Subclass Members Research Articles

USP35 mitigates endoplasmic reticulum stress-induced apoptosis by stabilizing RRBP1 in non-small cell lung cancer.

Deubiquitinating enzymes (DUBs) serve to maintain cellular homeostasis via protein ubiquitination and exert diverse regulatory functions in cancers and other diseases. Much progress has been made in characterizing biological roles of DUBs over the decades, yet the specific functions of many subclass members remain largely unexplored. It was not until recent years that the role of ubiquitin‐specific‐processing protease 35 (USP35) in cancers began to be understood. Here, we focus on delineating the roles and underlying mechanisms of USP35 in non‐small cell lung cancer (NSCLC). The isobaric tags for relative and absolute quantitation (iTRAQ) comparative proteomic approach were employed to identify differentially expressed proteins (DEPs) in H1299 cells induced by USP35 overexpression or silencing. Among the potential interactome of USP35, ribosome‐binding protein 1 (RRBP1), a membrane‐bound protein in endoplasmic reticulum (ER), captured our attentions. RRBP1 expression was found to positively correlate with USP35 levels in both genetically modified cells and human NSCLC tissues. Concordantly, both RRBP1 expression and USP35 expression were found to positively correlate with poor prognoses in lung adenocarcinoma patients. At the molecular level, USP35 was verified to directly interact with RRBP1 to prevent it from proteasomal‐dependent degradation. Functionally, USP35 alleviated ER stress‐induced cell apoptosis by stabilizing RRBP1 in NSCLC cells. Collectively, these findings indicate that USP35 plays a critical role in resisting ER stress‐induced cell death through deubiquitinating RRBP1, hence providing a rationale to target the USP35‐RRBP1 axis as an alternative therapeutic option for NSCLC.

Differential immune responses of immunoglobulin Z subclass members in antibacterial immunity in a zebrafish model.

Immunoglobulin Z (IgZ) or its equivalent immunoglobulin T (IgT) is a newly identified immunoglobulin (Ig) class from teleost fish. This Ig class is characterized by its involvement in mucosa-associated lymphoid tissues (MALTs) for mucosal defence against pathogen infection. Recently, several subclass members of IgZ/IgT, such as IgZ, IgZ2, Igτ1, Igτ2 and Igτ3, have been further identified from zebrafish, common carp and rainbow trout. However, the functional diversity and correlation among these subclasses remain uncertain. Here, we explored the differential immune reactions of the IgZ and IgZ2 subclasses in antibacterial immunity in a zebrafish model. IgZ was extensively distributed in the peripheral serum and skin/gill MALTs and showed a rapid induction upon bacterial infection. IgZ2 was specialized in skin/gill MALTs and showed a strong induction following IgZ production. Correspondingly, the IgZ+ B cells had a wider distribution in the systemic primary/secondary lymphoid tissues and MALTs than the IgZ2+ B cells, which were predominant in MALTs. IgZ and IgZ2 exhibited a complementary effect in antibacterial immunity by possessing differential abilities. That is, IgZ is preferentially involved in bactericidal reaction that is in part C1q-dependent, and IgZ2 participates in neutralization action through bacteria-coating activity. The production of IgZ largely depended on the αβ T/CD4+ T cells, whereas that of IgZ2 did not, suggesting the different dependencies of IgZ and IgZ2 on systemic immunity. Our findings demonstrate that the functional behaviour and mechanism of the IgZ/IgT family are more diverse than previously recognized and thus improve the current knowledge about this ancient Ig class.

Recent Developments in Cell Permeable Deubiquitinating Enzyme Activity-Based Probes.

Deubiquitinating enzymes (DUBs) function to remove or cleave ubiquitin from post-translationally modified protein substrates. There are about 100 known DUBs in the proteome, and their dysregulation has been implicated a number of disease states, but the specific function of many subclass members remains poorly understood. Activity-based probes (ABPs) react covalently with an active site residue to report on specific enzyme activity, and thus represent a powerful method to evaluate cellular and physiological enzyme function and dynamics. Ubiquitin-based ABPs, such as HA-Ub-VME, an epitope-tagged ubiquitin carrying a C-terminal reactive warhead, are the leading tool for “DUBome” activity profiling. However, these probes are generally cell membrane impermeable, limiting their use to isolated enzymes or lysates. Development of cell-permeable ABPs would allow engagement of DUB enzymes directly within the context of an intact live cell or organism, refining our understanding of physiological and pathological function, and greatly enhancing opportunities for translational research, including target engagement, imaging and biomarker discovery. This mini-review discusses recent developments in small molecule activity-based probes that target DUBs in live cells, and the unique applications of cell-permeable DUB activity-based probes vs. their traditional ubiquitin-based counterparts.

Normal and Aberrant NKL-Code in B-Cell Development and Lymphoma

Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation. Several members of the NKL subclass are deregulated in T-cell progenitors and support leukemogenesis. We have recently described particular expression patterns of nine NKL homeobox genes in early hematopoiesis and T-cell development. Here, we screened NKL homeobox gene activities in normal B-cell development and extended the NKL-code to include this lymphoid lineage. Analysis of public expression profiling datasets revealed that HHEX and NKX6-3 were the only members differentially active in naïve B-cells, germinal center B-cells, plasma cells and memory B-cells. Subsequent examination of different types of B-cell malignancies showed both aberrant overexpression of NKL-code members and ectopic activation of subclass members physiologically silent in lymphopoiesis including BARX2, DLX1, EMX2, NKX2-1, NKX2-2 and NKX3-2. Based on these findings we performed detailed studies of the B-cell specific NKL homeobox gene NKX6-3 which showed enhanced activity in patient subsets of follicular lymphoma, mantle cell lymphoma and diffuse large B-cell lymphoma (DLBCL), and in three DLBCL cell lines to serve as in vitro models. While excluding genomic and chromosomal rearrangements at the locus of NKX6-3 (8p11) promoter studies demonstrated that B-cell factors MYB and PAX5 activated NKX6-3 transcription. Furthermore, aberrant BMP7/SMAD1-signalling and deregulated expression of chromatin complex components AUTS2 and PCGF5 promoted NKX6-3 activation. Finally, NKL homeobox genes HHEX, HLX, MSX1 and NKX6-3 were expressed in B-cell progenitors and generated a regulatory gene network in cell lines which we propose may provide physiological support for NKL-code formation in early B-cell development. Together, we identified an NKL-code in B-cell development whose violation may deregulate differentiation and promote malignant transformation. DisclosuresNo relevant conflicts of interest to declare.

NKL homeobox gene activities in B-cell development and lymphomas.

Homeobox genes encode transcription factors which regulate basic processes in development and cell differentiation. Several members of the NKL subclass are deregulated in T-cell progenitors and support leukemogenesis. We have recently described particular expression patterns of nine NKL homeobox genes in early hematopoiesis and T-cell development. Here, we screened NKL homeobox gene activities in normal B-cell development and extended the NKL-code to include this lymphoid lineage. Analysis of public expression profiling datasets revealed that HHEX and NKX6-3 were the only members differentially active in naïve B-cells, germinal center B-cells, plasma cells and memory B-cells. Subsequent examination of different types of B-cell malignancies showed both aberrant overexpression of NKL-code members and ectopic activation of subclass members physiologically silent in lymphopoiesis including BARX2, DLX1, EMX2, NKX2-1, NKX2-2 and NKX3-2. Based on these findings we performed detailed studies of the B-cell specific NKL homeobox gene NKX6-3 which showed enhanced activity in patient subsets of follicular lymphoma, mantle cell lymphoma and diffuse large B-cell lymphoma (DLBCL), and in three DLBCL cell lines to serve as in vitro models. While excluding genomic and chromosomal rearrangements at the locus of NKX6-3 (8p11) promoter studies demonstrated that B-cell factors MYB and PAX5 activated NKX6-3 transcription. Furthermore, aberrant BMP7/SMAD1-signalling and deregulated expression of chromatin complex components AUTS2 and PCGF5 promoted NKX6-3 activation. Finally, NKL homeobox genes HHEX, HLX, MSX1 and NKX6-3 were expressed in B-cell progenitors and generated a regulatory gene network in cell lines which we propose may provide physiological support for NKL-code formation in early B-cell development. Together, we identified an NKL-code in B-cell development whose violation may deregulate differentiation and promote malignant transformation.

The Proline Variant of the W[F/L/M][T/S]R Cyclic Di-GMP Binding Motif Suppresses Dependence on Signal Association for Regulator Function.

Vibrio vulnificus is an estuarine bacterium and potent opportunistic human pathogen. It enters the food chain by asymptomatically colonizing a variety of marine organisms, most notably oysters. Expression of the brp-encoded extracellular polysaccharide, which enhances cell-surface adherence, is regulated by cyclic di-GMP (c-di-GMP) and the activator BrpT. The Vibrio cholerae and Vibrio parahaemolyticus homologs VpsT and CpsQ, directly bind c-di-GMP via a novel W[F/L/M][T/S]R motif, and c-di-GMP binding is absolutely required for activity. Notably, BrpT belongs to a distinct subclass of VpsT-like regulators that harbor a proline in the third position of the c-di-GMP binding motif (WLPR), and the impact of this change on activity is unknown. We show that the brp locus is organized as two linked operons with BrpT specifically binding to promoters upstream of brpA and brpH Expression data and structural modeling suggested that BrpT might be less dependent on c-di-GMP binding for activity than VpsT or CpsQ. We show that the affinity of BrpT for c-di-GMP is low and that signal binding is not a requisite for BrpT function. Furthermore, a BrpT mutant engineered to carry a canonical WLTR motif (BrpTP124T) bound c-di-GMP with high affinity and its activity was now c-di-GMP dependent. Conversely, introduction of the WLPR motif into VpsT suppressed its dependence on c-di-GMP for activity. This is the first demonstration of reduced dependence on signal association for regulator function within this motif family. Thus, BrpT defines a new class of VpsT-like transcriptional regulators, and the WLPR motif variant may similarly liberate the activity of other subclass members.IMPORTANCE A Vibrio genome may encode nearly 100 proteins that make, break, and bind c-di-GMP, underscoring its central role in the physiology of these bacteria. The activity of the biofilm regulators VpsT of V. cholerae and CpsQ of V. parahaemolyticus is regulated by the direct binding of c-di-GMP via a novel W[F/L/M][T/S]R motif. The V. vulnificus homolog, BrpT, bears an unusual WLPR variant and remains active at low intracellular c-di-GMP levels. This suggests that the WLPR motif may also liberate the activity of other members of this subclass. A single point mutation at the 3rd position of the motif was sufficient to moderate dependence on c-di-GMP binding for activator function, highlighting the simplicity with which complex bacterial signaling networks can be rewired.

Evolution and expression of BMP genes in flies

Bone morphogenetic proteins (BMPs) play key roles in development. In Drosophila melanogaster, there are three BMP-encoding genes: decapentaplegic (dpp), glass bottom boat (gbb) and screw (scw). dpp and gbb are found in all groups of insects. In contrast, the origin of scw via duplication of an ancestral gbb homologue is more recent, with new evidence placing it within the Diptera. Recent studies show that scw appeared basal to the Schizophora, since scw orthologues exist in aschizan cyclorrhaphan flies. In order to further localise the origin of scw, we have utilised new genomic resources for the nematoceran moth midge Clogmia albipunctata (Psychodidae). We identified the BMP subclass members dpp and gbb from an early embryonic transcriptome and show that their expression patterns in the blastoderm differ considerably from those seen in cyclorrhaphan flies. Further searches of the genome of C. albipunctata were unable to identify a scw-like gbb duplicate, but confirm the presence of dpp and gbb. Our phylogenetic analysis shows these to be clear orthologues of dpp and gbb from other non-cyclorrhaphan insects, with C. albipunctata gbb branching ancestrally to the cyclorrhaphan gbb/scw split. Furthermore, our analysis suggests that scw is absent from all Nematocera, including the Bibionomorpha. We conclude that the gbb/scw duplication occurred between the separation of the lineage leading to Brachycera and the origin of cyclorrhaphan flies 200–150 Ma ago.Electronic supplementary materialThe online version of this article (doi:10.1007/s00427-013-0445-9) contains supplementary material, which is available to authorized users.

Diversity and evolution of the small multidrug resistance protein family

BackgroundMembers of the small multidrug resistance (SMR) protein family are integral membrane proteins characterized by four α-helical transmembrane strands that confer resistance to a broad range of antiseptics and lipophilic quaternary ammonium compounds (QAC) in bacteria. Due to their short length and broad substrate profile, SMR proteins are suggested to be the progenitors for larger α-helical transporters such as the major facilitator superfamily (MFS) and drug/metabolite transporter (DMT) superfamily. To explore their evolutionary association with larger multidrug transporters, an extensive bioinformatics analysis of SMR sequences (> 300 Bacteria taxa) was performed to expand upon previous evolutionary studies of the SMR protein family and its origins.ResultsA thorough annotation of unidentified/putative SMR sequences was performed placing sequences into each of the three SMR protein subclass designations, namely small multidrug proteins (SMP), suppressor of groEL mutations (SUG), and paired small multidrug resistance (PSMR) using protein alignments and phylogenetic analysis. Examination of SMR subclass distribution within Bacteria and Archaea taxa identified specific Bacterial classes that uniquely encode for particular SMR subclass members. The extent of selective pressure acting upon each SMR subclass was determined by calculating the rate of synonymous to non-synonymous nucleotide substitutions using Syn-SCAN analysis. SUG and SMP subclasses are maintained under moderate selection pressure in comparison to integron and plasmid encoded SMR homologues. Conversely, PSMR sequences are maintained under lower levels of selection pressure, where one of the two PSMR pairs diverges in sequence more rapidly than the other. SMR genomic loci surveys identified potential SMR efflux substrates based on its gene association to putative operons that encode for genes regulating amino acid biogenesis and QAC-like metabolites. SMR subclass protein transmembrane domain alignments to Bacterial/Archaeal transporters (BAT), DMT, and MFS sequences supports SMR participation in multidrug transport evolution by identifying common TM domains.ConclusionBased on this study, PSMR sequences originated recently within both SUG and SMP clades through gene duplication events and it appears that SMR members may be evolving towards specific metabolite transport.

The role of class I HLH genes in neural development--have they been overlooked?

Helix-loop-helix (HLH) genes encode for transcription factors affecting a whole variety of developmental programs, including neurogenesis. At least seven functional classes (denoted I to VII) of HLH genes exist, (1) with subclass members exhibiting homo- and heterodimerisation for proper DNA binding and transcriptional regulation of downstream target genes. In the developing nervous system, members of class II, V and VI have been most extensively studied concerning their roles in neural programming. In contrast, the function of class I proteins (such as E12 and E47) is poorly defined and the orthodox view relegates them to general dimerisation duties that are necessary for the activity of the other classes. However, closer scrutiny of the spatiotemporal expression patterns of class I factors, combined with recent biochemical evidence, would suggest that class I proteins possess specific functions during early neural differentiation. This essay supports this possibility, in addition to putting forward the hypothesis that, outside their general dimerisation activity, class I genes have independent roles in regulating neurogenesis.

Supervisory control of families of linear set-point controllers. 2. Robustness

A simply structured high-level controller, called a supervisor, has recently been proposed in part I of this article (ibid., vol.41, 1996) for the purpose of orchestrating the switching of a sequence of candidate set-point controllers into feedback with an imprecisely modeled SISO process so as to cause the output of the process to approach and track a constant reference input. The process is assumed to be modeled by an SISO linear system whose transfer function is in the union of a number of subclasses, each subclass being small enough so that one of the candidate controllers would solve the set-point tracking problem, if the process transfer function was to be one of the subclass members. This paper proves that without any further modification, the same supervisor described in Part I can also perform this function in the face of norm-bounded unmodeled dynamics, and moreover that none of the signals within the overall system can grow without bound in response to bounded disturbance and noise inputs, whether they are constant or not.

Studies of Interviewer Variance for Attitudinal Variables

Abstract We studied the effects of the variable biases of interviewers on responses to a variety of items, mostly attitudinal, included in two surveys. Not satisfied with “testing statistical significance,” we measured this interviewer variance as a component of the total variance per respondent; thus s 2 =s a 2 + s b 2, where s a 2 is the “between interviewer” and s b 2 the “within interviewer” component; ρ = s a 2/s 2 is the proportion of the interviewer effect. The contribution of the interviewer variance to total survey errors is examined for its implications for the planning and interpretation of survey statistics. Among these we included subclass means and comparisons among pairs of subclasses. The data yielded important results: (1) We can obtain responses with low or moderate interviewer variance on highly “ambiguous” and “critical” attitudinal questions. The range of ρ's was mostly 0 to .07 in the first study, 0 to .05 in the second and their average is about .01 or .02; these effects are not generally higher than for most items in a good Census. (2) Nevertheless these seemingly small ρ's, combined with moderate workloads, can often increase the variance by factors as high as two or three, because the ratio of increase is where is the average number of interviews per interviewer. (3) The interviewer effects on subclass means were shown to be smaller in accord with where is average number of subclass members per interviewer. (4) In the comparisons of subclass means, the interviewer effects tend to zero, in accord with an additive model of the effects.

Studies of Interviewer Variance for Attitudinal Variables

Abstract We studied the effects of the variable biases of interviewers on responses to a variety of items, mostly attitudinal, included in two surveys. Not satisfied with “testing statistical significance,” we measured this interviewer variance as a component of the total variance per respondent; thus s 2 =s a 2 + s b 2, where s a 2 is the “between interviewer” and s b 2 the “within interviewer” component; ρ = s a 2/s 2 is the proportion of the interviewer effect. The contribution of the interviewer variance to total survey errors is examined for its implications for the planning and interpretation of survey statistics. Among these we included subclass means and comparisons among pairs of subclasses. The data yielded important results: (1) We can obtain responses with low or moderate interviewer variance on highly “ambiguous” and “critical” attitudinal questions. The range of ρ's was mostly 0 to .07 in the first study, 0 to .05 in the second and their average is about .01 or .02; these effects are not generally higher than for most items in a good Census. (2) Nevertheless these seemingly small ρ's, combined with moderate workloads, can often increase the variance by factors as high as two or three, because the ratio of increase is where is the average number of interviews per interviewer. (3) The interviewer effects on subclass means were shown to be smaller in accord with where is average number of subclass members per interviewer. (4) In the comparisons of subclass means, the interviewer effects tend to zero, in accord with an additive model of the effects.