Faculty Bibliography

Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo-electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two "half-channels" with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.

Discovery of the role of bacterial RNase J1 in termination of transcription suggests common allosteric principles and mechanistic congruency of termination between bacteria and eukaryotes, in which an unrelated RNase Xrn2/Rat1 plays a similar role.

RNA polymerase-binding RNA aptamers (RAPs) are natural RNA elements that control transcription in cis by directly contacting RNA polymerase. Many RAPs inhibit transcription by inducing Rho-dependent termination in Escherichia coli. Here, we studied the role of inhibitory RAPs (iRAPs) in modulation of antisense transcription (AT) using in silico and in vivo approaches. We revisited the antisense transcriptome in cells with impaired AT regulators (Rho, H-NS and RNaseIII) and searched for the presence of RAPs within antisense RNAs. Many of these RAPs were found at key genomic positions where they terminate AT. By exploring the activity of several RAPs both in a reporter system and in their natural genomic context, we confirmed their significant role in AT regulation. RAPs coordinate Rho activity at the antisense strand and terminate antisense transcripts. In some cases, they stimulated sense expression by alleviating ongoing transcriptional interference. Essentially, our data postulate RAPs as key determinants of Rho-mediated AT regulation in E. coli.

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.

Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.

Prokaryotes and eukaryotes alike endogenously generate the gaseous molecule hydrogen sulfide (H₂S). Bacterial H₂S acts as a cytoprotectant against antibiotics-induced stress and promotes redox homeostasis. In E. coli, endogenous H₂S production is primarily dependent on 3-mercaptopyruvate sulfurtransferase (3MST), encoded by mstA. Here, we show that cells lacking 3MST acquire a phenotypic suppressor mutation resulting in compensatory H₂S production and tolerance to antibiotics and oxidative stress. Using whole genome sequencing, we identified a non-synonymous mutation within an uncharacterized LacI-type transcription factor, ycjW. We then mapped regulatory targets of YcjW and discovered it controls the expression of carbohydrate metabolic genes and thiosulfate sulfurtransferase PspE. Induction of pspE expression in the suppressor strain provides an alternative mechanism for H₂S biosynthesis. Our results reveal a complex interaction between carbohydrate metabolism and H₂S production in bacteria and the role, a hitherto uncharacterized transcription factor, YcjW, plays in linking the two.

The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 Å and 4.9 Å, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub. Both structures show Dot1L binding to the same extended surface of the histone octamer. In yeast, this surface is used by silencing proteins involved in heterochromatin formation, explaining the mechanism of their competition with Dot1. These results provide a strong foundation for understanding conserved crosstalk between histone modifications found at actively transcribed genes and offer a general model of how ubiquitin might regulate the activity of chromatin enzymes.

Introduction: Dimethyl sulfoxide (DMSO), propylene glycol (PG) and miglyol (MG) are common organic solvents often used to dissolve neuro-pharmacological agents for in vivo assays in animal models of various pediatric brain disorders. Nonetheless, these compounds were reported to exhibit pharmacological and pathological effects on the central nervous system of their own. Here we report chronic effects of these solvents on behavior, motoric functions and brain morphology in young rats (P35-37), following neonatal exposure to one of the compounds (P6).
Method(s): Compounds were administered intraperitoneally (DMSO, 2 or 4 ml/kg; MG, 2 ml/kg) or per os (PG, 2.5 ml/kg) at concentrations considered safe and non-interfering with neuroscience research. Age/sex matched controls received phosphate-buffered saline (PBS). Animals were sacrificed following behavioral and locomotor assays (P40) and their brains were subjected to pathological analyses.
Result(s): Rats exposed to DMSO (n = 10; 4 ml/kg only), spent significantly more time in the open field center and traveled shorter distance (p < 0.05) vs. controls (n = 10). Rats exposed to DMSO and MG exhibited shorter social interactions vs. controls (p < 0.05). CatWalk gait analysis showed various disturbances (p < 0.05) in rats exposed to any of the three compounds (DMSO, 4 ml/kg only). Brain pathological analyses revealed increased expression of microglia (Iba-1+) and reactive astrocytes (GFAP+) in rats exposed to DMSO (p < 0.05).
Conclusion(s): Observed chronic behavioral, motoric and morphologic sequelae of neonatal exposure to DMSO, PG or MG at concentrations that are generally considered safe raise concerns about under-appreciated neuro-toxicity of these common organic solvents, which warrants further exploration in larger translational studies

The biological mediator hydrogen sulfide (H₂S) is produced by bacteria and has been shown to be cytoprotective against oxidative stress and to increase the sensitivity of various bacteria to a range of antibiotic drugs. Here we evaluated whether bacterial H₂S provides resistance against the immune response, using two bacterial species that are common sources of nosocomial infections, Escherichia coli and Staphylococcus aureus Elevations in H₂S increased the resistance of both species to immune-mediated killing. Clearance of infections with wild type and genetically H₂S-deficient E. coli and S. aureus was compared in vitro and in mouse models of abdominal sepsis and burn wound infection. Also, inhibitors of H₂S-producing enzymes were used to assess bacterial killing by leukocytes. We found that inhibition of bacterial H₂S production can increase susceptibility of both bacterial species to rapid killing by immune cells and can improve bacterial clearance after severe burn, an injury that increases susceptibility to opportunistic infections. These findings support the role of H₂S as a bacterial defense mechanism against the host response and implicate bacterial H₂S inhibition as a potential therapeutic intervention in the prevention or treatment of infections.