About 3 million people die every year as a result of infection with HIV, the virus that causes AIDS. Efforts to develop a vaccine against the virus are up against a particularly clever enemy: HIV assumes different guises even after entering the human body, going through rapid genetic changes that allow it to escape the immune system. In fact, HIV undergoes more changes while residing in the body of a single person than the flu virus has undergone since it was first discovered.

 

HIV-1, the type of HIV responsible for 90% of all infections, comes in two principal forms, called R5 and X4. These differ from each other significantly, both in the type of immune cells they attack and in the progression of the disease. R5 binds to proteins on the surface of the immune cells known as macrophages, weakening the immune system in the latent stage of the disease, when the person is a carrier of the virus but has not yet developed full-blown AIDS. By contrast, X4 attaches to the membranes of different immune system cells called T cells, causing these cells to die rapidly and bringing on the fatal stage of the syndrome, in which the immune system collapses completely. Yet for all the functional disparity between these two versions of the virus, when it comes to their constitutional sequence of amino acids – the building blocks of proteins – they differ in only one or two amino acids found in a certain protein on the viral envelope.

 

How does a change in a single amino acid affect the virus’s attack mechanisms to such an extent? In a study reported in the Proceedings of the National Academy of Sciences (PNAS), Prof. Jacob Anglister and research students Osnat Rosen and Michal Sharon of the Weizmann Institute’s Structural Biology Department used nuclear magnetic resonance (NMR) to closely examine the two versions of the virus. They focused on that segment of the envelope protein called V3 which is involved in the protein’s binding to target cell membranes – a critical stage in viral infection.

 

The scientists synthesized the sequence of amino acids making up the V3 protein segment in both versions and examined the structure produced when they bind to two different antibodies. They reasoned that in binding to an antibody, V3 would assume its natural spatial shape – the one that exists in the free virus and allows it to bind to various human cells. At first glance, the experiment seemed to produce two identical V3 structures, both shaped like a hairpin. However, a closer examination revealed a difference: Whereas the line-up of amino acids on one side of the hairpin was identical in both versions, on the other side, the orientation of X4 differed from that of R5. Consequently, the position of certain bonds between the amino acids on the two sides of the hairpin also changed. In addition, in X4, the amino acid side chains on one of the hairpin’s sides were pointed in the opposite direction to those of R5, resulting in significant differences in the protein’s shape and in the spatial organization of its binding site. Because of these variations in the shape of one crucial protein, the different versions of the virus latch on to different cells – macrophages in the case of R5, T cells in the case of X4 – and attack the immune system in different ways.

 

But what is it about the changes in the chemical sequence of the viral envelope protein that brings about the structural changes? And what makes this change so dramatic? To answer these questions, the scientists created a more complete picture of the spatial structure of V3, including the amino acid responsible for the differences between the two types of HIV. The picture revealed that this amino acid has a negative electric charge in R5, which allows it to interact with a positively charged amino acid on the opposite side of the hairpin, thereby stabilizing this structure. In contrast, a mutation in the X4 version causes the negatively charged amino acid to be replaced with a positively charged one, resulting in repulsion between the two parts of the hairpin: Here, the amino acids’ side chains, which carry the electric charge, turn on their axes and distance themselves from each other. Another mutation that can result in the transition of the virus from the R5 to the X4 form creates a sequence of three positively charged amino acids, which interacts with the electron clouds of another set of amino acids located diagonally across on the other side of the hairpin. The resulting structure is stable but different from the one characterizing the viral envelope protein in R5.

 

 

Modern hearing aids are a boon for a great many deaf people, but the quality of the sound they live with is still inferior to that of hearing people. It’s hard to fault the technology – the apparatus we’re born with for translating air vibrations into comprehensible sound is one of nature’s more complex examples of fine engineering. The first step to designing more efficient hearing aids, therefore, is to improve our understanding of the mechanisms that allow most of us to hear such a wide range of sounds, and distinguish among them.

 

Research by Dr. Itay Rousso of the Weizmann Institute’s Structural Biology Department, which recently appeared in the Proceedings of the National Academy of Sciences (PNAS), suggests that a crucial membrane in the inner ear helps to distinguish among different sound frequencies. This thin structure, called the tectorial membrane, communicates between two structures in the ear: the outer hair cells – which amplify sound in the form of mechanical vibrations – and the inner hair cells – which convert these mechanical vibrations to electrical signals and pass them on to the brain via the auditory nerve. Recent studies have shown that if certain genes for this membrane are missing or damaged, total deafness ensues.

 

Rousso and research student Rachel Gueta, together with researchers at the Ben-Gurion University of the Negev, wanted to explore the mechanical properties of the tectorial membrane. Using an atomic force microscope, which probes surfaces with a fine microscopic needle, they tested the resistance of the gel-like membrane at various points to assess precisely how rigid or flexible it was. To their surprise, the scientists found that the level of rigidity varies significantly along the length of the membrane: One end of the membrane can be up to ten times more rigid than the other. 

 

These differences occur in the part of the membrane that is in direct contact with the outer hair cells. Observation under a scanning electron microscope revealed that this variation is due to changes in the way the protein fibers are arranged: At one end they form a flimsy, net-like structure that allows the membrane to be flexible; at the other, rigid end the fibers are densely and uniformly packed.

 

The more rigid the tectorial membrane, the higher the frequency at which it can vibrate. Thus the flexible end of the membrane, which can respond to low frequency vibrations, is close to the hair cells that transmit low frequencies, and the rigid end is near hair cells that transmit high frequencies. This spatial separation, say the scientists, should translate into the ability to distinguish between sounds of different frequencies.

 

The new understanding of the mechanics of hearing may assist in the development of better hearing aids. Rousso, meanwhile, plans to continue exploring how variations in membrane rigidity affect hearing. The highest frequency our ears can register is a thousand times higher than the lowest, and Rousso intends to test the membrane under different physiological conditions to further understand how it functions, as well as to possibly shed light on the causes of certain hearing problems.   

 

Dr. Itay Rousso’s research is supported by the Clore Center for Biological Physics; the Helen and Martin Kimmel Center for Nanoscale Science; the Jeans-Jacques Brunschwig Fund for the Molecular Genetics of Cancer; the Estelle Funk Foundation; and the President’s Fund for Biomedical Research. Dr. Rousso is the incumbent of the Robert Edward and Roselyn Rich Manson Career Development Chair.

 

 

 

Prof. Ada Yonath of the  Weizmann Institute’s Structural Biology Department, working in collaboration with Prof. Lou Massa of the City University of New York and Nobel laureate Prof. Jerome Karle of the U.S. Naval Research Laboratory, recently took some significant steps toward getting to the bottom of this mystery. Yonath has been studying the workings of ribosomes for over 25 years, ever since she, together with scientists from the Max Planck Institutes in Germany, first crystallized them. Ribosomes are composed of a large number of protein molecules loosely bound to giant chains of nucleic acids known as ribosomal RNA. Using a technique called X-ray crystallography, the scientists bombarded the ribosome crystals with X-rays. Some sophisticated mathematical analysis of the scattering of the X-rays bouncing off the crystals revealed the ribosome’s three-dimensional structure in detail.

 

The team’s most recent research focuses on molecular “trucks” that transport the amino acids to the protein production line. Nobel laureate Prof. Aaron Klug and Prof. Alexander Rich (both members of the Weizmann Institute Board of Governors) solved the structure of these “trucks,” called tRNA, two decades ago. Though mainly composed of double-stranded RNA, their ends are single stranded and thus more flexible. All tRNA molecules look misleadingly alike, but they are highly specialized: Each can identify the messenger RNA (mRNA) segment carrying the instructions for a particular amino acid and bring it over to the ribosome. In action, one end of the tRNA molecule attaches to the mRNA as it carries the amino acid bound to its other end to the center of action. There, the protein segment’s bond to the growing chain is manufactured. 

 

Klug and Rich had discovered the structure of these molecules at rest, but it was clear that they undergo significant changes while working. These changes take place so quickly, however, that scientists, until now, had not been able to catch them in the act. Using a molecule that simulates tRNA, Yonath and her team were able to slow down the process and “freeze” the action at various stages

 

During protein manufacture, the tRNA molecules bind to the ribosome in pairs. One of the pair carries the new amino acid to be incorporated into the growing protein chain, which, in turn, is attached to the second. Once the bond is created, the free tRNA molecule disengages to make way for the next tRNA molecule carrying an amino acid to be attached.   

 

The research team found that while the tRNA molecule’s more stable part moves along with the protein-encoding mRNA, the flexible part – composed of the single-stranded RNA – rotates around a pivot formed by the junction between the two parts. With each rotation, a bond is formed between the amino acid at the end of the tRNA and the growing protein chain.

 

This rotation is facilitated by the shape of the ribosome. While the ribosome as a whole is asymmetrical, the section where the flexible ends of the “truck” dock is symmetrical, allowing new peptide bonds to form smoothly. By knowing the binding site for the first “truck,” the team was able to calculate the position of the second “truck,” which carries the growing peptide.

 

 

 

Adult stem cells, which are specific to each tissue type, function to maintain the body’s organs over the course of a lifetime, creating new cells to replace dead ones. Like their embryonic cousins, adult stem cells can renew themselves endlessly, and the process of differentiation, in which cells take on the characteristics of specific tissues, is inhibited. However, these exact properties make stem cells good candidates for turning cancerous. Recent theories propose that when an adult stem cell mutates, it can lose some of its properties of self-control while retaining its propensity for self-renewal – a dangerous combination that may lead to cancer.

 

In the past decade, scientists have discovered small amounts of stem cells, dubbed cancer stem cells, in many different types of cancer, including leukemia, breast cancer and brain tumors. These, they believe, are responsible for the continuous growth and post-treatment relapse of the cancer. Cancer stem cells are often especially resistant to standard chemotherapy drugs; they may survive treatment and eventually renew the cancer.

 

How, then, can cancer stem cells be targeted and destroyed? Recent research by Prof. David Givol and research student Hilah Gal of the Molecular Cell Biology Department, carried out together with Prof. Tsvee Lapidot of the Immunology Department and Prof. Eytan Domany of the Physics of Complex Systems Department, aimed to see what distinguishes cancer stem cells from other cancer cells and from healthy stem cells. Also participating in the research were Prof. Gideon Rechavi and his research team from the Sheba Medical Center, Tel Hashomer.

 

Upon comparing levels of gene expression in leukemia stem cells with those of non-stem leukemia cells, the scientists found about 400 genes that are expressed differently in the two types of cancer cell. In the cancer stem cells, for instance, certain genes involved in repairing mistakes in DNA were less active, possibly explaining the fact that these cells are more liable to accumulate harmful mutations. The team then compared the expression patterns of these 400 genes to those of healthy blood stem cells. About a third of them were common to both.

 

The trick now is to examine the patterns that distinguish the cancer stem cells from normal ones. If important patterns of activity that are unique to cancer stem cells can be identified, a way might be found to block these functions in the cancer stem cells without harming healthy adult stem cells. The team discovered that over half of the genes in cancer stem cells are expressed differently from those of adult stem cells, and these will hopefully provide a starting point in the search for promising drug targets. 

   

 

 

 

 

The most familiar image of DNA, the basic material of heredity, is that of two long strands of genetic “letters” strung together and twisted around each other in the famous double helix. But inside the cell nucleus, this double strand, which reaches over a meter in length when stretched out, is compressed into neat parcels. This DNA packaging is more than just a way of maintaining tidiness in the cell nucleus – it keeps much of our DNA locked away, preventing it from being easily copied.

 

The DNA strand is first packaged into “stackable” units called nucleosomes, each about 150 base pairs in length (base pairs being the “letters” that make up the genetic sequence), which are compressed into tiny spheres around proteins. The nucleosomes are strung, bead-like, along the entire chromosome, separated by free areas of about 20 base pairs. The precise location of the nucleosomes along the double strand plays an important role in the cell’s day-to-day function: Access to nucleosome-wrapped DNA is blocked for many proteins, including those responsible for some of life’s most basic processes. Among these excluded proteins are factors that initiate DNA replication, the transfer of genetic information from DNA to RNA and the repair of damaged DNA. In other words, the positioning of nucleosomes limits the genetic segments in which these processes can take place to the brief nucleosome-free areas.

 

What determines how and where a nucleosome will be positioned along the DNA sequence? Scientists have disagreed for years whether the placement of nucleosomes in live cells is controlled by the genetic sequence itself. In an article published recently in Nature, Dr. Eran Segal and research student Yair Field of the Computer Science and Applied Mathematics Department of the Weizmann Institute proved that the DNA sequence indeed encodes “zoning” – that is, information on where to place nucleosomes. They managed, together with colleagues from Northwestern University in Illinois, to crack the genetic code that sets the rules for where on the DNA strand the nucleosomes will be situated. After they successfully characterized this code, they were able to accurately predict a large number of nucleosome positions in yeast cells, purely on the basis of the DNA sequence. 

 

Segal and his colleagues accomplished this by examining around 200 different nucleosome sites on the DNA and asking whether their sequences had anything in common. Mathematical analysis revealed similarities between the nucleosome-bound sequences and eventually uncovered a specific “code word.” This “code word” consists of a periodic signal that appears every 10 bases on the sequence. The regular repetition of this signal helps the DNA segment to bend sharply into the spherical shape required to form a nucleosome. To identify this nucleosome positioning code, the research team used probabilistic models to characterize the sequences bound by nucleosomes; they then developed a computer algorithm to predict the organization of nucleosomes along an entire chromosome.

 

The team’s findings provided insight into another mystery that has long puzzled molecular bio-logists: How do cells direct the proteins that regulate genetic processes to their intended sites on the DNA, rather than to the many similar, but functionally irrelevant sites along the genomic sequence? The short binding sites do not themselves contain enough information for these proteins to discern among them. The scientists showed that basic information on the functional relevance of a binding site is at least partially written into the nucleosome code: The intended sites are found in nucleosome-free segments, thereby allowing them to be accessed by the proteins. In contrast, spurious binding sites with identical structures that could potentially sidetrack these proteins are conveniently situated in segments that form nucleosomes, and are thus mostly inaccessible. 

 

Since the packaging proteins that form the core of the nucleosome are among the most highly conserved throughout evolution, the scientists believe that the genetic code they identified should be found in many organisms, including humans. Several diseases, among them cancer, are typically accompanied or caused by mutations in the DNA, and such mutational processes may be influenced by the relative accessibility of the DNA to various proteins and by the organization of the DNA in the cell nucleus. The scientists believe, therefore, that the nucleosome positioning code they discovered may significantly aid researchers in their attempts to understand the mechanisms underlying many diseases.  

 

For Segal, the fact that computational modeling methods were crucial to these findings may have major implications for further research: “Often, a model yields insights that, in hindsight, could have been obtained by simple statistical analysis. In this work, our modeling approach played a key role in the discovery. It was only after we devised and applied our algorithm that we were able to obtain the biological insights that led to making successful predictions and proving that genomes do indeed encode, at least in part, nucleosome positions.”    

 
Dr. Eran Segal’s research is supported by the Willner Family Leadership Institute for the Weizmann Institute  of Science; the Arie and Ida Crown Memorial Charitable Fund; and the Estelle Funk Foundation.