The MPS1 family of protein kinases.

MPS1 protein kinases are found widely, but not ubiquitously, in eukaryotes. This family of potentially dual-specific protein kinases is among several that regulate a number of steps of mitosis. The most widely conserved MPS1 kinase functions involve activities at the kinetochore in both the chromosome attachment and the spindle checkpoint. MPS1 kinases also function at centrosomes. Beyond mitosis, MPS1 kinases have been implicated in development, cytokinesis, and several different signaling pathways. Family members are identified by virtue of a conserved C-terminal kinase domain, though the N-terminal domain is quite divergent. The kinase domain of the human enzyme has been crystallized, revealing an unusual ATP-binding pocket. The activity, level, and subcellular localization of Mps1 family members are tightly regulated during cell-cycle progression. The mitotic functions of Mps1 kinases and their overexpression in some tumors have prompted the identification of Mps1 inhibitors and their active development as anticancer drugs.

Dynamic reorganization of metabolic enzymes into intracellular bodies.

Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci--such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis--to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality--and dysfunctionality--of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable.

Stressing out over tRNA cleavage.

A conserved response to stress involves endonucleolytic cleavage of cytoplasmic transfer RNAs (tRNAs) by ribonucleases that are normally secreted or sequestered. Ribonuclease activation or release is an intriguing new aspect of cellular stress responses, with a potential impact on translation, apoptosis, cancer, and disease progression.

RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2'-O-methylation.

RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2'-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and 'free' pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2'-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.

Lipid metabolism has been good to me.

My career in research has flourished through hard work, supportive mentors, and outstanding mentees and collaborators. The Carman laboratory has contributed to the understanding of lipid metabolism through the isolation and characterization of key lipid biosynthetic enzymes as well as through the identification of the enzyme-encoding genes. Our findings from yeast have proven to be invaluable to understand regulatory mechanisms of human lipid metabolism. Several rewarding aspects of my career have been my service to the Journal of Biological Chemistry as an editorial board member and Associate Editor, the National Institutes of Health as a member of study sections, and national and international scientific meetings as an organizer. I advise early career scientists to not assume anything, acknowledge others' accomplishments, and pay it forward.

A structural perspective of human RNA polymerase III.

RNA polymerase III (Pol III) is a large multisubunit complex conserved in all eukaryotes that plays an essential role in producing a variety of short non-coding RNAs, such as tRNA, 5S rRNA and U6 snRNA transcripts. Pol III comprises of 17 subunits in both yeast and human with a 10-subunit core and seven peripheral subunits. Because of its size and complexity, Pol III has posed a formidable challenge to structural biologists. The first atomic cryogenic electron microscopy structure of yeast Pol III leading to the canonical view was reported in 2015. Within the last few years, the optimization of endogenous extract and purification procedure and the technical and methodological advances in cryogenic electron microscopy, together allow us to have a first look at the unprecedented details of human Pol III organization. Here, we look back on the structural studies of human Pol III and discuss them in the light of our current understanding of its role in eukaryotic transcription.

USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites.

USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked preference for ubiquitin-cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14's catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture.

Yeast GH30 Xylanase from Sugiyamaella lignohabitans Is a Glucuronoxylanase with Auxiliary Xylobiohydrolase Activity.

Xylanases are the enzymes that catalyze the breakdown of the main hemicellulose present in plant cell walls. They have attracted attention due to their biotechnological potential for the preparation of industrially interesting products from lignocellulose. While many xylanases have been characterized from bacteria and filamentous fungi, information on yeast xylanases is scarce and no yeast xylanase belonging to glycoside hydrolase (GH) family 30 has been described so far. Here, we cloned, expressed and characterized GH30 xylanase SlXyn30A from the yeast Sugiyamaella lignohabitans. The enzyme is active on glucuronoxylan (8.4 U/mg) and rhodymenan (linear ß-1,4-1,3-xylan) (3.1 U/mg) while its activity on arabinoxylan is very low (0.03 U/mg). From glucuronoxylan SlXyn30A releases a series of acidic xylooligosaccharides of general formula MeGlcA2Xyln. These products, which are typical for GH30-specific glucuronoxylanases, are subsequently shortened at the non-reducing end, from which xylobiose moieties are liberated. Xylobiohydrolase activity was also observed during the hydrolysis of various xylooligosaccharides. SlXyn30A thus expands the group of glucuronoxylanases/xylobiohydrolases which has been hitherto represented only by several fungal GH30-7 members.

Heterologous expression of 4α-glucanotransferase: overproduction and properties for industrial applications.

4α-Glucanotransferase (4α-GTase) is unique in its ability to form cyclic oligosaccharides, some of which are of industrial importance. Generally, low amount of enzymes is produced by or isolated from their natural sources: animals, plants, and microorganisms. Heterologous expressions of these enzymes, in an attempt to increase their production for applicable uses, have been widely studied since 1980s; however, the expressions are mostly performed in the prokaryotic bacteria, mostly Escherichia coli. Site-directed mutagenesis has added more value to these expressed enzymes to display the desired properties beneficial for their applications. The search for further suitable properties for food application leads to an extended research in expression by another group of host organism, the generally-recognized as safe host including the Bacillus and the eukaryotic yeast systems. Herein, our review focuses on two types of 4α-GTase: the cyclodextrin glycosyltransferase and amylomaltase. The updated studies on the general structure and properties of the two enzymes with emphasis on heterologous expression, mutagenesis for property improvement, and their industrial applications are provided.

Isolation and molecular identification of industrially important enzyme producer yeasts from tree barks and fruits.

This study aimed to identify the yeast strains associated with the tree bark samples collected from the Aegean and Marmara regions and from rotten fruit samples. Fifty-one yeast strains were successfully isolated and screened for their abilities to produce industrially important extracellular enzymes. Thirty isolates demonstrated ability to produce at least two different enzymes and were selected for subsequent molecular identification using sequence analysis of ITS region and D1/D2 domain of the 26S rDNA. The most prevalent strains belonged to Papiliotrema laurentii (%23), Papiliotrema terrestris (%13) and Candida membranifaciens (%10). Papiliotrema laurentii and Papiliotrema terrestris recorded the highest enzymatic activities for all the screened enzymes. To the best of our knowledge, this is the first report that identifies the yeast strains associated with the tree barks of Turkey and among the limited comprehensive studies that screened considerable number of isolates for their ability to produce several industrially important enzymes.

The safety of a Kluyveromyces lactis strain lineage for enzyme production.

Kluyveromyces lactis is broadly considered as a safe yeast in food and a suitable organism for the production of food enzymes. The K. lactis enzyme production strains of DSM are used to produce a variety of enzymes, for example beta-galactosidase (lactase), chymosin and esterase. All of these production strains are derived from the same lineage, meaning they all originate from the same ancestor strain after classical mutagenesis and/or genetic engineering. Four different enzyme preparations produced with strains within this lineage were toxicologically tested. These enzyme preparations were nontoxic in repeated-dose oral toxicity studies performed in rats and were non-genotoxic in vitro. These studies confirm the safety of the DSM K. lactis strains as a production platform for food enzymes, as well as the safety of the genetic modifications made to these strains through genetic engineering or classical mutagenesis. The outcome of the toxicity studies can be extended to other enzyme preparations produced by any strain from this lineage through read across. Therefore, no new toxicity studies are required for the safety evaluation, as long as the modifications made do not raise safety concerns. Consequently, this approach is in line with the public ambition to reduce animal toxicity studies.

Beta-glucosidase activity of wine yeasts and its impacts on wine volatiles and phenolics: A mini-review.

Beta-glucosidase is an important enzyme for the hydrolysis of grape glycosides in the course of winemaking. Yeasts are the main producers of ß-glucosidase in winemaking, therefore play an important role in determining wine aroma and flavour. This article discusses common methods for ß-glucosidase evaluation, the ß-glucosidase activity of different Saccharomyces and non- Saccharomyces yeasts and the influences of winemaking conditions, such as glucose and ethanol concentration, low pH environment, fermentation temperature and SO2 level, on their activity. This review further highlights the roles of ß-glucosidase in promoting the release of free volatile compounds especially terpenes and the modification of wine phenolic composition during the winemaking process. Furthermore, this review proposes future research direction in this area and guides wine professionals in yeast selection to improve wine quality.

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells.

The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer-polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions ("sticking") following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE's opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer-polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

Exploiting the Diversity of Saccharomycotina Yeasts To Engineer Biotin-Independent Growth of Saccharomyces cerevisiae.

Biotin, an important cofactor for carboxylases, is essential for all kingdoms of life. Since native biotin synthesis does not always suffice for fast growth and product formation, microbial cultivation in research and industry often requires supplementation of biotin. De novo biotin biosynthesis in yeasts is not fully understood, which hinders attempts to optimize the pathway in these industrially relevant microorganisms. Previous work based on laboratory evolution of Saccharomyces cerevisiae for biotin prototrophy identified Bio1, whose catalytic function remains unresolved, as a bottleneck in biotin synthesis. This study aimed at eliminating this bottleneck in the S. cerevisiae laboratory strain CEN.PK113-7D. A screening of 35 Saccharomycotina yeasts identified six species that grew fast without biotin supplementation. Overexpression of the S. cerevisiaeBIO1 (ScBIO1) ortholog isolated from one of these biotin prototrophs, Cyberlindnera fabianii, enabled fast growth of strain CEN.PK113-7D in biotin-free medium. Similar results were obtained by single overexpression of C. fabianii BIO1 (CfBIO1) in other laboratory and industrial S. cerevisiae strains. However, biotin prototrophy was restricted to aerobic conditions, probably reflecting the involvement of oxygen in the reaction catalyzed by the putative oxidoreductase CfBio1. In aerobic cultures on biotin-free medium, S. cerevisiae strains expressing CfBio1 showed a decreased susceptibility to contamination by biotin-auxotrophic S. cerevisiae This study illustrates how the vast Saccharomycotina genomic resources may be used to improve physiological characteristics of industrially relevant S. cerevisiaeIMPORTANCE The reported metabolic engineering strategy to enable optimal growth in the absence of biotin is of direct relevance for large-scale industrial applications of S. cerevisiae Important benefits of biotin prototrophy include cost reduction during the preparation of chemically defined industrial growth media as well as a lower susceptibility of biotin-prototrophic strains to contamination by auxotrophic microorganisms. The observed oxygen dependency of biotin synthesis by the engineered strains is relevant for further studies on the elucidation of fungal biotin biosynthesis pathways.

Catalytic Role of Gln202 in the Carboligation Reaction Mechanism of Yeast AHAS: A QM/MM Study.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate-dependent enzyme involved in the biosynthesis of valine, leucine, isoleucine, and lysine. Experimental evidence has shown that mutation of the Gln202 residue results in a decrease in the enzymatic activity, thus suggesting the main role of the carboligation catalyzed by AHAS. It has been postulated that this residue acts as an acid/base group, protonating the carbonyl oxygen from the 2-ketoacid substrate, during the carboligation reaction. However, previous studies have revealed that 2-ketoacid is not engaged in catalytically relevant interactions with ionizable groups that can act as an acid/base group during the catalysis. Therefore, it has been proposed that the carboligation reaction could occur through an intramolecular proton transfer without the assistance of an amino acid residue with acid-base properties. To decipher the role of Gln202, in this work, we studied the last two catalytic steps of the AHAS through quantum mechanics/molecular mechanics calculations using a full enzyme model of the wild-type AHAS and the Gln202Ala mutant. Our results indicate that the carboligation mechanism occurs through an intramolecular proton transfer that does not require the action of an additional acid-base group. The mechanism is composed of two steps in which the last one is rate-limiting. Our findings reveal that Gln202 stabilizes a catalytic water molecule in the reactive site through electrostatic contributions that are mostly relevant during the carboligation step, in agreement with experimental evidence. The catalytic water engages in intermolecular hydrogen bonds with the reacting species and makes a strong electronic contribution to the stabilization of the reaction intermediate (AL-ThDP).

The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men.

Protein lysine deacetylases have a pivotal role in numerous biological processes and can be divided into the Rpd3/Hda1 and sirtuin families, each having members in diverse organisms including prokaryotes. In vertebrates, the Rpd3/Hda1 family contains 11 members, traditionally referred to as histone deacetylases (HDAC) 1-11, which are further grouped into classes I, II and IV. Whereas most class I HDACs are subunits of multiprotein nuclear complexes that are crucial for transcriptional repression and epigenetic landscaping, class II members regulate cytoplasmic processes or function as signal transducers that shuttle between the cytoplasm and the nucleus. Little is known about class IV HDAC11, although its evolutionary conservation implies a fundamental role in various organisms.

Structural and biochemical analysis of DNA lesion-induced RNA polymerase II arrest.

Transcription, catalyzed by RNA polymerase II (Pol II) in eukaryotes, is the first step in gene expression. RNA Pol II is a 12-subunit enzyme complex regulated by many different transcription factors during transcription initiation, elongation, and termination. During elongation, Pol II encounters various types of obstacles that can cause transcriptional pausing and arrest. Through decades of research on transcriptional pausing, it is widely known that Pol II can distinguish between different types of obstacles by its active site. A major class of obstacles is DNA lesions. While some DNA lesions can cause transient transcriptional pausing, which can be bypassed by Pol II itself or with the help from other elongation factors, bulky DNA damage can cause prolonged transcriptional pausing and arrest, which signals for transcription coupled repair. Using biochemical and structural biology approaches, the outcomes of many different types of DNA lesions, DNA modifications, and DNA binding molecules to transcription were studied. In this mini review, we will describe the in vitro transcription assays with Pol II to investigate the impacts of various DNA lesions on transcriptional outcomes and the crystallization method of lesion-arrested Pol II complex. These methods can provide a general platform for the structural and biochemical analysis of Pol II transcriptional pausing and bypass mechanisms.

Analysis of RNA polymerase II ubiquitylation and proteasomal degradation.

Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.

In vitro reconstitution of yeast RNA polymerase II transcription initiation with high efficiency.

Transcription initiation can be reconstituted from highly purified general transcription factors (GTFs), RNA polymerase II (pol II), and promoter DNA. However, earlier biochemical reconstitution systems had a serious technical limitation, namely very poor initiation efficiency. Due to the poor efficiency of the reaction and trace amounts of proteins involved in the pre-initiation complex (PIC) assembly, detection of transcription and PIC formation was only possible by the synthesis of a radiolabeled transcript and by immunoblotting for PIC components on templates. Here we describe a transcription system that is capable of initiating transcription with >90% efficiency of template usage using homogeneous, active yeast components including TFIIA, TFIIB, TBP, TFIIE, TFIIF, TFIIH, Sub1, and pol II. The abundant specifically assembled PICs on promoter DNA can be separated from free general transcription factors (GTFs) and pol II by density gradient sedimentation, irrespective of the length of promoter DNA. The system is robust, and can be modified to accommodate many other transcription factors, and the resulting complexes can be analyzed by SDS-PAGE followed by Coomassie Blue staining. This technical advance now paves the way to conduct definitive biochemical and structural studies of the complete process of pol II initiation from the PIC, through promoter escape, and finally to productive elongation.