Heteromultimeric Complexes Research Articles

Analysis of Transmembrane β-Barrel Proteins by Native and Semi-native Polyacrylamide Gel Electrophoresis.

Membrane-embedded β-barrel proteins are important regulators of the outer membrane permeability barrier of Gram-negative bacteria. β-barrels are highly structured domains formed by a series of antiparallel β-strands. Each β-strand is locked in position by hydrogen bonds between its polypeptide backbone andthose of thetwo neighbouring strands in the barrel structure. Some transmembraneβ-barrel proteins form larger homo- or hetero-multimeric complexes that accomplish specific functions. In this chapter, we describe native and semi-native polyacrylamide gel electrophoresis (PAGE) methods to characterize the organization of transmembraneβ-barrel proteins. We illustrate blue native (BN)-PAGE as an analytical method to assess the formation of protein complexes. Furthermore, we describe a heat-modifiability assay via semi-native PAGE as arapid method to investigate the folding of transmembrane β-barrels.

Multimodal cotranslational interactions direct assembly of the human multi-tRNA synthetase complex

Amino acid ligation to cognate transfer RNAs (tRNAs) is catalyzed by aminoacyl-tRNA synthetases (aaRSs)-essential interpreters of the genetic code during translation. Mammalian cells harbor 20 cytoplasmic aaRSs, out of which 9 (in 8 proteins), with 3 non-aaRS proteins, AIMPs 1 to 3, form the ∼1.25-MDa multi-tRNA synthetase complex (MSC). The function of MSC remains uncertain, as does its mechanism of assembly. Constituents of multiprotein complexes encounter obstacles during assembly, including inappropriate interactions, topological constraints, premature degradation of unassembled subunits, and suboptimal stoichiometry. To facilitate orderly and efficient complex formation, some complexes are assembled cotranslationally by a mechanism in which a fully formed, mature protein binds a nascent partner as it emerges from the translating ribosome. Here, we show out of the 121 possible interaction events between the 11 MSC constituents, 15 are cotranslational. AIMPs are involved in the majority of these cotranslational interactions, suggesting they are not only critical for MSC structure but also for assembly. Unexpectedly, several cotranslational events involve more than the usual dyad of interacting proteins. We show two modes of cotranslational interaction, namely a "multisite" mechanism in which two or more mature proteins bind the same nascent peptide at distinct sites and a second "piggy-back" mechanism in which a mature protein carries a second fully formed protein and binds to a single site on an emerging peptide. Multimodal mechanisms of cotranslational interaction offer a diversity of pathways for ordered, piecewise assembly of small subcomplexes into larger heteromultimeric complexes such as the mammalian MSC.

Channels and Transporters in Astrocyte Volume Regulation in Health and Disease.

Astrocytes are the second most abundant cell type in the central nervous system and serve various functions, many of which maintain homeostasis of the intracellular milieu in the face of constant change. In order to accomplish these important functions, astrocytes must regulate their cell volume. In astrocytes, cell volume regulation involves multiple channels and transporters, including AQP4, TRPV4, TRPM4, VRAC, Na+/K+ ATPase, NKCC1 and Kir4.1. AQP4 is a bidirectional water channel directly involved in astrocyte cell volume regulation. AQP4 also forms heteromultimeric complexes with other channels and transporters involved in cell volume regulation. TRPV4, a mechanosensitive channel in involved in osmotic regulation in various cell types, forms a complex with AQP4 to decrease cell volume in response to cell swelling. TRPM4 also forms a complex with AQP4 and SUR1 in response to injury resulting in cell swelling. Another complex forms between Na+/K+ ATPase, AQP4, and mGluR5 to regulate the perisynaptic space. NKCC1 is a co-transporter involved in cell volume increases either independently through cotransport of water or a functional interaction with AQPs. VRAC is implicated in regulatory volume decreases and may also functionally interact with AQP4. Although Kir4.1 colocalizes with AQP4, its role in cell volume regulation is debated. In diseases where fluid/electrolyte homeostasis is disturbed such as stroke, ischemic injury, inflammation, traumatic brain injury and hydrocephalus, cell volume regulation is challenged, sometimes past the point of recovery. Thus, a greater understanding of signaling pathways which regulate transport proteins as well as the functional and physical interactions that exist between transporters will provide a basis for the development of pharmaceutical targets to treat these prevalent and often devastating diseases.

ATP-sensitive Potassium Channel Subunits in Neuroinflammation: Novel Drug Targets in Neurodegenerative Disorders.

Arachidonic acids and its metabolites modulate plenty of ligand-gated, voltage-dependent ion channels, and metabolically regulated potassium channels including ATP-sensitive potassium channels (KATP). KATP channels are hetero-multimeric complexes of sulfonylureas receptors (SUR1, SUR2A or SUR2B) and the pore-forming subunits (Kir6.1 and Kir6.2) likewise expressed in the pre-post synapsis of neurons and inflammatory cells, thereby affecting their proliferation and activity. KATP channels are involved in amyloid-β (Aβ)-induced pathology, therefore emerging as therapeutic targets against Alzheimer's and related diseases. The modulation of these channels can represent an innovative strategy for the treatment of neurodegenerative disorders; nevertheless, the currently available drugs are not selective for brain KATP channels and show contrasting effects. This phenomenon can be a consequence of the multiple physiological roles of the different varieties of KATP channels. Openings of cardiac and muscular KATP channel subunits, are protective against caspase-dependent atrophy in these tissues and some neurodegenerative disorders, whereas in some neuroinflammatory diseases, benefits can be obtained through the inhibition of neuronal KATP channel subunits. For example, glibenclamide exerts an anti-inflammatory effect in respiratory, digestive, urological, and central nervous system (CNS) diseases, as well as in ischemia-reperfusion injury associated with abnormal SUR1-Trpm4/TNF-α or SUR1-Trpm4/ Nos2/ROS signaling. Despite this strategy being promising, glibenclamide may have limited clinical efficacy due to its unselective blocking action of SUR2A/B subunits also expressed in cardiovascular apparatus with pro-arrhythmic effects and SUR1 expressed in pancreatic beta cells with hypoglycemic risk. Alternatively, neuronal selective dual modulators showing agonist/antagonist actions on KATP channels can be an option.

Slow and temperature-compensated autonomous disassembly of KaiB-KaiC complex.

KaiC is the central pacemaker of the circadian clock system in cyanobacteria and forms the core in the hetero-multimeric complexes, such as KaiB–KaiC and KaiA–KaiB–KaiC. Although the formation process and structure of the binary and ternary complexes have been studied extensively, their disassembly dynamics have remained elusive. In this study, we constructed an experimental system to directly measure the autonomous disassembly of the KaiB–KaiC complex under the condition where the dissociated KaiB cannot reassociate with KaiC. At 30°C, the dephosphorylated KaiB–KaiC complex disassembled with an apparent rate of 2.1±0.3 d–1, which was approximately twice the circadian frequency. Our present analysis using a series of KaiC mutants revealed that the apparent disassembly rate correlates with the frequency of the KaiC phosphorylation cycle in the presence of KaiA and KaiB and is robustly temperature-compensated with a Q10 value of 1.05±0.20. The autonomous cancellation of the interactions stabilizing the KaiB–KaiC interface is one of the important phenomena that provide a link between the molecular-scale and system-scale properties.

Biogenesis of RNA Polymerases in Yeast.

Eukaryotic RNA polymerases (RNA pols) transcriptional processes have been extensively investigated, and the structural analysis of eukaryotic RNA pols has been explored. However, the global assembly and biogenesis of these heteromultimeric complexes have been narrowly studied. Despite nuclear transcription being carried out by three RNA polymerases in eukaryotes (five in plants) with specificity in the synthesis of different RNA types, the biogenesis process has been proposed to be similar, at least for RNA pol II, to that of bacteria, which contains only one RNA pol. The formation of three different interacting subassembly complexes to conform the complete enzyme in the cytoplasm, prior to its nuclear import, has been assumed. In Saccharomyces cerevisiae, recent studies have examined in depth the biogenesis of RNA polymerases by characterizing some elements involved in the assembly of these multisubunit complexes, some of which are conserved in humans. This study reviews the latest studies governing the mechanisms and proteins described as being involved in the biogenesis of RNA polymerases in yeast.

Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4.

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators—calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein–protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

Zinc-Dependent Homomultimerization of Mutant Calreticulin Is Required for MPN Pathogenesis

Recurrent mutations in the ER chaperone calreticulin (CALR) are found in ~30% of MPNs. All known CALR mutations result in a +1-frameshift of the CALR reading frame that retains the N-domain and P-arm of wild-type CALR and generates a novel mutant-specific C-terminal domain encoded by an alternative reading frame. Multiple groups have previously demonstrated that mutant CALR (CALRMUT) physically binds to and activates the thrombopoietin receptor MPL and causes inappropriate receptor activation and downstream JAK-STAT signaling in a ligand-independent manner. These oncogenic activities of CALRMUT have been shown to be dependent on a threshold of basic amino acid residues in the mutant-specific C-terminus and on the lectin motif within the N-domain. In this study, we extended the analysis of the role of the N-domain in CALRMUT activity by performing a comprehensive alanine mutagenesis screen of all 177 non-alanine residues within the N-domain and assessed the ability of alanine-mutants to confer cytokine independent growth in Ba/F3-MPL cells. We identified two functional groups of residues as being important for CALRMUT activity: (i) Residues associated with CALR lectin motif. Consistent with previous work which had shown that Asp135 and Asp317 was essential for CALRMUT activity, we observed that loss of any of 4 residues within the lectin motif (Cys105, Lys111, Gly133 and Asp135) led to total impairment in the ability of CALRMUT to confer cytokine independence, bind MPL or elicit JAK-STAT signaling. Surprisingly, other residues implicated in lectin activity (Tyr109, Tyr128, Met131) had either only a partial effect or no effect on CALRMUT oncogenic activity, suggesting that different regions within the lectin motif of CALR have differential effects in facilitating MPL binding. (ii) Residues associated with zinc binding. We also identified a group of zinc-binding histidine residues (His99, His145 and His170) as being important for modulating CALRMUT activity. We observed that loss of any single histidine residue (1xHis) led to partial abrogation of cytokine independence in Ba/F3-MPL cells. Strikingly, combined loss of two (or more) histidines (2xHis) led to complete impairment in the capacity to confer cytokine independent growth or elicit JAK-STAT signaling, in conjunction with impaired ability to physically associate with Zn2+ by affinity chromatography. In addition, 2xHis-CALRMUT failed to bind MPL in pulldown assays or colocalize with MPL in FRET-based immunocytochemical analysis. We next investigated in greater detail how the zinc binding capacity of CALRMUT regulates its oncogenic activity. Previous studies have shown that CALRMUT forms homomultimers (Araki, Leukemia, 33:122), and that zinc is able to increase homomultimerization of recombinant wild-type CALR in vitro (Li, Biochemistry, 40:11193). We therefore tested the multimerization capacity of lectin-deficient or zinc binding-deficient CALRMUT by assessing the ability of heterologously-expressed FLAG-tagged CALRMUT species to complex and co-immunoprecipitate with a V5-tagged CALRMUT species in 293T cells. We observed that lectin-deficient CALRMUT and 1xHis-CALRMUT retained the capacity to form homomultimers, but the 2xHis- CALRMUT could not, suggesting that zinc binding was required for homomultimer formation. Treatment with ZnCl2 which increases free intracellular Zn2+ resulted in increased CALRMUT homomultimerization in a dose-dependent manner, as well as increased binding to MPL within heteromeric complexes. Finally, treatment with the zinc chelator TPEN led to decreased CALRMUT homomultimer formation, decreased CALRMUT-MPL heteromultimeric complexes and attenuated JAK-STAT signaling. Cumulatively, our data suggest that zinc is required for homomultimerization of CALRMUT, and is a necessary prerequisite event to facilitate binding to and activation of MPL in CALR-mutated MPNs. Disclosures Mullally: Janssen: Research Funding.

Determination of the Stoichiometry between α- and γ1 Subunits of the BK Channel Using LRET

Two families of accessory proteins, β and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca2+, the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb+3 as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.

A Novel Conserved Domain Mediates Dimerization of Protein Kinase D (PKD) Isoforms: DIMERIZATION IS ESSENTIAL FOR PKD-DEPENDENT REGULATION OF SECRETION AND INNATE IMMUNITY

Protein kinase D (PKD) isoforms are protein kinase C effectors in signaling pathways regulated by diacylglycerol. Important physiological processes (including secretion, immune responses, motility, and transcription) are placed under diacylglycerol control by the distinctive substrate specificity and subcellular distribution of PKDs. Potentially, broadly co-expressed PKD polypeptides may interact to generate homo- or heteromultimeric regulatory complexes. However, the frequency, molecular basis, regulatory significance, and physiological relevance of stable PKD-PKD interactions are largely unknown. Here, we demonstrate that mammalian PKDs 1-3 and the prototypical Caenorhabditis elegans PKD, DKF-2A, are exclusively (homo- or hetero-) dimers in cell extracts and intact cells. We discovered and characterized a novel, highly conserved N-terminal domain, comprising 92 amino acids, which mediates dimerization of PKD1, PKD2, and PKD3 monomers. A similar domain directs DKF-2A homodimerization. Dimerization occurred independently of properties of the regulatory and kinase domains of PKDs. Disruption of PKD dimerization abrogates secretion of PAUF, a protein carried in small trans-Golgi network-derived vesicles. In addition, disruption of DKF-2A homodimerization in C. elegans intestine impaired and degraded the immune defense of the intact animal against an ingested bacterial pathogen. Finally, dimerization was indispensable for the strong, dominant negative effect of catalytically inactive PKDs. Overall, the structural integrity and function of the novel dimerization domain are essential for PKD-mediated regulation of a key aspect of cell physiology, secretion, and innate immunity in vivo.

Mechanism of allele specific assembly and disruption of master regulator transcription factor complexes of NF-KBp50, NF-KBp65 and HIF1a on a non-coding FAS SNP

A challenging question in genetics is to understand the molecular function of non-coding variants of the genome. By using differential EMSA, ChIP and functional genome analysis, we have found that changes in transcription factors (TF) apparent binding affinity and dissociation rates are responsible for allele specific assembly or disruption of master TFs: we observed that NF-KBp50, NF-KBp65 and HIF1a bind with an affinity of up to 10 fold better to the C-allele than to the T-allele of rs7901656 both in vivo and in vitro. Furthermore, we showed that NF-KBp50, p65 and HIF1a form higher order heteromultimeric complexes overlapping rs7901656, implying synergism of action among TFs governing cellular response to infection and hypoxia. With rs7901656 on the FAS gene as a paradigm, we show how allele specific transcription factor complex assembly and disruption by a causal variant contributes to disease and phenotypic diversity. This finding provides the highly needed mechanistic insight into how the molecular etiology of regulatory SNPs can be understood in functional terms.

Intermolecular Interactions of Cardiac Transcription Factors NKX2.5 and TBX5.

Heart development in mammalian systems is controlled by combinatorial interactions of master cardiac transcription factors such as TBX5 and NKX2.5. They bind to promoters/enhancers of downstream targets as homo- or heteromultimeric complexes. They physically interact and synergistically regulate their target genes. To elucidate the molecular basis of the intermolecular interactions, a heterodimer and a homodimer of NKX2.5 and TBX5 were studied using X-ray crystallography. Here we report a crystal structure of human NKX2.5 and TBX5 DNA binding domains in a complex with a 19 bp target DNA and a crystal structure of TBX5 homodimer. The ternary complex structure of NKX2.5 and TBX5 with the target DNA shows physical interactions between the two proteins through Lys158 (NKX2.5), Asp140 (TBX5), and Pro142 (TBX5), residues that are highly conserved in TBX and NKX families across species. Extensive homodimeric interactions were observed between the TBX5 proteins in both crystal structures. In particular, in the crystal structure of TBX5 protein that includes the N-terminal and DNA binding domains, intermolecular interactions were mediated by the N-terminal domain of the protein. The N-terminal domain of TBX5 was predicted to be "intrinsically unstructured", and in one of the two molecules in an asymmetric unit, the N-terminal domain assumes a β-strand conformation bridging two β-sheets from the two molecules. The structures reported here may represent general mechanisms for combinatorial interactions among transcription factors regulating developmental processes.

Transient Expression of Secretory IgA In Planta is Optimal Using a Multi-Gene Vector and may be Further Enhanced by Improving Joining Chain Incorporation.

Secretory IgA (sIgA) is a crucial antibody in host defense at mucosal surfaces. It is a promising antibody isotype in a variety of therapeutic settings such as passive vaccination and treatment of inflammatory disorders. However, heterologous production of this heteromultimeric protein complex is still suboptimal. The challenge is the coordinate expression of the four required polypeptides; the alpha heavy chain, the light chain, the joining chain, and part of the polymeric-Ig-receptor called the secretory component, in a 4:4:1:1 ratio. We evaluated the transient expression of three sIgAκ variants, harboring the heavy chain isotype α1, α2m1, or α2m2, of the clinical antibody Ustekinumab in planta. Ustekinumab is directed against the p40 subunit that is shared by the pro-inflammatory cytokines interleukin (IL)-12 and IL-23. A sIgA variant of this antibody may enable localized treatment of inflammatory bowel disease. Of the three different sIgA variants we obtained the highest yield with sIgA1κ reaching up to 373 μg sIgA/mg total soluble protein. The use of a multi-cassette vector containing all four expression cassettes was most efficient. However, not the expression strategy, but the incorporation of the joining chain turned out to be the limiting step for sIgA production. Our data demonstrate that transient expression in planta is suitable for the economic production of heteromultimeric protein complexes such as sIgA.

Different KChIPs Compete for Heteromultimeric Assembly with Pore-Forming Kv4 Subunits

Auxiliary Kv channel-interacting proteins 1–4 (KChIPs1–4) coassemble with pore-forming Kv4 α-subunits to form channel complexes underlying somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form variable channel complexes that can exhibit distinct biophysical properties for modulation of neural function. In this study, we use single-molecule subunit counting by total internal reflection fluorescence microscopy in combinations with electrophysiology and biochemistry to investigate whether different isoforms of auxiliary KChIPs, KChIP4a, and KChIP4bl, can compete for binding of Kv4.3 to coassemble heteromultimeric channel complexes for modulation of channel function. To count the number of photobleaching steps solely from cell membrane, we take advantage of a membrane tethered k-ras-CAAX peptide that anchors cytosolic KChIP4 proteins to the surface for reduction of background noise. Single-molecule subunit counting reveals that the number of KChIP4 isoforms in Kv4.3-KChIP4 complexes can vary depending on the KChIP4 expression level. Increasing the amount of KChIP4bl gradually reduces bleaching steps of KChIP4a isoform proteins, and vice versa. Further analysis of channel gating kinetics from different Kv4-KChIP4 subunit compositions confirms that both KChIP4a and KChIP4bl can modulate the channel complex function upon coassembly. Taken together, our findings show that auxiliary KChIPs can heteroassemble with Kv4 in a competitive manner to form heteromultimeric Kv4-KChIP4 channel complexes that are biophysically distinct and regulated under physiological or pathological conditions.

A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels

ATP-sensitive potassium (KATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of KATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.

Cellulosome stoichiometry in Clostridium cellulolyticum is regulated by selective RNA processing and stabilization.

The mechanism, physiological relevance and evolutionary implication of selective RNA processing and stabilization (SRPS) remain elusive. Here we report the genome-wide maps of transcriptional start sites (TSs) and post-transcriptional processed sites (PSs) for Clostridium cellulolyticum. The PS-associated genes are preferably associated with subunits of heteromultimeric protein complexes, and the intergenic PSs (iPSs) are enriched in operons exhibiting highly skewed transcript-abundance landscape. Stem-loop structures associated with those iPSs located at 3′ termini of highly transcribed genes exhibit folding free energy negatively correlated with transcript-abundance ratio of flanking genes. In the cellulosome-encoding cip-cel operon, iPSs and stem-loops precisely regulate structure and abundance of the subunit-encoding transcripts processed from a primary polycistronic RNA, quantitatively specifying cellulosome stoichiometry. Moreover, cellulosome evolution is shaped by the number, position and biophysical nature of TSs, iPSs and stem-loops. Our findings unveil a genome-wide RNA-encoded strategy controlling in vivo stoichiometry of protein complexes.

Protein complexes in bacteria.

Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies.

Total and isoform-specific quantitative assessment of circulating fibulin-1 using selected reaction monitoring MS and time-resolved immunofluorometry.

Targeted proteomics using SRM-MS combined with stable-isotope dilution has emerged as a promising quantitative technique for the study of circulating protein biomarkers. The purpose of this study was to develop and characterize robust quantitative assays for the emerging cardiovascular biomarker fibulin-1 and its circulating isoforms in human plasma. We used bioinformatics analysis to predict total and isoform-specific tryptic peptides for absolute quantitation using SRM-MS. Fibulin-1 was quantitated in plasma by nanoflow-LC-SRM-MS in undepleted plasma and time-resolved immunofluorometric assay (TRIFMA). Both methods were validated and compared to a commercial ELISA (CircuLex). Molecular size determination was performed under native conditions by SEC analysis coupled to SRM-MS and TRIFMA. Absolute quantitation of total fibulin-1, isoforms -1C, and -1D was performed by SRM-MS. Fibulin-1C was the most abundant isoform in plasma. Circulating fibulin-1 isoforms were homo -or hetero multimeric complexes (range 318-364 kDa). Good correlation was obtained between SRM-MS and TRIFMA but not CircuLex. For biomarker studies using smaller cohorts, SRM-MS provides an alternative measure of total and specific fibulin-1 isoforms in undepleted plasma. For larger cohorts TRIFMA provides a faster platform for fibulin-1 quantitation in plasma. While the correlation between these methods was acceptable, low correlation was obtained between the commercial CircuLex assay and SRM-MS or TRIFMA.

Dynamic Subunit Stoichiometry of Kv4.3-KChIP4A Channel Complexes Visualized by Single-Molecule Subunit Counting

Auxiliary Kv channel-interacting proteins 1-4 (KChIPs1-4) co-assemble with pore-forming Kv4 α-subunits to underlie somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form dynamic channel complexes that can exhibit distinct biophysical properties for modulation of neural function. To test this hypothesis, we utilized single molecule subunit counting to investigate whether different isoforms of auxiliary KChIPs such as KChIP4a and KChIP4bl can compete for binding of Kv4.3 to co-assemble hetero-multimeric channel complexes. Single-molecule imaging subunit counting revealed that the number of KChIP4 proteins in Kv4.3-KChIP channel complexes can vary depending on KChIP4 expression level. Increasing amount of KChIP4bl gradually reduces the bleaching steps of GFP for KChIP4a proteins and vice-versa. To further demonstrate Kv4 gating affected by different KChIP4 subunit stoichiometry, we generated two tandem constructs to mimic the situations of KChIP4a half-occupied channel complexes (KChIP4a-2xKv4.3) and KChIP4a saturated channel complexes (KChIP4a-Kv4.3) expressed in Xenopus oocytes. Gating kinetics of KChIP4a-2xKv4.3 co-expressed with KChIP4bl (to mimic channel complex like Kv4.3:KChIP4a:KChIP4bl with the ratio of 4:2:2) shows that both KChIP4a and KChIP4bl can simultaneously modulate the function of channel complexes upon co-assembly. The significance of dynamic channel complexes was further investigated in hippocampal neurons from kainic acid seizure model in rats by detecting a shift of expression profile from KChIP4bl to KChIP4a. Our preliminary findings demonstrate that auxiliary KChIPs can hetero-assemble with Kv4 in a competitive manner to form hetero-multimeric Kv4-KChIP4 complexes that are biophysically distinct and dynamically regulated under pathological conditions.

Timely binding of IHF and Fis to DARS2 regulates ATP-DnaA production and replication initiation.

In Escherichia coli, the ATP-bound form of DnaA (ATP–DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP–DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP–DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP–DnaA was fully active in replication initiation and underwent DnaA–ATP hydrolysis. ADP–DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP–DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP–DnaA production, thereby promoting timely initiation. Moreover, we show that IHF–DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP–DnaA and replication initiation in coordination with the cell cycle and growth phase.