O-GlcNAc protein modification in plants: Evolution and function

The role in plants of posttranslational modification of proteins with O-linked N-acetylglucosamine and the evolution and function of O-GlcNAc transferases responsible for this modification are reviewed. Phylogenetic analysis of eukaryotic O-GlcNAc transferases (OGTs) leads us to propose that plants have two distinct OGTs, SEC- and SPY-like, that originated in prokaryotes. Animals and some fungi have a SEC-like enzyme while plants have both. Green algae and some members of the Apicomplexa and amoebozoa have the SPY-like enzyme. Interestingly the progenitor of the Apicomplexa lineage likely had a photosynthetic plastid that persists in a degenerated form in some species, raising the possibility that plant SPY-like OGTs are derived from a photosynthetic endosymbiont. OGTs have multiple tetratricopeptide repeats (TPRs) that within the SEC- and SPY-like classes exhibit evidence of strong selective pressure on specific repeats, suggesting that the function of these repeats is conserved. SPY-like and SEC-like OGTs have both unique and overlapping roles in the plant. The phenotypes of sec and spy single and double mutants indicate that O-GlcNAc modification is essential and that it affects diverse plant processes including response to hormones and environmental signals, circadian rhythms, development, intercellular transport and virus infection. The mechanistic details of how O-GlcNAc modification affects these processes are largely unknown. A major impediment to understanding this is the lack of knowledge of the identities of the modified proteins.     

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

Lactobacillus casei L ‐lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6‐bisphosphate (FBP), respectively, and exhibits unusually high pH‐dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 Å resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q‐axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra‐helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L ‐lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P‐axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q‐axis related dimer through the rigid NAD‐binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH‐dependent allosteric equilibrium. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Polypyrimidine tract‐binding protein homologues from Arabidopsis underlie regulatory circuits based on alternative splicing and downstream control

Alternative splicing (AS) of precursor mRNAs is a widespread phenomenon in plants; however, many questions, especially regarding its regulation and functional implications, remain to be elucidated. In vertebrates, polypyrimidine tract‐binding proteins (PTBs) have been identified as key splicing factors influencing splice site selection and orchestrating coordinated splicing programmes during developmental processes. Here, we analysed three PTB homologues from Arabidopsis thaliana and provide evidence for their gene regulatory potential based on AS and a splicing‐independent mechanism. Our data reveal that Arabidopsis PTB homologues are subject to extensive auto‐ and cross‐regulation via AS‐coupled nonsense‐mediated decay, thereby establishing a basis for interlinking their expression. Furthermore, the multiple modes of action of Arabidopsis PTB homologues are reflected in their subcellular localization in the nucleus, cytosol and processing bodies. This work provides insight into the regulation of AS in plants and highlights the regulatory potential of the multifunctional plant PTB homologues, which might have important implications in diverse biological processes.     

Protein folding pathways and state transitions described by classical equations of motion of an elastic network model

Protein topology defined by the matrix of residue contacts has proved to be a fruitful basis for the study of protein dynamics. The widely implemented coarse-grained elastic network model of backbone fluctuations has been used to describe crystallographic temperature factors, allosteric couplings, and some aspects of the folding pathway. In the present study, we develop a model of protein dynamics based on the classical equations of motion of a damped network model (DNM) that describes the folding path from a completely unfolded state to the native conformation through a single-well potential derived purely from the native conformation. The kinetic energy gained through the collapse of the protein chain is dissipated through a friction term in the equations of motion that models the water bath. This approach is completely general and sufficiently fast that it can be applied to large proteins. Folding pathways for various proteins of different classes are described and shown to correlate with experimental observations and molecular dynamics and Monte Carlo simulations. Allosteric transitions between alternative protein structures are also modeled within the DNM through an asymmetric double-well potential.     

微生物细胞工厂中多基因表达的控制策略

微生物代谢工程和合成生物学是当今微生物技术领域研究的热点,微生物的生长速度快、容易进行大规模培养;遗传背景清楚、遗传操作简便可靠等性质使其在与人类生活相关的多个领域中起到重要的作用。微生物细胞工厂是指人工设计的能够进行物质生产的微生物代谢体系。许多微生物细胞工厂的构建由于引入多个基因或整条代谢途径,而可能导致代谢失衡、部分代谢中间产物积累等问题,需要使用一定的调控策略加以控制。以下对涉及多个基因作用的微生物细胞工厂中所使用的调控策略,分为若干层次进行了总结和探讨,并对今后多基因控制策略的发展方向进行了预测与展望。