Methanospirillum hungatei cells and flagella structure

The archaeal motility structure (flagellum /archaellum)

During an initial structural and proteomic survey of the model archaeal microbe Methanospirillum hungatei we identified proteins comprising the attached cellular flagellum. Little was previously known about these archaeal appendages and we therefore characterized them and obtained the first atomic-level structure of a model archaeal flagellum. The findings reveal properties distinct from the bacterial flagellar counterpart regarding subunit size, post translational modifications and tertiary structure. Experiments are continuing to further characterize the post translational glycan modifications to the M. hungatei flagellum in collaborations by the Gunsalus and Loo Laboratories of the UCLA-DOE Institute.

https://www.nature.com/articles/nmicrobiol2016222

Genome-wide transcriptomics in Chlamydomonas reinhardtii during day-night cycles reveals new metabolic patterns in light and stress response (Merchant lab)

A day in the life of Chlamydomonas

The unicellular green alga Chlamydomonas reinhardtii excels at acclimating to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among new findings is the observation that Chlamydomonas exhibits low respiratory activity at night and relies instead on fermentative metabolism; we propose that the ferredoxin FDX9 acts as the electron donor to fermentative hydrogenases. The light stress responsive genes PSBS, LHCSR1 and LHCSR3 all show an acute response to light at dawn under abrupt dark-to light transitions. LHCSR3 genes also exhibits a later burst in expression in the middle of the day in response to higher light intensities. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with co-expression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.

Figure 1: Heatmap representing gene expression for 11,306 differentially expressed genes over the diurnal cycle. Chlamydomonas cells grown under light-dark cycles accumulate biomass as a direct consequence of photosynthesis during the day, and divide shortly after dusk.

Figure 2: Diagram of Chlamydomonas cell division progression over the diurnal cycle.
Cells grow in volume and accumulate biomass during the day, using photosynthesis to fuel growth. Around dusk, cells will initiate the mitotic division and yield two daughter cells per mother cell. At night, daughter cells will regenerate their flagella, which were resorbed before cell division, and enter the quiescent phase of the cell cycle until the following dawn. 

Development of a new enzyme reagent that labels proteins with peptides via a specific lysine-isopeptide bond at high yield (Clubb lab)

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, few enzyme-catalyzed methods are currently available to selectively conjugate peptides to internal sites within proteins. To overcome this problem, the Clubb, Loo and Ton-That laboratories have developed a new enzyme reagent that labels proteins with peptides via a specific lysine-isopeptide bond at high yield. The new tool may be useful in creating antibody drug conjugates.

McConnell SA, Amer BR, Muroski J, Fu J, Loo RO, Loo JA, Osipiuk J, Ton-That J and Clubb RT. Protein labeling via a specific lysine-isopeptide bond using the pilin polymerizing sortase from Corynebacterium diphtheria. Journal of the American Chemical Society 140 2018; 8420-8423.

New method to create stable enzyme coated B. subtilis cells for biotechnological applications.

Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. The Clubb group developed a unique two-step procedure that enables the construction of enzyme coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3 days. The results of this work could aid the development of whole cell display systems that have useful biotechnological applications.

Huang GL, Gosschalk JE, Kim YS and Clubb RT. Stabilizing displayed proteins on vegetative Bacillus subtilis cells. Applied Microbiology and Biotechnology 102 2018; 6547-6565

UCLA Scientists Test New Strategy That Could Help Fight Ovarian Cancer

 

Approach borrows from technique of inhibiting protein fibers that cause Alzheimer’s and Parkinson’s diseases
* High-grade serous ovarian cancers are aggressive tumors, and most patients relapse despite standard therapy
* UCLA researchers developed a peptide that penetrates cancer cells and stops their growth by restoring the protective function of the p53 protein
* Scientists hope to test this therapeutic approach to treat women with ovarian cancer in the near future

UCLA scientists have developed a promising novel method to treat gynecologic tumors. The approach focuses on a protein called p53, which is commonly mutated in women who have high-grade serous ovarian cancer, the deadliest form of reproductive cancer. In many women with the disease, the cancer is very advanced by the time it is diagnosed and is therefore difficult to treat.

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International collaboration co-led by Prof. David Eisenberg elucidates the mechanism of safe storage and action of the potent human toxin MBP-1

Eosinophils are white blood cells that are part of the body’s innate immune defense against pathogens. Once an infection occurs, eosinophils are activated and migrate to the infected tissue where they contribute to kill the invading microorganisms (bacteria, viruses or helminths) by secreting a number of toxic proteins, including the Major Basic Protein (MBP-1).
While eosinophils are typically maintained at very low numbers in blood, in certain diseases, such as allergies, bronchial asthma, eosinophilic esophagitis or other eosinophilic syndromes, a highly increased number of cells is present. In these diseases, eosinophils can be aberrantly activated and infiltrate organs where, by releasing MBP-1 and other toxins, they can generate substantial tissue damage.
The Eisenberg lab co-led an international collaboration of scientists from over 10 different institutions to elucidate the details of how the powerful MBP-1 toxin is safely stored inside the eosinophil cell as well as the mechanism of its toxicity upon extracellular release. The findings were published this month in Molecular Cell.

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Crowdsourcing the phase problem

Successful preliminary studies suggest that with further development the crowdsourcing approach could fill a gap in current crystallographic methods by making it possible to extract meaningful information in cases where limited resolution might otherwise prevent initial phasing.

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Structure and identification of a pterin dehydratase-like protein as a ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) assembly factor in the α-carboxysome.

Nicole Wheatley in the Yeates lab recently received her Ph.D. degree after her success in discovering a RuBisCO chaperone, dubbed alpha-carboxysome RiBisCO assembly factor (acRAF). Carboxysomes are bacterial microcompartments that assist in the fixation of carbon dioxide from the atmosphere. RuBisCO is the enzyme that catalyzes the fixation, and is estimated to be the most abundant enzyme on earth.

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Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3.

The Bowie lab uncovered the structural basis for autosomal dominant polycystic kidney disease, the most common genetic disorder leading to end-stage renal failure in humans. The team, lead by Catherine Leettola discovered the identities of the pair of proteins which interact normally in healthy patients but fail to interact in patients affected by the disease (ANKS3 and ANKS6).

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Protein crystal structure obtained at 2.9 Å resolution from injecting bacterial cells into an X-ray free-electron laser beam.

The Eisenberg lab led an international team of 22 scientists in obtaining a 2.9 angstrom resolution protein crystal structure by injecting bacterial cells into an X-ray free-electron laser beam. Their accomplishment was remarkable because unlike the ∼100,000 biological structures determined by X-ray crystallography to date, the macromolecule under study was not extracted from the cells that produced it.

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