Hamid Mirzaei

Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

A database of mass spectrometric assays for the yeast proteome

To the Editor: The current most widely used mass spectrometry (MS)-based proteomic methods sample the available proteome in a quasi-random manner. In each analysis only a subset of the proteins contained in a sample are measured, and replicate analyses sample partly overlapping proteome segments. In addition, these analyses are biased toward abundant proteins1,2. This limits the feasibility of consistently and comprehensively measuring defined sets of proteins (such as proteins constituting signaling or metabolic networks) across different samples, thus precluding the generation of complete datasets to support mathematical modeling of biological processes. To overcome these limitations we previously proposed a targeted proteomic strategy1,2 based on a MS technique called selected reaction monitoring (SRM, also referred to as multiple reaction monitoring (MRM)). This strategy can be used to generate specific, quantitative MS assays for the proteins of interest, which can be then applied for protein detection and quantification in multiple biological samples. First, for each target protein, proteotypic peptides (peptides that unambiguously represent these proteins and are preferentially detectable by MS (ref. 3)) are selected. Then precursor/fragment ion relationships are established, which identify each proteotypic peptide. These consist of pairs of massto-charge ratio (m/z) values that are selected by the two mass analyzers of a triple quadrupole (QQQ) mass spectrometer to isolate the targeted peptide ion and corresponding, diagnostic fragment ion(s). The detector then counts the analytes matching the defined relationship(s) and returns a signal intensity over the chromatographic time. These relationships, termed SRM (or MRM) transitions, therefore effectively constitute MS assays that identify a peptide and, by inference, the corresponding protein in a complex digest. The assays are quantitatively accurate, in particular if isotopically labeled standards are used. SRM measurements result in higher sensitivity (low-attomolar4) and specificity compared to other MS-based proteomic techniques. Yeast proteins spanning all ranges of abundance, from 1.3 million to <100 copies/cell can be identified and quantified by SRM in unfractionated whole yeast digests (P.P. et al., unpublished data). However, despite these favorable characteristics, SRM has not been broadly applied in proteomics, because of the effort required for establishing SRM assays for every protein. The initial selection of optimal proteotypic peptides is complicated by MS signal responses that vary greatly for different tryptic peptides of the same protein. In addition, for each proteotypic peptide, predominant fragment ions have to be selected that define the SRM transitions. As most fragment ion spectra are generated on instruments other than QQQ mass spectrometers, and operating conditions are often poorly documented, the commonly accessible fragment ion spectra can only be a starting point for establishing optimal Figure 1 | Categorization of the yeast proteins represented in MRMAtlas by function and cellular abundance. (a) Grouping of open reading frames by biological processes in S. cerevisiae is according to the Gene Ontology database nomenclature. The same ORF can map to more than one Gene Ontology biological process. (b) Yeast protein abundances in MRMAtlas are derived from a published dataset5. Organelle organization or genesis

Phosphorylation-Regulated Binding of RNA Polymerase II to Fibrous Polymers of Low-Complexity Domains

The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD.

A Gain-of-Function Mutation in DHT Synthesis in Castration-Resistant Prostate Cancer

Growth of prostate cancer cells is dependent upon androgen stimulation of the androgen receptor (AR). Dihydrotestosterone (DHT), the most potent androgen, is usually synthesized in the prostate from testosterone secreted by the testis. Following chemical or surgical castration, prostate cancers usually shrink owing to testosterone deprivation. However, tumors often recur, forming castration-resistant prostate cancer (CRPC). Here, we show that CRPC sometimes expresses a gain-of-stability mutation that leads to a gain-of-function in 3β-hydroxysteroid dehydrogenase type 1 (3βHSD1), which catalyzes the initial rate-limiting step in conversion of the adrenal-derived steroid dehydroepiandrosterone to DHT. The mutation (N367T) does not affect catalytic function, but it renders the enzyme resistant to ubiquitination and degradation, leading to profound accumulation. Whereas dehydroepiandrosterone conversion to DHT is usually very limited, expression of 367T accelerates this conversion and provides the DHT necessary to activate the AR. We suggest that 3βHSD1 is a valid target for the treatment of CRPC.

P7C3 Neuroprotective Chemicals Function by Activating the Rate-Limiting Enzyme in NAD Salvage

The P7C3 class of aminopropyl carbazole chemicals fosters the survival of neurons in a variety of rodent models of neurodegeneration or nerve cell injury. To uncover its mechanism of action, an active derivative of P7C3 was modified to contain both a benzophenone for photocrosslinking and an alkyne for CLICK chemistry. This derivative was found to bind nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme involved in the conversion of nicotinamide into nicotinamide adenine dinucleotide (NAD). Administration of active P7C3 chemicals to cells treated with doxorubicin, which induces NAD depletion, led to a rebound in intracellular levels of NAD and concomitant protection from doxorubicin-mediated toxicity. Active P7C3 variants likewise enhanced the activity of the purified NAMPT enzyme, providing further evidence that they act by increasing NAD levels through its NAMPT-mediated salvage.

Sulfur Amino Acids Regulate Translational Capacity and Metabolic Homeostasis through Modulation of tRNA Thiolation

Protein translation is an energetically demanding process that must be regulated in response to changes in nutrient availability. Herein, we report that intracellular methionine and cysteine availability directly controls the thiolation status of wobble-uridine (U34) nucleotides present on lysine, glutamine, or glutamate tRNAs to regulate cellular translational capacity and metabolic homeostasis. tRNA thiolation is important for growth under nutritionally challenging environments and required for efficient translation of genes enriched in lysine, glutamine, and glutamate codons, which are enriched in proteins important for translation and growth-specific processes. tRNA thiolation is downregulated during sulfur starvation in order to decrease sulfur consumption and growth, and its absence leads to a compensatory increase in enzymes involved in methionine, cysteine, and lysine biosynthesis. Thus, tRNA thiolation enables cells to modulate translational capacity according to the availability of sulfur amino acids, establishing a functional significance for this conserved tRNA nucleotide modification in cell growth control.

Comparative Evaluation of Current Peptide Production Platforms Used in Absolute Quantification in Proteomics

Absolute quantification of peptides by mass spectrometry requires a reference, frequently using heavy isotope-coded peptides as internal standards. These peptides have traditionally been generated by chemical stepwise synthesis. Recently a new way to supply such peptides was described in which nucleotide sequences coding for the respective peptides are concatenated into a synthetic gene (QconCAT). These QconCATs are then expressed to produce a polypeptide consisting of concatenated peptides, purified, quantified by various methods, and then digested to yield the final internal standard peptides. Although both of these methods for peptide production are routinely used for absolute quantifications, there is currently no information regarding the accuracy of the quantifications made in each case. In this study, we used sets of synthetic and biological peptides in parallel to evaluate the accuracy of either method. We also addressed some technical issues regarding the preparation and proper utilization of such standard peptides. Twenty-five peptides derived from the Caenorhabditis elegans proteome were selected for this study. Twenty-four were successfully chemically synthesized. Five QconCAT genes were designed, each a concatenation of the same 25 peptides but each in separate, different randomized order, and expressed via in vitro translation reactions that contained heavy isotope-labeled lysine and arginine. Three of the five QconCATs were successfully produced. Different digestion conditions, including various detergents and incubation conditions, were tested to find those optimal for the generation of a reproducible and accurate reference sample mixture. All three QconCAT polypeptides were then digested using the optimized conditions and then mixed in a 1:1 ratio with their synthetic counterparts. Multireaction monitoring mass spectrometry was then used for quantification. Results showed that the digestion protocol had a significant impact on equimolarity of final peptides, confirming the need for optimization. Under optimal conditions, however, most QconCAT peptides were produced at an equimolar ratio. A few QconCAT-derived peptides were largely overestimated due to problems with solubilization or stability of the synthetic peptides. Although the order in which the peptide sequences appeared in the QconCAT sequence proved to affect the success rate of in vitro translation, it did not significantly affect the final peptide yields. Overall neither the chemical synthesis nor the recombinant genetic approach proved to be superior as a method for the production of reference peptides for absolute quantification.

Nuclear export inhibition through covalent conjugation and hydrolysis of Leptomycin B by CRM1.

The polyketide natural product Leptomycin B inhibits nuclear export mediated by the karyopherin protein chromosomal region maintenance 1 (CRM1). Here, we present 1.8- to 2.0-Å-resolution crystal structures of CRM1 bound to Leptomycin B and related inhibitors Anguinomycin A and Ratjadone A. Structural and complementary chemical analyses reveal an unexpected mechanism of inhibition involving covalent conjugation and CRM1-mediated hydrolysis of the natural products’ lactone rings. Furthermore, mutagenesis reveals the mechanism of hydrolysis by CRM1. The nuclear export signal (NES)-binding groove of CRM1 is able to drive a chemical reaction in addition to binding protein cargos for transport through the nuclear pore complex.

Type III Effector VopC Mediates Invasion for Vibrio Species

Vibrio spp. are associated with infections caused by contaminated food and water. A type III secretion system (T3SS2) is a shared feature of all clinical isolates of V. parahaemolyticus and some V. cholerae strains. Despite its being responsible for enterotoxicity, no molecular mechanism has been determined for the T3SS2-dependent pathogenicity. Here, we show that although Vibrio spp. are typically thought of as extracellular pathogens, the T3SS2 of Vibrio mediates host cell invasion, vacuole formation, and replication of intracellular bacteria. The catalytically active effector VopC is critical for Vibrio T3SS2-mediated invasion. There are other marine bacteria encoding VopC homologs associated with a T3SS; therefore, we predict that these bacteria are also likely to use T3SS-mediated invasion as part of their pathogenesis mechanisms. These findings suggest a new molecular paradigm for Vibrio pathogenicity and modify our view of the roles of T3SS effectors that are translocated during infection.

Marker for type VI secretion system effectors.

Significance The recently discovered type VI secretion system (T6SS) is used by Gram-negative bacteria to deliver effector proteins into both eukaryotic and prokaryotic neighboring cells to mediate virulence and competition, respectively. Even though several T6SS effector families have been described, many T6SSs are not associated with known effectors. In this work, we report the discovery of a conserved motif named MIX (marker for type six effectors) that is often located near the T6SS genome neighborhood and is found in numerous proteins from diverse Proteobacteria, among them several T6SS effectors. We show that the MIX motif can be used as a marker to identify new T6SS effectors, thereby significantly enlarging the list of known T6SS effector families. Bacteria use diverse mechanisms to kill, manipulate, and compete with other cells. The recently discovered type VI secretion system (T6SS) is widespread in bacterial pathogens and used to deliver virulence effector proteins into target cells. Using comparative proteomics, we identified two previously unidentified T6SS effectors that contained a conserved motif. Bioinformatic analyses revealed that this N-terminal motif, named MIX (marker for type six effectors), is found in numerous polymorphic bacterial proteins that are primarily located in the T6SS genome neighborhood. We demonstrate that several MIX-containing proteins are T6SS effectors and that they are not required for T6SS activity. Thus, we propose that MIX-containing proteins are T6SS effectors. Our findings allow for the identification of numerous uncharacterized T6SS effectors that will undoubtedly lead to the discovery of new biological mechanisms.