Nonessential Protein Research Articles

Compromised CDK12 activity causes dependency on the high activity of O-GlcNAc transferase.

O-GlcNAc transferase (OGT) coordinates with regulators of transcription, including cyclin-dependent kinase 12 (CDK12), the major transcription elongation kinase. Here, we use inhibitor- and knockdown-based strategies to show that co-targeting of OGT and CDK12 is toxic to prostate cancer cells. OGT catalyzes all nucleocytoplasmic O-GlcNAcylation and due to its essentiality in higher eukaryotes, it is not an ideal drug target. Our glycoproteomics-data revealed that short-term CDK12 inhibition induces hyper-O-GlcNAcylation of the spliceosome-machinery in different models of prostate cancer. By integrating our glycoproteomics-, gene essentiality- and clinical-data from CDK12 mutant prostate cancer patients, we identify the non-essential serine-arginine protein kinase 1 (SRPK1) as a synthetic lethal partner with CDK12-inactivation. Both normal and cancer cells become highly sensitive against inhibitors of OGT and SRPK1 if they have lowered activity of CDK12. Inactivating mutations in CDK12 are enriched in aggressive prostate cancer, and we propose that these patients would benefit from therapy targeting the spliceosome.

Systematic analysis of the Candida albicans kinome reveals environmentally contingent protein kinase-mediated regulation of filamentation and biofilm formation in vitro and in vivo.

Protein kinases are critical regulatory proteins in both prokaryotes and eukaryotes. Accordingly, protein kinases represent a common drug target for a wide range of human diseases. Therefore, understanding protein kinase function in human pathogens such as the fungus Candida albicans is likely to extend our knowledge of its pathobiology and identify new potential therapies. To facilitate the study of C. albicans protein kinases, we constructed a library of 99 non-essential protein kinase homozygous deletion mutants marked with barcodes in the widely used SN genetic background. Here, we describe the construction of this library and the characterization of the competitive fitness of the protein kinase mutants under 11 different growth and stress conditions. We also screened the library for protein kinase mutants with altered filamentation and biofilm formation, two critical virulence traits of C. albicans. An extensive network of protein kinases governs these virulence traits in a manner highly dependent on the specific environmental conditions. Studies on specific protein kinases revealed that (i) the cell wall integrity MAPK pathway plays a condition-dependent role in filament initiation and elongation; (ii) the hyper-osmolar glycerol MAPK pathway is required for both filamentation and biofilm formation, particularly in the setting of in vivo catheter infection; and (iii) Sok1 is dispensable for filamentation in hypoxic environments at the basal level of a biofilm but is required for filamentation in normoxia. In addition to providing a new genetic resource for the community, these observations emphasize the environmentally contingent function of C. albicans protein kinases.IMPORTANCECandida albicans is one of the most common causes of fungal disease in humans for which new therapies are needed. Protein kinases are key regulatory proteins and are increasingly targeted by drugs for the treatment of a wide range of diseases. Understanding protein kinase function in C. albicans pathogenesis may facilitate the development of new antifungal drugs. Here, we describe a new library of 99 protein kinase deletion mutants to facilitate the study of protein kinases. Furthermore, we show that the function of protein kinases in two virulence-related processes, filamentation and biofilm formation, is dependent on the specific environmental conditions.

CDK12-inactivation-induced MYC signaling causes dependency on the splicing kinase SRPK1.

Inactivation of cyclin-dependent kinase 12 (CDK12) characterizes an aggressive sub-group of castration-resistant prostate cancer (CRPC). Hyper-activation of MYC transcription factor is sufficient to confer the CRPC phenotype. Here, we show that loss of CDK12 promotes MYC activity, which renders the cells dependent on the otherwise non-essential splicing regulatory kinase SRSF protein kinase 1 (SRPK1). High MYC expression is associated with increased levels of SRPK1 in patient samples, and overexpression of MYC sensitizes prostate cancer cells to SRPK1 inhibition using pharmacological and genetic strategies. We show that Endovion (SCO-101), a compound currently in clinical trials against pancreatic cancer, phenocopies the effects of the well-characterized SRPK1 inhibitor SRPIN340 on nascent transcription. This is the first study to show that Endovion is an SRPK1 inhibitor. Inhibition of SRPK1 with either of the compounds promotes transcription elongation, and transcriptionally activates the unfolded protein response. In brief, here we discover that CDK12 inactivation promotes MYC signaling in an SRPK1-dependent manner, and show that the clinical grade compound Endovion selectively targets the cells with CDK12 inactivation.

High Expression of KIFC1 in Glioma Correlates with Poor Prognosis.

Kinesin family member C1 (KIFC1), a non-essential kinesin-like motor protein, has been found to serve a crucial role in supernumerary centrosome clustering and the progression of several human cancer types. However, the role of KIFC1 in glioma has been rarely reported. Thus, the present study aimed to investigate the role of KIFC1 in glioma progression. Online bioinformatics analysis was performed to determine the association between KIFC1 expression and clinical outcomes in glioma. Immunohistochemical staining was conducted to analyze the expression levels of KIFC1 in glioma and normal brain tissues. Furthermore, KIFC1 expression was knocked in the glioma cell lines, U251 and U87MG, and the functional roles of KIFC1 in cell proliferation, invasion and migration were analyzed using cell multiplication, wound healing and Transwell invasion assays, respectively. The autophagic flux and expression levels matrix metalloproteinase-2 (MMP2) were also determined using imaging flow cytometry, western blotting and a gelation zymography assay. The results revealed that KIFC1 expression levels were significantly upregulated in glioma tissues compared with normal brain tissues, and the expression levels were positively associated with tumor grade. Patients with glioma with low KIFC1 expression levels had a more favorable prognosis compared with patients with high KIFC1 expression levels. In vitro, KIFC1 knockdown not only inhibited the proliferation, migration and invasion of glioma cells, but also increased the autophagic flux and downregulated the expression levels of MMP2. Upregulation of KIFC1 expression may promote glioma progression and KIFC1 may serve as a potential prognostic biomarker and possible therapeutic target for glioma.

The Prp19C/NTC subunit Syf2 and the Prp19C/NTC-associated protein Cwc15 function in TREX occupancy and transcription elongation.

The Prp19 complex (Prp19C), also named NineTeen Complex (NTC), is conserved from yeast to human and functions in many different processes such as genome stability, splicing, and transcription elongation. In the latter, Prp19C ensures TREX occupancy at transcribed genes. TREX, in turn, couples transcription to nuclear mRNA export by recruiting the mRNA exporter to transcribed genes and consequently to nascent mRNAs. Here, we assess the function of the nonessential Prp19C subunit Syf2 and the nonessential Prp19C-associated protein Cwc15 in the interaction of Prp19C and TREX with the transcription machinery, Prp19C and TREX occupancy, and transcription elongation. Whereas both proteins are important for Prp19C-TREX interaction, Syf2 is needed for full Prp19C occupancy, and Cwc15 is important for the interaction of Prp19C with RNA polymerase II and TREX occupancy. These partially overlapping functions are corroborated by a genetic interaction between Δcwc15 and Δsyf2 Finally, Cwc15 also interacts genetically with the transcription elongation factor Dst1 and functions in transcription elongation. In summary, we uncover novel roles of the Prp19C component Syf2 and the Prp19C-associated protein Cwc15 in Prp19C's function in transcription elongation.

Herpes Simplex Virus 1 Glycoprotein B from a Hyperfusogenic Virus Mediates Enhanced Cell-Cell Fusion.

Herpes simplex virus 1 (HSV-1) causes significant morbidity and death in humans worldwide. Herpes simplex virus 1 has a complex fusion mechanism that is incompletely understood. The HSV-1 strain ANG has notable fusion and entry activities that distinguish it from wild type. HSV-1 ANG virions fused with the Vero cell surface at 4 °C and also entered cells more efficiently at 15 °C, relative to wild type HSV-1 strain KOS virions, consistent with a hyperfusogenic phenotype. Understanding the molecular basis for the unique entry and fusion activities of HSV-1 strain ANG will help decipher the HSV fusion reaction and entry process. Sequencing of HSV-1 ANG genes revealed multiple changes in gB, gC, gD, gH, and gL proteins relative to wild type HSV-1 strains. The ANG UL45 gene sequence, which codes for a non-essential envelope protein, was identical to wild type KOS. HSV-1 ANG gB, gD, and gH/gL were necessary and sufficient to mediate cell-cell fusion in a virus-free reporter assay. ANG gB, when expressed with wild type KOS gD and gH/gL, increased membrane fusion, suggesting that ANG gB has hyperfusogenic cell-cell fusion activity. Replacing the KOS gD, gH, or gL with the corresponding ANG alleles did not enhance cell-cell fusion. The novel mutations in the ANG fusion and entry glycoproteins provide a platform for dissecting the cascade of interactions that culminate in HSV fusion and entry.

Multiple Rounds of In Vivo Random Mutagenesis and Selection in Vibrio natriegens Result in Substantial Increases in REE Binding Capacity.

Rare earth elements (REE) are essential ingredients in many modern technologies, yet their purification remains either environmentally harmful or economically unviable. Adsorption, or biosorption, of REE onto bacterial cell membranes offers a sustainable alternative to traditional solvent extraction methods. But in order for biosorption-based REE purification to compete economically, the capacity and specificity of biosorption sites must be enhanced. Although there have been some recent advances in characterizing the genetics of REE-biosorption, the variety and complexity of bacterial membrane surface sites make targeted genetic engineering difficult. Here, we propose using multiple rounds of in vivo random mutagenesis induced by the MP6 plasmid combined with plate-throughput REE-biosorption screening to improve a microbe's capacity and selectivity for biosorbing REE. We engineered a strain of Vibrio natriegens capable of biosorbing 210% more dysprosium compared to the wild-type and produced selectivity improvements of up to 50% between the lightest (lanthanum) and heaviest (lutetium) REE. We believe that mutations we observed in ABC transporters as well as a nonessential protein in the BAM outer membrane β-barrel protein insertion complex likely contribute to some─but almost certainly not all─of the biosorption changes we observed. Given the ease of finding significant biosorption mutants, these results highlight just how many genes likely contribute to biosorption as well as the power of random mutagenesis in identifying genes of interest and optimizing a biological system for a task.

Supply of essential and nonessential amino acids, proteins, antioxidants, iron and zinc from the main varieties of beans consumed in Mexico and their potential for biofortification

The objective of the present study was to characterize the contribution of essential and non-essential amino acids, protein, iron (Fe), and zinc (Zn) concentration, and antioxidant activity of the main bean varieties produced and consumed in Mexico. 23 varieties of beans were selected, and their amino acid profile, Fe and Zn concentration, protein and antioxidant activity were evaluated. The data obtained were subjected to an analysis of variance, test of separation of means and a correlation analysis. The results obtained indicate that the beans analyzed are an excellent source of essential amino acids (Histidine, threonine, valine, methionine, lysine, isoleucine, leucine and phenylalanine) and non-essential amino acids (Aspartic acid, serine, glutamic acid, glycine, alanine, proline, cysteine, tyrosine and arginine). The most prominent amino acids in Mexican bean varieties were: Glutamic acid, proline, aspartic acid, serine, lysine, and leucine. Regarding antioxidant activity, the most outstanding concentrations ranged from 91.18 to 96.76% inhibition. The bean varieties with the highest accumulation of amino acids, proteins, Fe, Zn and antioxidant activity were: 1) Black-eyed (23); 2) Peruvian (154); 3) Flor de Junio (150); 4) Pinto Saltillo (155). Finally, it was found that the common bean (Phaseolus vulgaris L.) is an excellent vehicle for the biofortification of Fe and Zn, which can contribute significantly to combat malnutrition problems and health of vulnerable communities in the urban and rural sectors of Mexico, as well as in the developing countries of the world.

Discovery of Molecular Glue Degraders via Isogenic Morphological Profiling.

Molecular glue degraders (MGDs) are small molecules that degrade proteins of interest via the ubiquitin-proteasome system. While MGDs were historically discovered serendipitously, approaches for MGD discovery now include cell-viability-based drug screens or data mining of public transcriptomics and drug response datasets. These approaches, however, have target spaces restricted to the essential proteins. Here we develop a high-throughput workflow for MGD discovery that also reaches the nonessential proteome. This workflow begins with the rapid synthesis of a compound library by sulfur(VI) fluoride exchange chemistry coupled to a morphological profiling assay in isogenic cell lines that vary in levels of the E3 ligase CRBN. By comparing the morphological changes induced by compound treatment across the isogenic cell lines, we were able to identify FL2-14 as a CRBN-dependent MGD targeting the nonessential protein GSPT2. We envision that this workflow would contribute to the discovery and characterization of MGDs that target a wider range of proteins.

Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system

Current manufacturing processes for recombinant adeno-associated viruses (rAAVs) have less-than-desired yields and produce significant amounts of empty capsids. The increasing demandand the high cost of goods for rAAV-based gene therapies motivate development of more efficient manufacturing processes. Recently, the US Food and Drug Administration (FDA) approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS), a technology that demonstrated production of high titers of full capsids. This work presents a first mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV maturation in the BEVS. The model predictions are successfully validated for in-house and literature experimental measurements of the vector genome and of structural and non-structural proteins collected during rAAV manufacturing in the BEVS with the TwoBac and ThreeBac constructs. A model-based analysis of the process is carried out to identify the bottlenecks that limit full capsid formation. Vector genome amplification is found to be the limiting step for rAAV production in Sf9 cells using either the TwoBac or ThreeBac system. In turn, vector genome amplification is hindered by limiting Rep78 levels. Transgene and non-essential baculovirus protein expression in the insect cell during rAAV manufacturing also negatively influences the rAAV production yields.

Abstract LB017: Potential role of kinesin family member C1 (KIFC1) in melanoma

Abstract According to the Surveillance, Epidemiology, and End Results (SEER) analyses, based on 2017-2019 data, approximately ~2.1 percent of men and women will be diagnosed with melanoma at some point during their lifetime. Melanoma can be fatal if not surgically removed at an early stage, with the 5-year survival being a dismal 29.8% for a stage IV melanoma. Melanoma mortality is primarily due to the distant organ metastases and its resistance to treatment, even with newly developed immunotherapy and/or targeted therapies (BRAF and MEK inhibitors). Thus, the discovery of novel melanoma targets is crucial as they may be able to provide alternative treatment options or be used alone or in combination with current therapies. KIFC1 (kinesin family member C1; also known as HSET, an ortholog in Drosophila melanogaster), a nonessential kinesin motor protein, has an established role in centrosome clustering which is utilized by cancer cells to evade apoptosis and mitotic arrest. This occurs when replicating cells harbor additional centrosomes, a hallmark of cancer. The requirement of KIFC1 in mitosis of cancer cells harboring abnormal centrosomes, but not that of normal cells, makes it a potential drug target for cancer management. The goal of this study was to determine the role and significance of KIFC1 in melanoma. Differential expression analysis of GTEX and TCGA RNA-seq data identified significantly higher levels of KIFC1 in both primary and metastatic melanoma as compared to normal skin. In addition, higher expression of KIFC1 was found to be associated with poorer overall survival of melanoma patients. Furthermore, a quantitative protein estimation with the automated ProteinSimple Jess capillary western blotting system demonstrated higher KIFC1 expression in several melanoma cell lines when compared to normal melanocytes. In addition, we also found significantly increased expression of KIFC1 mRNA in mouse melanoma tumors obtained from the genetically engineered BrafV600E/PtenNULL mice, as compared to the normal skin. Next, we determined the effect of a small molecule KIFC1 inhibitor, AZ82 on A375, G361 and Hs294T melanoma cells. AZ82 is an ATP-competitive inhibitor that binds the KIFC1-microtubule complex, blocking the release of ADP and negating its role as a motor protein. Employing RealTime-Glo MT cell viability assay as well as the endpoint XTT growth assay, we found that AZ82 treatment resulted in a significant growth inhibition of human melanoma cells at 2-3.5 µM concentrations. Additionally, AZ82 treatments resulted in a significant decrease in clonogenic survival in all three human melanoma cell lines. Collectively, our data suggests a potential role of KIFC1 in melanoma progression, and merits further investigation to determine the roles and functional and therapeutic significance of KIFC1 in melanoma. Citation Format: Davis S. Mau, Mary A. Ndiaye, Gabriella R. Zaemisch, Chandra K. Singh, Jess Vera, Sean R. McIlwain, Nihal Ahmad. Potential role of kinesin family member C1 (KIFC1) in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB017.

A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in calcium-induced Toxoplasma gondii egress.

Fundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.

Coral and it's symbionts responses to the typical global marine pollutant BaP by 4D-Proteomics approach

The symbiosis of corals, zooxanthellae, and microbes is the foundation of the coral reef ecosystem. In addition to global warming, marine pollutants are another important factor causing the breakdown of coral symbiosis. Benzo(a)pyrene (BaP) is a globally widespread marine environmental pollutant that poses a severe threat to marine ecosystems. However, responses of coral symbionts to global marine pollutant stress remain unclear. In this study, we selected Acropora formosa as the target coral to explore its response to 50 μg L−1 BaP stress using diaPASEF proteomics and 16s rRNA microbiome analysis. The results showed that: 1) the coral symbionts were sensitive to BaP stress; 2) the photosynthetic system of zooxanthellae was crucial for the balance of symbiotic relationships; 3) the destruction of the photosynthetic system induced a zooxanthellae hypoxic stress response; 4) corals adapted to BaP stress by promoting non-essential protein degradation and changing energy metabolism strategies; 5) symbiotic bacteria showed strong adaptability to BaP. This study not only fills the gap in understanding the response mechanism of coral symbionts under BaP stress, but also provides fundamental data for coral reef protection strategies.

Dopey proteins are essential but overlooked regulators of membrane trafficking.

Dopey family proteins play crucial roles in diverse processes from morphogenesis to neural function and are conserved from yeast to mammals. Understanding the mechanisms behind these critical functions could have major clinical significance, as dysregulation of Dopey proteins has been linked to the cognitive defects in Down syndrome, as well as neurological diseases. Dopey proteins form a complex with the non-essential GEF-like protein Mon2 and an essential lipid flippase from the P4-ATPase family. Different combinations of Dopey, Mon2 and flippases have been linked to regulating membrane remodeling, from endosomal recycling to extracellular vesicle formation, through their interactions with lipids and other membrane trafficking regulators, such as ARL1, SNX3 and the kinesin-1 light chain KLC2. Despite these important functions and their likely clinical significance, Dopey proteins remain understudied and their roles elusive. Here, we review the major scientific discoveries relating to Dopey proteins and detail key open questions regarding their function to draw attention to these fascinating enigmas.

Effects of non-essential protein on D-glucose to control diabetes: DFT approach.

Diabetes is a disease found in every 1 out of 4 people in the world. The glucose molecule is one of the sources of energy in the body and the lack of the digestion of glucose causes diabetes type 1 and type 2. Arginine and cysteine are nonessential amino acids that contain sulfur and help maintain the metabolisms of humans. We explored the glucose-arginine (Glc-arg) and glucose-cysteine (Glc-cys) molecules by finding their structural properties, electronic properties, chemical reactivity, mechanical strength, and transport properties because these non-essential amino acid molecules inhibit glucose-stimulated insulin secretion. Density functional theory (DFT) has been implemented to study all the properties of Glc-arg and Glc-cys using SIESTA software. Glucose-arginine (Glc-arg) inhibits a large percentage of glucose secretion and shows high chemical reactivity.

Short communication: Postpartum plasma amino acid concentration in primi- and multiparous Holstein cows

A better understanding of changes in plasma amino acid (AA) concentrations during the transition period may help optimize nutrition to improve health, welfare, and production in dairy cows. The aim of this study was to assess changes in plasma AA during the transition period in Holstein cows and first-lactation heifers. Blood samples for AA analysis were collected from the tail vein/artery of 9 multi- and 8 primiparous cows 3 weeks prior to and at weeks 1, 2, and 3 post-calving. Concentration of the sum of essential AA (EAA) was similar between primiparous and multiparous cows before calving but was 17.0% lower in multiparous vs. primiparous cows postpartum, which was related to both greater milk production and imbalance in estimated metabolizable protein supply vs. demand in the multiparous group of cows. In both parity groups, EAA concentrations declined by 20.0% from week 1 to week 3 postpartum. The sum of non-essential protein and non-protein AA increased linearly in the weeks after calving and was similar between primi- and multiparous cows. Compared with prepartum, plasma His concentration decreased 13.1% postpartum in both primiparous and multiparous cows but primiparous cows had 7.6% greater plasma His concentration than multiparous cows pre- and postpartum. Methionine and Lys concentrations were not affected by parity, but concentrations of both AA decreased (22.0% and 24.5%, respectively) after calving. Overall, plasma concentration of EAA decreased after calving, indicating a negative EAA balance (supply vs. demand) around parturition. Lower postpartum plasma concentrations of EAA in multiparous cows likely reflect an overall negative EAA balance due to decreased supply and increased demand to sustain greater milk production, compared with primiparous cows.

Fluctuation in amino acids content in Triticum aestivum L. cultivars as an indicator on the impact of post-emergence herbicides in controlling weeds.

This study was carried out in a demonstrated field in El-Sharkia Governorate, Egypt, during the winter of season 2020 to evaluate the leverage of four post-emergence herbicides i.e., tribenuron-methyl, clodinafop- propargyl, pyroxsulam and pinoxaden compared to control on total protein and amino acid contents in three wheat cultivars (Shandwel 1, Giza 171, and Sakha 95). Generally, the use of foliar herbicides led to a significant decrease in essential, non-essential amino acids and protein contents. However, tribenuran-methyl herbicide signifcantly increased the levels of proline, glycine, arginine, and histidine, but cystine and threonine not affected as compared to control. On the other hand, foliar herbicide application was significantly increased physiological , biochemical parameters and yield of Shandweel cultivar as compared to the other varieties. The physiological and biochemical models of dual-herbicide-tolerant wheat cultivars add to our understanding of the crop. In recent agricultural systems, herbicide tolerant plants are important for long-term weed management. Therefore, the study recommended the safely usage of Tribenuran-methyl as foliar herbicide in weed managment.

Rice (Oryza sativa) TIR1 and 5'adamantyl-IAA Significantly Improve the Auxin-Inducible Degron System in Schizosaccharomyces pombe.

The auxin-inducible degron (AID) system is a powerful tool to induce targeted degradation of proteins in eukaryotic model organisms. The efficiency of the existing Schizosaccharomyces pombe AID system is limited due to the fusion of the F-box protein TIR1 protein to the SCF component, Skp1 (Skp1-TIR1). Here, we report an improved AID system for S. pombe that uses the TIR1 from Oryza sativa (OsTIR1) not fused to Skp1. Furthermore, we demonstrate that degradation efficiency can be improved by pairing an OsTIR1 auxin-binding site mutant, OsTIR1F74A, with an auxin analogue, 5′adamantyl-IAA (AID2). We provide evidence for the enhanced functionality of the OsTIR1 AID and AID2 systems by application to the essential DNA replication factor Mcm4 and to a non-essential recombination protein, Rad52. Unlike AID, no detectable auxin-independent depletion of AID-tagged proteins was observed using AID2.

DrpB (YedR) Is a Nonessential Cell Division Protein in Escherichia coli.

We report that the small Escherichia coli membrane protein DrpB (formerly YedR) is involved in cell division. We discovered DrpB in a screen for multicopy suppressors of a ΔftsEX mutation that prevents divisome assembly when cells are plated on low ionic strength medium, such as lysogeny broth without NaCl. Characterization of DrpB revealed that (i) translation initiates at an ATG annotated as codon 22 rather than the GTG annotated as codon 1, (ii) DrpB localizes to the septal ring when cells are grown in medium of low ionic strength but localization is greatly reduced in medium of high ionic strength, (iii) overproduction of DrpB in a ΔftsEX mutant background improves recruitment of the septal peptidoglycan synthase FtsI, implying multicopy suppression works by rescuing septal ring assembly, (iv) a ΔdrpB mutant divides quite normally, but a ΔdrpB ΔdedD double mutant has a strong division and viability defect, albeit only in medium of high ionic strength, and (v) DrpB homologs are found in E. coli and a few closely related enteric bacteria, but not outside this group. In sum, DrpB is a poorly conserved nonessential division protein that improves the efficiency of cytokinesis under suboptimal conditions. Proteins like DrpB are likely to be a widespread feature of the bacterial cell division apparatus, but they are easily overlooked because mutants lack obvious shape defects.IMPORTANCE A thorough understanding of bacterial cell division requires identifying and characterizing all of the proteins that participate in this process. Our discovery of DrpB brings us one step closer to this goal in E. coli.

The High Osmolarity Glycerol Mitogen-Activated Protein Kinase regulates glucose catabolite repression in filamentous fungi.

The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.