C16orf72/HAPSTR1 is a molecular rheostat in an integrated network of stress response pathways.

All cells contain specialized signaling pathways that enable adaptation to specific molecular stressors. Yet, whether these pathways are centrally regulated in complex physiological stress states remains unclear. Using genome-scale fitness screening data, we quantified the stress phenotype of 739 cancer cell lines, each representing a unique combination of intrinsic tumor stresses. Integrating dependency and stress perturbation transcriptomic data, we illuminated a network of genes with vital functions spanning diverse stress contexts. Analyses for central regulators of this network nominated C16orf72/HAPSTR1, an evolutionarily ancient gene critical for the fitness of cells reliant on multiple stress response pathways. We found that HAPSTR1 plays a pleiotropic role in cellular stress signaling, functioning to titrate various specialized cell-autonomous and paracrine stress response programs. This function, while dispensable to unstressed cells and nematodes, is essential for resilience in the presence of stressors ranging from DNA damage to starvation and proteotoxicity. Mechanistically, diverse stresses induce HAPSTR1, which encodes a protein expressed as two equally abundant isoforms. Perfectly conserved residues in a domain shared between HAPSTR1 isoforms mediate oligomerization and binding to the ubiquitin ligase HUWE1. We show that HUWE1 is a required cofactor for HAPSTR1 to control stress signaling and that, in turn, HUWE1 feeds back to ubiquitinate and destabilize HAPSTR1. Altogether, we propose that HAPSTR1 is a central rheostat in a network of pathways responsible for cellular adaptability, the modulation of which may have broad utility in human disease.

Engineering a Hyperstable Yersinia pestis Outer Membrane Protein Ail Using Thermodynamic Design.

Development of viable therapeutics to effectively combat tier I pneumopathogens such as Yersinia pestis requires a thorough understanding of proteins vital for pathogenicity. The host invasion protein Ail, although indispensable for Yersinia pathogenesis, has evaded detailed characterization, as it is an outer membrane protein with intrinsically low stability and high aggregation propensity. Here, we identify molecular elements of the metastable Ail structure that considerably alter protein-lipid and intraprotein thermodynamics. In addition, we find that four residues Q50, L88, L92, and A94 contribute additively to the lowered stability of Ail, and their conserved substitution is sufficient to re-engineer Ail to Out14, a thermodynamically hyperstable low-aggregation variant with a functional scaffold. Interestingly, Ail also shows two (parallel) folding pathways, which has not yet been reported for ß-barrel membrane proteins. Additionally, we identify the molecular mechanism of enhanced thermodynamic stability of Out14. We show that this enhanced stability of Out14 is due to a favorable change in the nonpolar accessible surface, and the accumulation of a kinetically accelerated off-pathway folding intermediate, which is absent in wild-type Ail. Such engineered hyperstable Ail ß-barrels can be harnessed for targeted drug screening and developing medical countermeasures against Yersiniae. Application of similar strategies will help design effective translational therapeutics to combat biopathogens.

Rate and grade transition control using PI controller based supervisory and regulatory layers: Diacetone alcohol process case study.

Product rate and grade transitions are two significant operations undertaken by process plants to operate in uncertain environments. Rate transitions, typically dictated by economic considerations, are often implemented in open loop via a throughput manipulator (TPM). The absence of feedback of product rate to manipulate the TPM results in an offset during rate/grade transitions as well as in presence of feed-side disturbances. In this work, we explore use of a simple PI controller based supervisory control layer that guides the plantwide regulatory layer to achieve product rate targets during grade transition or in the presence of feed grade disturbances. While such a layer cannot incorporate optimal operation and constraint handling explicitly as in model predictive control, it obviates the need for advanced process control elements that medium-to-small scale industries can ill-afford. The PI based supervisory-regulatory control structure is illustrated using simulated case studies on a prototypical process for diacetone alcohol production comprising of a reactor followed by two distillation columns and a recycle.

UBR7 acts as a histone chaperone for post-nucleosomal histone H3.

Histone chaperones modulate the stability of histones beginning from histone synthesis, through incorporation into DNA, and during recycling during transcription and replication. Following histone removal from DNA, chaperones regulate histone storage and degradation. Here, we demonstrate that UBR7 is a histone H3.1 chaperone that modulates the supply of pre-existing post-nucleosomal histone complexes. We demonstrate that UBR7 binds to post-nucleosomal H3K4me3 and H3K9me3 histones via its UBR box and PHD. UBR7 binds to the non-nucleosomal histone chaperone NASP. In the absence of UBR7, the pool of NASP-bound post-nucleosomal histones accumulate and chromatin is depleted of H3K4me3-modified histones. We propose that the interaction of UBR7 with NASP and histones opposes the histone storage functions of NASP and that UBR7 promotes reincorporation of post-nucleosomal H3 complexes.

NOTCH1-driven UBR7 stimulates nucleotide biosynthesis to promote T cell acute lymphoblastic leukemia.

Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) is the most divergent member of UBR box-containing E3 ubiquitin ligases/recognins that mediate the proteasomal degradation of its substrates through the N-end rule. Here, we used a proteomic approach and found phosphoribosyl pyrophosphate synthetases (PRPSs), the essential enzymes for nucleotide biosynthesis, as strong interacting partners of UBR7. UBR7 stabilizes PRPS catalytic subunits by mediating the polyubiquitination-directed degradation of PRPS-associated protein (PRPSAP), the negative regulator of PRPS. Loss of UBR7 leads to nucleotide biosynthesis defects. We define UBR7 as a transcriptional target of NOTCH1 and show that UBR7 is overexpressed in NOTCH1-driven T cell acute lymphoblastic leukemia (T-ALL). Impaired nucleotide biosynthesis caused by UBR7 depletion was concomitant with the attenuated cell proliferation and oncogenic potential of T-ALL. Collectively, these results establish UBR7 as a critical regulator of nucleotide metabolism through the regulation of the PRPS enzyme complex and uncover a metabolic vulnerability in NOTCH1-driven T-ALL.

Performance assessment of pulsating floc blanket clarifiers and conventional clariflocculators in pilot-scale models.

Continuous upflow pilot plants based on conventional clariflocculation (CC) and pulsating floc blanket clarification (PFBC) technologies were designed and fabricated for a capacity to treat about 8,000 L/day, to understand the fundamental differences in their functioning and assess their relative performance, especially under low turbidity conditions. Influent turbidity varying from 2 to 10 NTU was treated using coagulant alum, and efficiency of CC and PFBC in terms of average turbidity removal was found to be 23% and 48%, respectively. On observing this vast difference, it was postulated that total residual aluminum should also be lower in water treated from PFBC. Experiments and MLR analysis confirmed the hypothesis, with residual aluminum ranging from 0.055 to 0.040 mg/L and 0.036 mg/L to below detectable levels for CC and PFBC, respectively. These findings are of high significance, since minimization of residual aluminum in drinking water is a priority of WHO due to its reported neurotoxicity and can be complied with simple replacement of CC with PFBC. PRACTITIONER POINTS: Pulsating floc blanket clarifier (PFBC) performed better than conventional clariflocculator (CC) in terms of turbidity removal. Pulsating floc blanket allowed more effective utilization of coagulant alum, resulting in significantly lower residual aluminum in clarified water. Turbidity levels of influent and effluent are related to residual aluminum in treated water. PFBCs are more compact and modular, and can facilitate a good alternative to CCs.

Evolutionary selection of a 19-stranded mitochondrial ß-barrel scaffold bears structural and functional significance.

Transmembrane ß-barrels of eukaryotic outer mitochondrial membranes (OMMs) are major channels of communication between the cytosol and mitochondria and are indispensable for cellular homeostasis. A structurally intriguing exception to all known transmembrane ß-barrels is the unique odd-stranded, i.e. 19-stranded, structures found solely in the OMM. The molecular origins of this 19-stranded structure and its associated functional significance are unclear. In humans, the most abundant OMM transporter is the voltage-dependent anion channel. Here, using the human voltage-dependent anion channel as our template scaffold, we designed and engineered odd- and even-stranded structures of smaller (V216, V217, V218) and larger (V220, V221) barrel diameters. Determination of the structure, dynamics, and energetics of these engineered structures in bilayer membranes reveals that the 19-stranded barrel surprisingly holds modest to low stability in a lipid-dependent manner. However, we demonstrate that this structurally metastable protein possesses superior voltage-gated channel regulation, efficient mitochondrial targeting, and in vivo cell survival, with lipid-modulated stability, all of which supersede the occurrence of a metastable 19-stranded scaffold. We propose that the unique structural adaptation of these transmembrane transporters exclusively in mitochondria bears strong evolutionary basis and is functionally significant for homeostasis.

Shape memory alloy actuation of non-bonded piezo sensor configuration for bone diagnosis and impedance based analysis.

In the recent years, there has been a growing interest in research community towards the application of smart materials for bio-medical structural health monitoring. Amongst the smart materials, directly bonded piezo sensors (DBPS), based on the electro-mechanical impedance (EMI) technique, have been successfully employed for the above purpose. The principle behind the EMI technique is that high frequency excitations (typically > 30 kHz) generated by a surface bonded PZT patch are used to detect changes in structural drive point impedance caused by cracks or any other type of damage. Bone healing and damage have been shown to be successfully monitored using the DBPS. However, in most of the diagnostic cases of live human and animal subjects, directly bonding a PZT patch is always an irritant or hazard for a live subject. To circumvent direct bonding, the authors have developed and experimentally demonstrated a non-bonded piezo sensor (NBPS) configuration as a good alternative to DBPS while maintaining the effectiveness of measurement well within discernible limits. This paper presents further improvement in the NBPS configuration aiming at autonomous operation of the gripping mechanism using shape memory alloy (SMA) wires. The experiments are performed on replicas of femur bone in healthy and osteoporosis state. This paper shows the effective use of SMA clamping for bone identification and its damage assessment in comparison to earlier mechanical gripping using jubilee clamps. This paper also covers impedance based identification applied to SMA and clamp based NBPS configurations. In place of raw admittance signatures, effective drive point impedance is utilized for the purpose of bone diagnostics which provides a more realistic assessment of the condition of bone.

Numerical evaluation of nonbonded piezo sensor for biomedical diagnostics using electromechanical impedance technique.

Directly bonded piezo sensor, conventionally employed in the electromechanical impedance (EMI) technique, although a proven candidate for structural health monitoring, is severely constrained in its application in the biomedical field due to its bonding requirement. In contrast, nonbonded piezo sensor (NBPS) provides a viable platform to assess the condition of human bones, tissues, and other biomedical subjects using the EMI technique without inflicting pain or irritation to the skin. The name NBPS was coined to emphasize that there was no direct bonding between the PZT patch and the live subject; instead, the PZT patch was bonded to a supporting medium, which maintains the mechanical interaction between the PZT patch and the subject. However, there are several aspects in the analysis of NBPS configuration that cannot be addressed completely through experimental study due to measurement constraints, cost, and time. For example, experimentally changing the density of bone continuously to study the osteoporosis effect is a tedious task warranting large number of specimens. This paper presents a detailed parametric study based on finite element method covering condition monitoring of human bones using the NBPS configuration. It is for the first time that 3D analysis for specimen identification and damage detection in bones using NBPS has been carried out. In addition to the validation of the numerical model against the previously established experimental studies involving bones, quantification of the extent of damage and its localization has been investigated. The density changes due to osteoporosis in bones are comprehensively investigated by the NBPS including the quantification aspect of osteoporosis/damage. Definite acquisition of bone signature and detection of physiological changes in bones are achieved even with the presence of skin, muscle, and fat layers on the bone.

Hydrophobic Mismatch Modulates Stability and Plasticity of Human Mitochondrial VDAC2.

The human mitochondrial outer membrane protein voltage-dependent anion channel isoform 2 (hVDAC2) is a ß-barrel metabolite flux channel that is indispensable for cell survival. It is well established that physical forces imposed on a transmembrane protein by its surrounding lipid environment decide protein structure and stability. Yet, how the mitochondrial membrane and protein-lipid interplay together regulate hVDAC2 stability is unknown. Here, we combine experimental biophysical investigations of protein stability with all-atom molecular dynamics simulations to study the effect of the most abundant mitochondrial phosphocholine (PC) lipids on hVDAC2. We demonstrate experimentally that increasing the PC lipid acyl chain length from diC14:0 to diC18:0-PC has a nonlinear effect on the ß-barrel. We show that protein stability is highest in diC16:0-PC, which exhibits a negative mismatch with the hVDAC2 barrel. Our simulations also reveal that structural rigidity of hVDAC2 is highest under optimal negative mismatch provided by diC16:0-PC bilayers. Further, we validate our observations by altering the physical properties of PC membranes indirectly using cholesterol. We propose that VDAC plasticity and stability in the mitochondrial outer membrane are modulated by physical properties of the bilayer.

Inheritance of CENP-A Nucleosomes during DNA Replication Requires HJURP.

Centromeric chromatin defines the site of kinetochore formation and ensures faithful chromosome segregation. Centromeric identity is epigenetically specified by the incorporation of CENP-A nucleosomes. DNA replication presents a challenge for inheritance of centromeric identity because nucleosomes are removed to allow for replication fork progression. Despite this challenge, CENP-A nucleosomes are stably retained through S phase. We used BioID to identify proteins transiently associated with CENP-A during DNA replication. We found that during S phase, HJURP transiently associates with centromeres and binds to pre-existing CENP-A, suggesting a distinct role for HJURP in CENP-A retention. We demonstrate that HJURP is required for centromeric nucleosome inheritance during S phase. HJURP co-purifies with the MCM2-7 helicase complex and, together with the MCM2 subunit, binds CENP-A simultaneously. Therefore, pre-existing CENP-A nucleosomes require an S phase function of the HJURP chaperone and interaction with MCM2 to ensure faithful inheritance of centromere identity through DNA replication.

Posttranslational mechanisms controlling centromere function and assembly.

Accurate chromosome segregation is critical to ensure the faithful inheritance of the genome during cell division. Human chromosomes distinguish the location of the centromere from general chromatin by the selective assembly of CENP-A containing nucleosomes at the active centromere. The location of centromeres in most higher eukaryotes is determined epigenetically, independent of DNA sequence. CENP-A containing centromeric chromatin provides the foundation for assembly of the kinetochore that mediates chromosome attachment to the microtubule spindle and controls cell cycle progression in mitosis. Here we review recent work demonstrating the role of posttranslational modifications on centromere function and CENP-A inheritance via the direct modification of the CENP-A nucleosome and pre-nucleosomal complexes, the modification of the CENP-A deposition machinery and the modification of histones within existing centromeres.

Posttranslational modifications of CENP-A: marks of distinction.

Centromeres are specialized chromosome domain that serve as the site for kinetochore assembly and microtubule attachment during cell division, to ensure proper segregation of chromosomes. In higher eukaryotes, the identity of active centromeres is marked by the presence of CENP-A (centromeric protein-A), a histone H3 variant. CENP-A forms a centromere-specific nucleosome that acts as a foundation for centromere assembly and function. The posttranslational modification (PTM) of histone proteins is a major mechanism regulating the function of chromatin. While a few CENP-A site-specific modifications are shared with histone H3, the majority are specific to CENP-A-containing nucleosomes, indicating that modification of these residues contribute to centromere-specific function. CENP-A undergoes posttranslational modifications including phosphorylation, acetylation, methylation, and ubiquitylation. Work from many laboratories have uncovered the importance of these CENP-A modifications in its deposition at centromeres, protein stability, and recruitment of the CCAN (constitutive centromere-associated network). Here, we discuss the PTMs of CENP-A and their biological relevance.

Epidermal Growth Factor Receptor activation promotes ADA3 acetylation through the AKT-p300 pathway.

The ADA3 (Alteration/Deficiency in Activation 3) protein is an essential adaptor component of several Lysine Acetyltransferase (KAT) complexes involved in chromatin modifications. Previously, we and others have demonstrated a crucial role of ADA3 in cell cycle progression and in maintenance of genomic stability. Recently, we have shown that acetylation of ADA3 is key to its role in cell cycle progression. Here, we demonstrate that AKT activation downstream of Epidermal Growth Factor Receptor (EGFR) family proteins stimulation leads to phosphorylation of p300, which in turn promotes the acetylation of ADA3. Inhibition of upstream receptor tyrosine kinases (RTKs), HER1 (EGFR)/HER2 by lapatinib and the accompanying reduction of phospho-AKT levels led to a decrease in p300 phosphorylation and ADA3 protein levels. The p300/PCAF inhibitor garcinol also destabilized the ADA3 protein in a proteasome-dependent manner and an ADA3 mutant with K→R mutations exhibited a marked increase in half-life, consistent with opposite role of acetylation and ubiquitination of ADA3 on shared lysine residues. ADA3 knockdown led to cell cycle inhibitory effects, as well as apoptosis similar to those induced by lapatinib treatment of HER2+ breast cancer cells, as seen by accumulation of CDK inhibitor p27, reduction in mitotic marker pH3(S10), and a decrease in the S-phase marker PCNA, as well as the appearance of cleaved PARP. Taken together our results reveal a novel RTK-AKT-p300-ADA3 signaling pathway involved in growth factor-induced cell cycle progression.

ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC.

BACKGROUND: We have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized. METHODS: We overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients. RESULTS: Overexpression of ADA3 in ER- hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 and c-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients' outcome was independent of tumor grade, stage and size, and ER status. CONCLUSION: ADA3 overexpression enhances cell proliferation that is associated with increased expression of c-MYC. Expression patterns with respect to ADA3/c-MYC can divide patients into four significantly different subgroups, with c-MYC High and ADA3 Low status independently predicting poor survival in patients.

Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation.

Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation.

A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function.

Alteration/Deficiency in Activation 3 (ADA3) Protein, a Cell Cycle Regulator, Associates with the Centromere through CENP-B and Regulates Chromosome Segregation.

ADA3 (alteration/deficiency in activation 3) is a conserved component of several transcriptional co-activator and histone acetyltransferase (HAT) complexes. Recently, we generated Ada3 knock-out mice and demonstrated that deletion of Ada3 leads to early embryonic lethality. The use of Ada3(FL/FL) mouse embryonic fibroblasts with deletion of Ada3 using adenovirus Cre showed a critical role of ADA3 in cell cycle progression through mitosis. Here, we demonstrate an association of ADA3 with the higher order repeat region of the α-satellite region on human X chromosome centromeres that is consistent with its role in mitosis. Given the role of centromere proteins (CENPs) in mitosis, we next analyzed whether ADA3 associates with the centromere through CENPs. Both an in vivo proximity ligation assay and immunofluorescence studies confirmed the association of ADA3 with CENP-B protein, a highly conserved centromeric protein that binds to the 17-bp DNA sequences on α-satellite DNA. Deletional analysis showed that ADA3 directly associates with CENP-B through its N terminus, and a CENP-B binding-deficient mutant of ADA3 was incompetent in cell proliferation rescue. Notably, knockdown of ADA3 decreased binding of CENP-B onto the centromeres, suggesting that ADA3 is required for the loading of CENP-B onto the centromeres. Finally, we show that deletion of Ada3 from Ada3(FL/FL) mouse embryonic fibroblasts exhibited various chromosome segregation defects. Taken together, we demonstrate a novel ADA3 interaction with CENP-B-centromere that may account for its previously known function in mitosis. This study, together with its known function in maintaining genomic stability and its mislocalization in cancers, suggests an important role of ADA3 in mitosis.