JUPITER, Fla.—Quality control is a rather pivotal facet of the pharmaceutical industry. Drugs must be safe and efficacious, have minimal side effects and be manufactured from quality products. They must reach high standards in the clinic, and face even stricter evaluation from regulatory authorities before they ever reach the commercial market. And as it turns out, the human body is also a fan of quality control, as a Florida team from The Scripps Research Institute has discovered that ribosomal assembly features its own quality control steps in the manufacture of proteins.
The discovery was published Sept. 7 online ahead of print in a study titled “The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation,” which appeared in Molecular Cell.
Ribosomes work to read messenger RNA (mRNA) and translate it into proteins, which are assembled from amino acids. Nearly 200 proteins known as assembly factors, plus four RNA molecules and 78 ribosomal proteins that comprise a mature ribosome, are involved in ribosome assembly.
“We used genetic and biochemical experiments to show that bypassing this step in translation-like cycle produces defects,” said Homa Ghalei, the first author of the study. “Our results show that this cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.”
“We now understand how this complicated assembly process works, and we can finally show that this really is a quality control mechanism—a significant advance over a discovery we made back in 2012, which we were never able to prove,” said Katrin Karbstein, an associate professor in the Department of Integrative Structural and Computational Biology, who led the study. “But even then, it seemed to be the most logical conclusion.”
As the authors explain in the paper’s abstract, “the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance.” Those subunits, known as 40S ribosomal subunits, bind to other subunits to produce larger ribosomes, which are referred to as 80S-like ribosomes and do not produce proteins.
Such defects as mentioned above are a serious issue, as there is a known link between defects in ribosome assembly and cancer, as well as other diseases. As noted on the Karbstein lab’s webpage, “when cells cannot assemble the number of ribosomes needed for normal growth, or if the ribosomes that are assembled are compromised, diseases such as Diamond-Blackfan anemia, 5q-syndrome and congenital asplenia occur. These are also associated with a ~30-fold higher cancer incidence.”
The webpage explores that cancer connection further, explaining that “Conversion of the stable translationally inactive assembly intermediate in Figure 1 to mature ribosomes is initiated by the dissociation of the assembly factor Ltv1 (Strunk et al., Cell 2012). We have recently shown that this dissociation is mediated by the casein kinase 1d homolog Hrr25 (Ghalei et al., in revision). Importantly, Hrr25 is not essential when Ltv1 is deleted from yeast, demonstrating that the essential function of Hrr25 lies in ribosome assembly, and not any of the other cellular processes including DNA repair, cell cycle progression, etc. Excitingly, the role of Hrr25/CK1δ is conserved in human cells. In collaboration with the Cleveland and Roush labs, we have shown that a small molecule currently in development against triple-negative breast cancer works through the CK1δ/Ltv1 circuit. This validates the ribosome assembly pathway as a novel cancer drug target.”
“With important cellular machines like ribosomes, it makes sense that mechanisms exist to make sure things work correctly,” remarked Karbstein. “We’ve confirmed for the first time that such a quality control function exists for these small ribosomal subunits that, in essence, do a test run but don’t produce a protein—without it, mistakes in RNA translation would produce dangerous mutated proteins.”
Other authors of the study include Juliette Trepreau, Jason C. Collins, Hari Bhaskaran and Bethany S. Strunk, all of TSRI.
Opening the door to a ‘functional cure’ for HIV
JUPITER, FLA.—In findings that open the door to a completely different approach to curing HIV infections, scientists from the Florida campus of The Scripps Research Institute (TSRI) have for the first time shown that a novel compound effectively suppresses production of the virus in chronically infected cells and prevents viral rebound, even when those infected cells are subjected to vigorous stimulation. The study was led by Susana Valente, a TSRI associate professor and published online Oct. 17 before print in the journal Cell Reports.
“No other antiretroviral used in the clinic today is able to completely suppress viral production in infected cells in vivo,” Valente said. “When combining this drug with the standard cocktail of antiretrovirals used to suppress infection in humanized mouse models of HIV-1 infection, our study found a drastic reduction in virus RNA present—it is really the proof of concept for a ‘functional cure.’”
Valente, a pioneer in this new approach, calls it “Block-and-Lock”—the approach blocks reactivation of the virus in cells, even during treatment interruptions, and locks HIV into durable state of latency.
Valente and her colleagues use a derivative of a natural compound called didehydro-Cortistatin A (dCA), which blocks replication in HIV-infected cells by inhibiting the viral transcriptional activator, called Tat, halting viral production, reactivation and replenishment of the latent viral reservoir.
“Combining dCA with antiretroviral therapy accelerates HIV-1 suppression and prevents viral rebound after treatment interruption, even during strong cellular activation,” Valente said. “It’s important to note that our study uses the maximum tolerable dose of the drug, with virtually no side effects.”
The scientists studied the combination therapy in a mouse model of HIV latency and persistence. Once the combined treatment regimen was halted, viral rebound was delayed up to 19 days, compared with just seven days in mouse models receiving only antiretroviral treatment.
“This demonstrates the potential of ‘block-and-lock’ strategies,” said Cari F. Kessing, a TSRI research associate and co-first author of the study. “This study shows that a ‘functional cure’ approach can succeed in reducing residual virus in the blood during anti-retroviral treatment and limiting viral rebound during treatment interruption.”
“In half of the dCA-treated mice, the virus was undetectable for 16 days after all treatment was halted,” said the University of North Carolina’s Christopher Nixon, another first author.
“We blocked Tat, and the cell’s machinery did the rest,” added Chuan Li, a TSRI research associate and a co-author of the study. “The result was that the HIV promoter becomes repressed.”
Valente pointed out that the animal models were exposed to just a single month of treatment. “That’s a relatively short period of time,” she said. “We think longer treatments will result in longer, or even permanent, rebound delays. The question is how long? We’re studying that now.”