No abstract

HSP101 belongs to the ClpB protein subfamily whose members promote the renaturation of protein aggregates and are essential for the induction of thermotolerance. We found that maize HSP101 accumulated in mature kernels in the absence of heat stress. At optimal temperatures, HSP101 disappeared within the first 3 days after imbibition, although its levels increased in response to heat shock. In embryonic cells, HSP101 concentrated in the nucleus and in some nucleoli. Hsp101 maps near the umc132 and npi280 markers on chromosome 6. Five maize hsp101-m-:: Mu1 alleles were isolated. Mutants were null for HSP101 and defective in both induced and basal thermotolerance. Moreover, during the first 3 days after imbibition, primary roots grew faster in the mutants at optimal temperature. Thus, HSP101 is a nucleus-localized protein that, in addition to its role in thermotolerance, negatively influences the growth rate of the primary root. HSP101 is dispensable for proper embryo and whole plant development in the absence of heat stress.

Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by RED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.The bleomycins (BLMs) 3 (1, 2) are important clinically used hybrid peptide-polyketide antitumor compounds. Combined with other agents, the BLMs are used clinically for the treatment of several types of tumors and marketed under the trade name Blenoxane, with BLM A2 and B2 as the principle constituents (3). Early development of drug resistance and cumulative pulmonary toxicity are the major limitations of BLMs in chemotherapy (3, 4). Therefore, it is an important research goal to develop strategies to produce novel BLM analogs by microbial fermentation, particularly those unavailable or extremely difficult to prepare by chemical synthesis.The BLMs are thought to exert their biological effects through a sequence-selective, metal-dependent oxidative cleavage of DNA and RNA in the presence of oxygen (5, 6). They can be dissected into four functional domains: (i) the metal-binding domain, which consists of the pyrimidoblamic acid subunit along with the adjacent ␤-hydroxyl histidine; (ii) the bithiazole and C-terminal amine, which confers the majority of BLM-DNA affinity; (iii) the (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid linker subunit, which plays an important role in the efficiency of DNA cleavage by BLMs; and (iv) the carbamoylated disaccharide moiety (4). The exact functional role of the sugars and the attached carbamoyl group remains controversial. They possibly contribute to cell recognition, cellular uptake of BLMs, metal ion coordination, and/or DNA affinity (4, 6).The investigation of biosynthetic pathways and generation of new natural product analogs by genetic engineering critically depends on the availability of tools for recombinant DNA work in the producing organism. In general, this can be achieved by the development of an expedient genetic system for ...

Retigabine (RTG) is the first approved anti-epileptic drug that acts via activation of voltage-gated potassium channels, targeting KCNQ channels that underlie the neuronal M-current. RTG exhibits little specificity between KCNQ2-5 as a result of conservation of a Trp residue in the pore domain that binds to the drug. The RTG analogue ICA-069673 ('ICA73') exhibits much stronger effects on KCNQ2 channels, including a large hyperpolarizing shift of the voltage-dependence of activation, an ∼2-fold enhancement of peak current and pronounced subtype specificity for KCNQ2 over KCNQ3. Based on ICA73 sensitivity of chimeric constructs of the transmembrane segments of KCNQ2 and KCNQ3, this drug appears to interact with the KCNQ2 voltage sensor (S1-S4) rather than the pore region targeted by RTG. KCNQ2 point mutants in the voltage sensor were generated based on KCNQ2/KCNQ3 sequence differences, and screened for ICA73 sensitivity. These experiments reveal that KCNQ2 residues F168 and A181 in the S3 segment are essential determinants of ICA73 subtype specificity. Mutations at either position in KCNQ2 abolish the ICA73-mediated gating shift, but preserve RTG sensitivity. Interestingly, A181P mutant channels show little ICA73-mediated gating shift but retain current potentiation by the drug. Mutations (L198F and P211A), which introduce these critical KCNQ2 residues at corresponding positions in KCNQ3, transplant partial ICA73 sensitivity. These findings demonstrate that RTG and ICA73 act via distinct mechanisms, and also reveal specific residues that underlie subtype specificity of KCNQ channel openers.

Kv1.2 is a prominent voltage-gated potassium channel that influences action potential generation and propagation in the central nervous system. We explored multi-protein complexes containing Kv1.2 using mass spectrometry followed by screening for effects on Kv1.2. We report that Slc7a5, a neutral amino acid transporter, has a profound impact on Kv1.2. Co-expression with Slc7a5 reduces total Kv1.2 protein, and dramatically hyperpolarizes the voltage-dependence of activation by −47 mV. These effects are attenuated by expression of Slc3a2, a known binding partner of Slc7a5. The profound Slc7a5-mediated current suppression is partly explained by a combination of gating effects including accelerated inactivation and a hyperpolarizing shift of channel activation, causing channels to accumulate in a non-conducting state. Two recently reported Slc7a5 mutations linked to neurodevelopmental delay exhibit a localization defect and have attenuated effects on Kv1.2. In addition, epilepsy-linked gain-of-function Kv1.2 mutants exhibit enhanced sensitivity to Slc7a5.