), we reproduced the observed phase-separation properties. In addition, LASSI revealed a deviation in the experimental measurement, which was independently identified as a previously unknown intramolecular interaction. Thus, thermodynamic properties of the individual PRM/SH3 interactions allow us to model the phase-separation behavior of the SLP65/CIN85 system faithfully.

Clinical Outcomes of 111 Patients with Early Onset Idiopathic Scoliosis (EOIS) Receiving Brace Treatment: A Longitudinal Retrospective Cohort Study.

Introduction: Bracing is one of the first-line treatment for early-onset idiopathic scoliosis (EOIS) to control curves from progression. This study aimed to explore the determinants that govern bracing effectiveness in EOIS. Methods: One hundred and eleven patients with EOIS (mean age of 8.6 ± 1.25 at diagnosis) received bracing treatment and had a final follow-up beyond skeletal maturity were identified from records between 1988 and 2021. Demographic data and clinical features of spinal curvature were obtained for correlation analyses to determine the associations between curve outcomes and clinical features. Results: Most patients were female (85.6%) and had a major curve on the left side (67%). The mean baseline Cobb angle of major curves was 21.73 ± 7.92°, with a mean Cobb angle progression of 18.05 ± 19.11°. The average bracing duration was 5.3 ± 1.9 years. Only 26 (23.4%) of them underwent surgery. The final Cobb angle and curve progression at the final follow-up with a Cobb angle of ≥50° were positively correlated with the initial Cobb angle (r = 0.206 and r = 0.313, respectively) and negatively correlated with maturity parameters. The lumbar curve type was found to correlate with a smaller final Cobb angle. Conclusions: The majority of patients had a final Cobb angle < 50°, which was considered a successful bracing outcome. The final Cobb angle correlated with the initial Cobb angle and curve types observed in EOIS.

The G285S mutation in nsP1 is sufficient to render Sindbis virus as a stable vector for gene delivery.

Neuroscience, gene therapy, and vaccine have all benefited from the increased use of viral vectors. Sindbis virus (SINV) is a notable candidate among these vectors. However, viral vectors commonly suffer from a loss of expression of the transgene, especially RNA viral vectors. In this study, we used a directed evolution approach by continuous passage of selection to identify adaptive mutations that help SINV to stably express exogenous genes. As a result, we found two adaptive mutations that are located at aa 285 (G to S) of nsP1 and aa 422 (D to G) of nsP2, respectively. Further study showed that G285S was sufficient for SINV to stabilize the expression of the inserted gene, while D422G was not. Combined with AlphaFold2 and sequence alignment with the genus Alphavirus, we found that G285S is conserved. Based on this mutation, we constructed a new vector for the applications in neural circuits mapping. Our results indicated that the mutant SINV maintained its anterograde transsynaptic transmission property. In addition, when the transgene was replaced by another gene, granulocyte-macrophage colony-stimulating factor (GM-CSF), the vector still showed stable expression of the inserted gene. Hence, using SINV as an example, we have demonstrated an efficient approach to greatly augment the gene delivery capacity of viral vectors, which will be useful to neuroscience and oncolytic therapy.

Clean clothes or dirty clothes? Outbreak investigation of carbapenem-resistant Acinetobacter baumannii related to laundry contamination through multilocus sequence typing (MLST).

OBJECTIVE: To investigate the source in an outbreak of carbapenem-resistant Acinetobacter baumannii (CRA) in a general hospital due to contamination of a laundry evaporative cooler and the laundry environment using multilocus sequence typing (MLST). METHODS: For CRA culture, clinical samples were collected from infected patients and close contacts, and environmental sampling was performed in patient surroundings and laundry facilities. MLST was used for the molecular typing of representative CRA isolates. Bacterial isolates with identical sequence types were considered epidemiologically linked and attributable to the same source. OXA genes in Acinetobacter baumannii were detected using polymerase chain reaction (PCR). RESULTS: In total, 58 patients were affected in this outbreak. The mean patient age was 75.3, and 50% were female. The most common diagnoses at admission were skin and soft-tissue infection (n = 12, 20.7%) and pneumonia (n = 12, 20.7%). OXA-23 was positive in 64.7% of isolates. A CRA isolate from the evaporative cooler in the laundry was identical to that of 11 patients across 3 wards, belonging to ST345. Isolates from 3 laundry linen racks were identical to those of 7 patients from 3 wards, classified as ST1145. Isolates found on another linen rack and a pajama shelf were identical to isolates from 3 other patients from 2 wards, belonging to ST2207. There was no significant difference between sequence type distributions of clinical and environmental isolates (P = .12), indicating high likelihood of CRA originating from the same source. CONCLUSIONS: MLST confirmed that contamination of the laundry evaporative cooler and surrounding environment caused a polyclonal CRA hospital outbreak. Hospital laundry is an important area for infection control and outbreak investigations of CRA.

A methyl-TROSY approach for NMR studies of high-molecular-weight DNA with application to the nucleosome core particle.

The development of methyl-transverse relaxation-optimized spectroscopy (methyl-TROSY)-based NMR methods, in concert with robust strategies for incorporation of methyl-group probes of structure and dynamics into the protein of interest, has facilitated quantitative studies of high-molecular-weight protein complexes. Here we develop a one-pot in vitro reaction for producing NMR quantities of methyl-labeled DNA at the C5 and N6 positions of cytosine (5mC) and adenine (6mA) nucleobases, respectively, enabling the study of high-molecular-weight DNA molecules using TROSY approaches originally developed for protein applications. Our biosynthetic strategy exploits the large number of naturally available methyltransferases to specifically methylate DNA at a desired number of sites that serve as probes of structure and dynamics. We illustrate the methodology with studies of the 153-base pair Widom DNA molecule that is simultaneously methyl-labeled at five sites, showing that high-quality 13C-1H spectra can be recorded on 100 µM samples in a few minutes. NMR spin relaxation studies of labeled methyl groups in both DNA and the H2B histone protein component of the 200-kDa nucleosome core particle (NCP) establish that methyl groups at 5mC and 6mA positions are, in general, more rigid than Ile, Leu, and Val methyl probes in protein side chains. Studies focusing on histone H2B of NCPs wrapped with either wild-type DNA or DNA methylated at all 26 CpG sites highlight the utility of NMR in investigating the structural dynamics of the NCP and how its histone core is affected through DNA methylation, an important regulator of transcription.

Confronting the Invisible: Assignment of Protein 1HN Chemical Shifts in Cases of Extreme Broadening.

NMR studies of intrinsically disordered proteins (IDPs) at neutral pH values are hampered by the rapid exchange of backbone amide protons with solvent. Although exchange rates can be modulated by changes in pH, interactions between IDPs that lead to phase separation sometimes only occur at neutral pH values or higher, where backbone amide-based experiments fail. Here we describe a simple NMR experiment for measuring amide proton chemical shifts in cases where 1HN spectra cannot be obtained. The approach uses a weak 1H B1 field, searching for elusive 1HN resonance frequencies that become encoded in the intensities of cross-peaks in three-dimensional 1Hα-detect spectra. Applications to the CAPRIN1 protein in both dilute- and phase-separated states highlight the utility of the method, establishing that accurate 1HN chemical shifts can be obtained even in cases where solvent hydrogen exchange rates are on the order of 1500 s-1.

Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness.

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers.

NMR Experiments for Studies of Dilute and Condensed Protein Phases: Application to the Phase-Separating Protein CAPRIN1.

Intrinsically disordered proteins (IDPs) or regions of intrinsic disorder in otherwise folded proteins (IDRs) play important roles in many different biological processes, including formation of biological condensates via liquid-liquid phase separation. NMR spectroscopy is a powerful tool for obtaining site-specific structural and dynamical information on IDPs/IDRs, and recent efforts have focused on the development of experiments for atomic-resolution studies of these molecules. These include triple-resonance experiments that are based on 13CO-direct detection of magnetization, exploiting increased sensitivity of cryogenically cooled probes. In order to evaluate the different classes of experiment for studies of IDRs or IDPs in both dilute and phase-separated environments, in particular at neutral and higher pHs where many of these proteins phase separate, we compared 13CO-detect versus 1Hα-detect experiments, showing that significant sensitivity gains are achieved via proton detection under the conditions of our experiments. A suite of 1Hα-detect experiments was subsequently developed for studies of IDPs/IDRs and applied to the dilute phase of a 103-residue disordered region of CAPRIN1 that phase separates at neutral pH. Residue-specific chemical shifts derived from our study enable the accurate prediction of the importance of the N-terminal Arg-containing region of this construct for promoting phase separation relative to other Arg-rich stretches of sequence, subsequently confirmed by mutagenesis. Our study emphasizes that the sequence positions of key residues can be a critical factor in controlling phase separation and highlights the unique role of NMR in establishing the relations between amino acid sequence and phase-separation propensity.

Eliminating Structural Barriers: The Impact of Unrestricted Access on Hepatitis C Treatment Uptake Among People Living With Human Immunodeficiency Virus.

BACKGROUND: High costs of direct-acting antivirals (DAAs) have led health-care insurers to limit access worldwide. Using a natural experiment, we evaluated the impact of removing fibrosis stage restrictions on hepatitis C (HCV) treatment initiation rates among people living with human immunodeficiency virus (HIV), and then examined who was left to be treated. METHODS: Using data from the Canadian HIV-HCV Coinfection Cohort, we applied a difference-in-differences approach. Changes in treatment initiation rates following the removal of fibrosis stage restrictions were assessed using a negative binomial regression with generalized estimating equations. The policy change was then specifically assessed among people who inject drugs (PWID). We then identified the characteristics of participants who remained to be treated using a modified Poisson regression. RESULTS: Between 2010-2018, there were a total of 585 HCV initiations among 1130 eligible participants. After removing fibrosis stage restrictions, DAA initiations increased by 1.8-fold (95% confidence interval [CI] 1.3-2.4) controlling for time-invariant differences and secular trends. Among PWID the impact appeared even stronger, with an adjusted incidence rate ratio of 3.6 (95% CI 1.8-7.4). However, this increased treatment uptake was not sustained. At 1 year following universal access, treatment rates declined to 0.8 (95% CI .5-1.1). Marginalized participants (PWID and those of indigenous ethnicity) and those disengaged from care were more likely to remain HCV RNA positive. CONCLUSIONS: After the removal of fibrosis restrictions, HCV treatment initiations nearly doubled immediately, but this treatment rate was not sustained. To meet the World Health Organization elimination targets, the minimization of structural barriers and adoption of tailored interventions are needed to engage and treat all vulnerable populations.

Sensitivity-Enhanced Four-Dimensional Amide-Amide Correlation NMR Experiments for Sequential Assignment of Proline-Rich Disordered Proteins.

Proline is prevalent in intrinsically disordered proteins (IDPs). NMR assignment of proline-rich IDPs is a challenge due to low dispersion of chemical shifts. We propose here new sensitivity-enhanced 4D NMR experiments that correlate two pairs of amide resonances that are either consecutive (NH i-1, NH i) or flanking a proline at position i-1 (NH i-2, NH i). The maximum 2-fold enhancement of sensitivity is achieved by employing two coherence order-selective (COS) transfers incorporated unconventionally into the pulse sequence. Each COS transfer confers an enhancement over amplitude-modulated transfer by a factor of √2 specifically when transverse relaxation is slow. The experiments connect amide resonances over a long fragment of sequence interspersed with proline. When this method was applied to the proline-rich region of B cell adaptor protein SLP-65 (pH 6.0) and α-synuclein (pH 7.4), which contain a total of 52 and 5 prolines, respectively, 99% and 92% of their nonprolyl amide resonances have been successfully assigned, demonstrating its robustness to address the assignment problem in large proline-rich IDPs.

Patient profiles as organizing HIV clinicians' ART adherence management: a qualitative analysis.

The effectiveness of antiretroviral therapy (ART) depends on optimal clinical management and patient adherence. Little is known about patient characteristics that clinicians consider in the management of ART adherence. Exploring this issue, five focus groups were conducted with 31 HIV-clinicians from across France. A qualitative typological analysis suggests that clinician management of patient adherence is based on characteristics that coalesce into seven patient profiles. For the "passive" patient, described as taking ART exactly as prescribed without questioning their doctor's expertise, a directive and simple management style was preferred. The "misleading" patient is characterized as concerned with social desirability and as reporting no adherence difficulties for fear of displeasing their doctor. If clinical outcomes are suboptimal, the clinicians' strategy is to remind them of the importance of open patient-clinician communication. The "stoic" patient is described as requesting and adequately taking the most potent ART available. Here, clinicians emphasize assessment of side effects, which the patient may minimize. The "hedonistic" patient's festive lifestyle and sexual risk-taking are seen as compromising adherence; with them, clinicians stress the patient's responsibility for their own health and that of their sexual partners. The "obsessive" patient is portrayed as having an irrational fear of ART failure and an inability to distinguish illusory from genuine adherence barriers. With this patient, clinicians seek to identify the latter. The "overburdened" patient is recognized as coping with life priorities that interfere with adherence and, with them, a forgiving ART is favored. The "underprivileged" patient is presented as having limited education, income and housing. In this case, clinicians seek to improve the patient's living conditions and access to care. These results shed light on HIV clinicians' ART adherence management. The value of these profiles for HIV care and patients should be investigated.

Developing a patient-reported outcome measure for HIV care on perceived barriers to antiretroviral adherence: assessing the needs of HIV clinicians through qualitative analysis.

PURPOSE: To identify HIV clinicians' needs for the clinical use of a new patient-reported outcome measure (PRO) on barriers to antiretroviral therapy (ART) adherence. METHODS: In 2015, five focus groups with 31 clinicians from France were transcribed, coded with Atlas.ti, and submitted to a typological analysis. RESULTS: The analysis identified seven patient profiles, each tied to distinct barriers to adherence and to specific needs for the PRO's content, data collection and transmission. Clinicians preferred, for the patient who is: (1) 'passive,' that the PRO collect information on ART knowledge, to ensure that the prescription's instructions are being respected; (2) 'misleading,' that it be able to detect adherence to ART and socially desirable responses; (3) 'stoic,' that questions challenge the patient to recognize treatment-specific side effects; (4) 'hedonistic,' that the PRO contains content on lifestyle and risk-taking; (5) 'obsessive,' that the PRO captures quality of life and stressful life events; (6) 'overburdened,' that the PRO provides information on the person's home environment, socioeconomic status and cultural constraints. For all or most patient profiles, the clinicians wished that the PRO be completed, minimally, prior to the medical consultation and to receive alerts, under varying conditions, when problematic scores were detected. Depending on the profile, there was preference for the inclusion of open-ended questions and transmission of cross-sectional, periodic or longitudinal PRO data. CONCLUSION: Overall, this study's findings suggest that to support the clinical management of ART adherence, our PRO must meet the needs of a wide variety of patients and must perform multiple functions.

Cocaine/crack use is not associated with fibrosis progression measured by AST-to-Platelet Ratio Index in HIV-HCV co-infected patients: a cohort study.

BACKGROUND: Cocaine and crack use has been associated with HIV and HCV infections, but its consequences on HCV progression have not been well established. We analyzed the impact of cocaine/crack use on liver fibrosis progression in a cohort of HIV-HCV co-infected patients. METHODS: A Canadian multicenter prospective cohort study followed 1238 HIV-HCV co-infected persons every 6 months between 2003 and 2013. Data were analyzed from 573 patients with positive HCV RNA, not on HCV treatment, without significant liver fibrosis (AST-to-Platelet Ratio Index (APRI) <1.5) or history of end-stage liver disease at baseline, and having at least two study visits. Recent cocaine/crack use was defined as use within 6 months of cohort entry. Incidence rates of progression to significant fibrosis (APRI ≥ 1.5) were determined according to recent cocaine/crack use. Cox Proportional Hazards models were used to assess the association between time-updated cocaine/crack use and progression to APRI ≥ 1.5 adjusting for age, sex, HCV duration, baseline ln(APRI), and time-updated alcohol abuse, history of other drug use and CD4+ cell count. RESULTS: At baseline, 211 persons (37%) were recent cocaine/crack users and 501 (87%) ever used cocaine/crack. Recent users did not differ from non-recent users on gender, age, and CD4+ T-cell count. Over 1599 person-years of follow up (522 PY in recent users, 887 PY in previous users and 190 PY in never users),158 (28%) persons developed significant fibrosis (9.9/100 PY; 95% CI, 8.3-11.4); 56 (27%) recent users (10.7/100 PY; 7.9-13.5), 81 (28%) previous users (9.1/100 PY; 7.1-11.1), and 21 (29%) never users (11.1/100 PY; 6.3-15.8). There was no association between ever having used or time-updated cocaine/crack use and progression to APRI ≥ 1.5 (adjusted HR (95%CI): 0.96 (0.58, 1.57) and 0.88;(0.63-1.25), respectively). CONCLUSIONS: We could not find evidence that cocaine/crack use is associated with progression to advanced liver fibrosis in our prospective study of HIV-HCV co-infected patients.

PAK4 Phosphorylates p53 at Serine 215 to Promote Liver Cancer Metastasis.

PAK4 kinase contributes to signaling pathways controlling cancer cell transformation, invasion, and survival, but its clinicopathological impact has begun to emerge only recently. Here we report that PAK4 overexpression in hepatocellular carcinoma (HCC) conveys aggressive metastatic properties. A novel nuclear splice isoform of PAK4 lacking exon 2 sequences was isolated as part of our studies. By stably overexpressing or silencing PAK4 in HCC cells, we showed that it was critical for their migration. Mechanistic investigations in this setting revealed that PAK4 directly phosphorylated p53 at S215, which not only attenuated transcriptional transactivation activity but also inhibited p53-mediated suppression of HCC cell invasion. Taken together, our results showed how PAK4 overexpression in HCC promotes metastatic invasion by regulating p53 phosphorylation. Cancer Res; 76(19); 5732-42. ©2016 AACR.

The adaptor protein CIN85 assembles intracellular signaling clusters for B cell activation.

The adaptor molecule Cbl-interacting protein of 85 kD (CIN85) regulates signaling from a number of cell surface receptors, such as growth factor receptors and antigen receptors on lymphocytes. Because of its multidomain structure, CIN85 is thought to act as a classical adaptor protein that connects functionally distinct components of a given signaling pathway through diverse protein domains. However, we found that in B lymphocytes, CIN85 functions to oligomerize SLP-65, which is the central effector protein of the B cell receptor (BCR). Therefore, CIN85 trimerizes through a carboxyl-terminal, coiled-coil domain. The multiple Src homology 3 (SH3) domains of trimeric CIN85 molecules associated with multiple SLP-65 molecules, which recruited further CIN85 trimers, thereby perpetuating the oligomerization process. Formation of this oligomeric signaling complex in resting B cells rendered the cells poised for the efficient initiation of intracellular signaling upon BCR stimulation. Our data suggest that the functionality of signaling cascades does not rely solely on the qualitative linkage of their various components but requires a critical number of effectors to become concentrated in signaling complexes.

Structural characterization of eRF1 mutants indicate a complex mechanism of stop codon recognition.

Eukarya translation termination requires the stop codon recognizing protein eRF1. In contrast to the multiple proteins required for translation termination in Bacteria, eRF1 retains the ability to recognize all three of the stop codons. The details of the mechanism that eRF1 uses to recognize stop codons has remained elusive. This study describes the structural effects of mutations in the eRF1 N-domain that have previously been shown to alter stop codon recognition specificity. Here, we propose a model of eRF1 binding to the pre-translation termination ribosomal complex that is based in part on our solution NMR structures of the wild-type and mutant eRF1 N-domains. Since structural perturbations induced by these mutations were spread throughout the protein structure, residual dipolar coupling (RDC) data were recorded to establish the long-range effects of the specific mutations, E55Q, Y125F, Q(122)FM(Y)F(126). RDCs were recorded on (15)N-labeled eRF1 N-domain weakly aligned in either 5% w/v n-octyl-penta (ethylene glycol)/octanol (C8E5) or the filamentous phage Pf1. These data indicate that the mutations alter the conformation and dynamics of the GTS loop that is distant from the mutation sites. We propose that the GTS loop forms a switch that is key for the multiple codon recognition capability of eRF1.

Macromolecular assembly of the adaptor SLP-65 at intracellular vesicles in resting B cells.

The traditional view of how intracellular effector proteins are recruited to the B cell antigen receptor (BCR) complex at the plasma membrane is based on the occurrence of direct protein-protein interactions, as exemplified by the recruitment of the tyrosine kinase Syk (spleen tyrosine kinase) to phosphorylated motifs in BCR signaling subunits. By contrast, the subcellular targeting of the cytosolic adaptor protein SLP-65 (Src homology 2 domain-containing leukocyte adaptor protein of 65 kD), which serves as a proximal Syk substrate, is unclear. We showed that SLP-65 activation required its association at vesicular compartments in resting B cells. A module of ~50 amino acid residues located at the amino terminus of SLP-65 anchored SLP-65 to the vesicles. Nuclear magnetic resonance spectroscopy showed that the SLP-65 amino terminus was structurally disordered in solution but could bind in a structured manner to noncharged lipid components of cellular membranes. Our finding that preformed vesicular signaling scaffolds are required for B cell activation indicates that vesicles may deliver preassembled signaling cargo to sites of BCR activation.

IPA-3 inhibits the growth of liver cancer cells by suppressing PAK1 and NF-κB activation.

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the in vivo tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.

The two PPX-GppA homologues from Mycobacterium tuberculosis have distinct biochemical activities.

Inorganic polyphosphate (poly-P), guanosine pentaphosphate (pppGpp) and guanosine tetraphosphate (ppGpp) are ubiquitous in bacteria. These molecules play a variety of important physiological roles associated with stress resistance, persistence, and virulence. In the bacterial pathogen Mycobacterium tuberculosis, the identities of the proteins responsible for the metabolism of polyphosphate and (p)ppGpp remain to be fully established. M. tuberculosis encodes two PPX-GppA homologues, Rv0496 (MTB-PPX1) and Rv1026, which share significant sequence similarity with bacterial exopolyphosphatase (PPX) and guanosine pentaphosphate 5'-phosphohydrolase (GPP) proteins. Here we delineate the respective biochemical activities of the Rv0496 and Rv1026 proteins and benchmark these against the activities of the PPX and GPP proteins from Escherichia coli. We demonstrate that Rv0496 functions as an exopolyphosphatase, showing a distinct preference for relatively short-chain poly-P substrates. In contrast, Rv1026 has no detectable exopolyphosphatase activities. Analogous to the E. coli PPX and GPP enzymes, the exopolyphosphatase activities of Rv0496 are inhibited by pppGpp and, to a lesser extent, by ppGpp alarmones, which are produced during the bacterial stringent response. However, neither Rv0496 nor Rv1026 have the ability to hydrolyze pppGpp to ppGpp; a reaction catalyzed by E. coli PPX and GPP. Both the Rv0496 and Rv1026 proteins have modest ATPase and to a lesser extent ADPase activities. pppGpp alarmones inhibit the ATPase activities of Rv1026 and, to a lesser extent, the ATPase activities of Rv0496. We conclude that PPX-GppA family proteins may not possess all the catalytic activities implied by their name and may play distinct biochemical roles involved in polyphosphate and (p)ppGpp metabolic pathways.

AMPK promotes p53 acetylation via phosphorylation and inactivation of SIRT1 in liver cancer cells.

AMP-activated protein kinase (AMPK), a biologic sensor for cellular energy status, has been shown to act upstream and downstream of known tumor suppressors. However, whether AMPK itself plays a tumor suppressor role in cancer remains unclear. Here, we found that the α2 catalytic subunit isoform of AMPK is significantly downregulated in hepatocellular carcinoma (HCC). Clinicopathologic analysis revealed that underexpression of AMPK-α2 was statistically associated with an undifferentiated cellular phenotype and poor patient prognosis. Loss of AMPK-α2 in HCC cells rendered them more tumorigenic than control cells both in vitro and in vivo. Mechanistically, ectopic expression of AMPK enhanced the acetylation and stability of p53 in HCC cells. The p53 deacetylase, SIRT1, was phosphorylated and inactivated by AMPK at Thr344, promoting p53 acetylation and apoptosis of HCC cells. Taken together, our findings suggest that underexpression of AMPK is frequently observed in HCC, and that inactivation of AMPK promotes hepatocarcinogenesis by destabilizing p53 in a SIRT1-dependent manner.