CKAP5

Researchers have identified a new approach to controlling bacterial infections. The team found a way to turn on a vital bacterial defense mechanism to fight and manage bacterial infections. The defense system, called cyclic oligonucleotide-based antiphage signaling system (CBASS), is a natural mechanism used by certain bacteria to protect themselves from viral attacks. Bacteria self-destruct as a means to prevent the spread of virus to other bacterial cells in the population. Researchers at the Icahn School of Medicine at Mount Sinai have identified a new approach to controlling bacterial infections.The findings were described in the February 6 online issue of Nature Structural & Molecular Biology. The team found a way to turn on a vital bacterial defense mechanism to fight and manage bacterial infections. The defense system, called cyclic oligonucleotide-based antiphage signaling system (CBASS), is a natural mechanism used by certain bacteria to protect themselves from viral attacks. Bacteria self-destruct as a means to prevent the spread of virus to other bacterial cells in the population. "We wanted to see how the bacterial self-killing CBASS system is activated and whether it can be leveraged to limit bacterial infections," says co-senior author Aneel Aggarwal, PhD, Professor of Pharmacological Sciences at Icahn Mount Sinai. "This is a fresh approach to tackling bacterial infections, a significant concern in hospitals and other settings. It's essential to find new tools for fighting antibiotic resistance. In the war against superbugs, we need to constantly innovate and expand our toolkit to stay ahead of evolving drug resistance." According to a 2019 report by the Centers for Disease Control and Prevention, more than 2.8 million antimicrobial-resistant infections occur in the United States each year, with over 35,000 people dying as a result. As part of the experiments, the researchers studied how "Cap5," or CBASS-associated protein 5, is activated for DNA degradation and how it could be used to control bacterial infections through a combination of structural analysis and various biophysical, biochemical, and cellular assays. Cap5 is a key protein that becomes activated by cyclic nucleotides (small signaling molecules) to destroy the bacterial cell's own DNA. "In our study, we started by identifying which of the many cyclic nucleotides could activate the effector Cap5 of the CBASS system," says co-senior author Olga Rechkoblit, PhD, Assistant Professor of Pharmacological Sciences at Icahn Mount Sinai. "Once we figured that out, we looked closely at the structure of Cap5 when it's bound to these small signaling molecules. Then, with expert help from Daniela Sciaky, PhD, a researcher at Icahn Mount Sinai, we showed that by adding these special molecules to the bacteria's environment, these molecules could potentially be used to eliminate the bacteria." The researchers found that determining the structure of Cap5 with cyclic nucleotides posed a technical challenge, requiring expert help from Dale F. Kreitler, PhD, AMX Beamline Scientist at Brookhaven National Laboratory. It was achieved by using micro-focused synchrotron X-ray radiation at the same facility. Micro-focused synchrotron X-ray radiation is a type of X-ray radiation that is not only produced using a specific type of particle accelerator (synchrotron) but is also carefully concentrated or focused on a tiny area for more detailed imaging or analysis. Next, the researchers will explore how their discoveries apply to other types of bacteria and assess whether their method can be used to manage infections caused by various harmful bacteria. The paper is titled "Activation of CBASS-Cap5 endonuclease immune effector by cyclic nucleotides." Other authors who contributed to this work are Angeliki Buku, PhD, and Jithesh Kottur, PhD, both with Icahn Mount Sinai. The work was funded by National Institutes of Health grants R35-GM131780, P41GM111244, KP1605010, P30 GM124165, S10OD021527, GM103310, and by the Simons Foundation grant SF349247.

Kinesin proteins eat away at microtubules, shortening them and pulling chromosomes to opposite spindle poles during cell division in most organisms. However, fission yeast lacks these proteins, and yet undergoes the same process. A recent study indicated that in fission yeast, non-kinesin proteins of the TOG/XMAP215 family promote microtubule shortening through a process known as microtubule catastrophe. Cell division, i.e., the process through which a parent cell divides into two daughter cells, is fundamental to the growth, repair, and reproduction of living organisms. During cell division, chromosomes are pulled towards opposite spindle poles through the shortening of molecular ropes known as microtubules. Microtubules, which are composed of the protein tubulin, shorten with the help of "Pac-man"-like molecules that eat away at their tips. In most organisms, these Pac-man-like molecules are a group of proteins from the kinesin-13 subfamily that lead to microtubule shortening. Interestingly, during cell division in fission yeast, microtubule shortening also occurs in the absence of kinesin-13 proteins, suggesting the involvement of other proteins. Recently, a team of researchers including Professor Masamitsu Sato and Ph.D student Yuichi Murase from the Department of Life Science and Medical Bioscience at Waseda University, Mika Toya from the Global Center of Science and Engineering at Waseda University, Junichiro Yajima from the Department of Life Sciences at the University of Tokyo, and Takahiro Hamada from the Department of Bioscience, Okayama University of Science conducted a study to identify these non-kinesin proteins. It is known that proteins from the TOG/XMAP215 family such as Dis1 are involved in microtubule elongation in certain species. However, previous findings have indicated that even in cells without the Dis1 protein, the length of the microtubules is similar to that of normal cells. Elaborating further, Sato says, "Twenty years back, researchers considered TOG proteins as conventional microtubule stabilizers and believed that kinesin is the only factor responsible for microtubule shortening to carry chromosomes. But my observation through a microscope indicated that Dis1 acted like a de-stabilizer, making me curious about how exactly microtubule shortening occurs in yeast." In this study, which was published online on November 26, 2022, in Communications Biology, the team tested their hypothesis that Dis1 might be involved in microtubule shortening in yeast. To arrive at their findings, the team conducted in vitro and in vivo experiments in yeast cells. They captured images of microtubules in yeast via fluorescence microscopy and examined how Dis1 modified microtubule length. Images captured within intervals of five seconds depicted that Dis1 promotes microtubule shortening through microtubule catastrophe -- a process in which growing microtubules suddenly switch into a rapidly shortening state. Before being split and pulled, chromosomes are held in place by microtubules emanating from the opposite poles. The team observed that Dis1 collects near the tip of the microtubule at the point in the chromosome where microtubules attach, known as the kinetochore. This suggests that Dis1 causes microtubule catastrophe by remaining anchored to the microtubule-kinetochore junction, thereby causing a sudden shortening of microtubules. To test whether Dis1 is indeed involved in chromosome pulling, the team created artificial Dis1 oligomers which were then tethered to the chromosome arm region, to induce artificial chromosome pulling in yeast. The team observed smooth and frequent pulling of chromosomal arms by the microtubules connected to the artificial Dis1 kinetochore towards the spindle poles. The interaction of Dis1 with the microtubules in fission yeast is unique and unexpected since TOG/XMAP215 proteins are known to be involved in microtubule polymerization, the process that leads to microtubule elongation in many organisms. What are the long-term applications of these findings? Sato muses, "We envision that the molecular device created in this study can be a platform for the artificial chromosome segregation machinery, which might contribute to therapy for diseases that are caused by defects in chromosome segregation. Alternatively, it could be used for the construction of artificial cells that can segregate genetic materials to descendants independently."