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A protein complex that maintains order in the cell

Researchers believe that the defective transport of proteins can be linked with diseases such as Alzheimer’s. Prof. Dr. Elke Deuerling and Dr. Martin Gamerdinger, molecular biologists from the University of Konstanz, have now discovered what is necessary to prevent erroneous protein transport.

Proteins are the building blocks of life. The production and transport of proteins in the cells follows complex patterns. Specific signals in the proteins and transport factors ensure that proteins arrive safely at their intended destinations, for example the endoplasmic reticulum (ER) or the mitochondria. A team of researchers headed up by Professor Elke Deuerling in the Department of Molecular Microbiology at the University of Konstanz, has now discovered that successful protein transport and correct sorting requires not only the signal recognition particle (SRP), which stimulates the transport of specific proteins to the ER, but also the nascent polypeptide-associated complex (NAC). Little has previously been known about the role of NAC in the cell. "Many different functions have been postulated for NAC, including a role in the co-translational transport of proteins into the ER. However, data have been quite contradictory," says Prof. Deuerling, speaking of previous scientific knowledge of NAC. Together with Dr. Martin Gamerdinger, Prof. Deuerling has elucidated the effect and importance of the complex in protein sorting and transport.

Glossary

  • A gene is a hereditary unit which has effects on the traits and thus on the phenotype of an organism. Part on the DNA which contains genetic information for the synthesis of a protein or functional RNA (e.g. tRNA).
  • Being lytic is the feature of a bacteriophage leading to the destruction (lysis) of the host cell upon infection.
  • Mitosis is the technical term for cell division, after the preceding doubling of DNA (replication) each daughter cell contains an entire set of chromosomes.
  • Pathogenity is the ability to cause a disease. One differentiates between human, animal, and plant pathogens which specifically cause a disease in either humans, animals or plants.
  • A protein is a high-molecular complex made up of amino acids. The proteins perform a wide variety of activities in the cells and represent more than 50% of organic mass.
  • The ribosome is a cellular component composed of ribosomal RNA and protein. It is the site of protein synthesis using mRNA as a template.
  • Ribonucleic acid (abbr. RNA) is a normally single-stranded nucleic acid, which is very similar to DNA. It also consists of a sugar-phosphate backbone and a sequence of four bases. However, the sugar is a ribose and instead of thymine, RNA contains uracil. RNA has got various forms and functions; e.g. it serves as template during protein synthesis and it also constitutes the genome of RNA viruses.
  • Genetic sequences are successions of the bases adenine, thymine, guanine, and cytosine on the DNA (or uracil instead of thymine in the case of RNA).
  • Translation in a biological context is the process in which the base sequence of mRNA is translated into the amino acid sequence of a protein. This process takes place in the ribosomes. Based on a single mRNA molecule, many protein molecules can be synthesised.
  • Caenorhabditis elegans is a tiny roundworm which is a good model organism for genetics and developmental biology. Its genome, consisting of only six chromosomes, was the first one of a multicellular organism to be completely sequenced in 1998. C. elegans has always exactly 959 cells at full-grown size. It has a short generation time of about three days and is easy to grow at high densities.
  • Biochemistry is the study of the chemical processes in living organisms. Therefore it touches the studies of chemistry and biology as well as physiology.
  • Molecular biology deals with the structure, biosynthesis and function of DNA and RNA and their interaction with each other and with proteins. Molecular data can lead to an improved understanding of the reasons for diseases and can help to improve the mode of action of drugs.
  • Expression means the biosynthesis of a gene product. Usually, DNA is transcribed into mRNA and subsequently translated into proteins.
  • Molecular means: at the level of molecules.
  • Alzheimer's disease (also called Morbus Alzheimer) is a slowly progressing dementia that manifests itself in an increasing reduction of brain functions. This disease mainly affects older people. It is primarily caused by intracellular deposits of a fragment of amyloid precursor protein (APP). This leads to a proceeding loss of neurons and therfore to a loss of brain mass. At the beginning of this disease, the concerned people only show a marginal obliviousness. In later stages, speech, the ability to reason and memory are mainly affected. In the end, the concerned people lose their entire sanity and personality.
  • Fluorescence is the spontanous emission of light in a certain wavelength after excitation of a molecule with light of another wavelength.
  • A mitochondrion is an organelle in the cytoplasm of eukaryotic cells with a double-membraned organization. Also called „power station of the cell", mitochondria generate most of the cell's supply of adenosine triphosphate (ATP), the source of chemical energy. There are many mitochondria in cells (like heart and skeletal muscle cells), which require large amounts of energy.
  • Viruses possess either a DNA or RNA genome, whereas the DNA or RNA can occur as single or double stranded molecule. In the case of single stranded RNA, the genetic information becomes replicated after a definite period of time. Double stranded RNAs (dsRNAs) do not exist in eukaryotes. Thus, eukaryotic cells detect viral dsRNA as foreign molecules and abolish them. RNA interference (RNAi) is a complex system within living cells of plants and animals that causes gene silencing. Thus, plants and animals have the possibility to protect themselves against diseases caused by RNA viruses. In research, this mechanism is used to silence particular genes. Small chemically synthesized RNAs (small interfering RNAs, siRNAs) are introduced into cells. They are complementary to the mRNA of particular genes. The assembly of siRNA and the appropriate mRNA forms ds RNA molecules that further on become degraded. Due to the specific effects of this gene silencing mechanism, it is possible to discover the functions of silenced genes. Currently siRNAs are used in research studies to investigate their therapeutic capability in HIV, parkinson, cancer and other diseases.
The molecular biologists Dr. Martin Gamerdinger and Prof. Dr. Elke Deuerling are studying the role of the nascent polypeptide-associated complex (NAC) in protein transport. © University of Konstanz

Elke Deuerling and her team of researchers are concentrating on studying ribosome-associated chaperones, to which NAC also belongs. Their main interest is in elucidating the function of this complex at the ribosomes. Ribosomes generally have a high intrinsic affinity to the translocation pore of the ER membrane, regardless of whether they are active or whether they synthesise a protein with a signalling sequence required for the specific SRP-mediated transport of proteins into the ER. The researchers assumed that this could only be achieved by an inhibitory process that prevented unspecific ribosome binding. "We wanted to find out whether NAC had this particular function and how it affects cellular transport processes," said Prof. Deuerling.

Dr. Gamerdinger decided to study the function of the NAC protein complex in the animal model. It soon became evident that NAC played a crucial role in regulating protein transport. "Simultaneously lowering the expression of the NAC and SRP genes provided the answer that the scientists were seeking. "It led to a greatly potentiated ER stress response, and it showed us that NAC has a decisive effect on the transport of proteins to the ER," said Dr. Martin Gamerdinger.

The nematode C. elegans brings the result the scientists were seeking

Dr. Gamerdinger used an experimental set-up with Caenorhabditis elegans (C. elegans) as an experimental animal to show that NAC exerts an important regulatory function in the co-translational transport of proteins to the ER. "We chose C. elegans as a model organism because it cannot survive without NAC. Therefore, we simply used RNA interference methods to reduce the quantity of NAC in adult animals," said Prof. Deuerling. This experimental approach helped the researchers to study NAC-related defects in living animals on the molecular level as well as using biochemical methods. "Another reason for choosing C. elegans was that adult C. elegans is a post-mitotic organism that does not undergo programmed cell death in stress situations. We were thus able to precisely analyse the primary effects of NAC depletion," said Dr. Gamerdinger.

The nematode C. elegans is particularly well suited as a model organism. © University of Konstanz / Martin Gamerdinger

The absence of NAC leads to stress in the ER. This happens because ribosomes tend to bind unspecifically to the ER membrane. Without NAC, the proteins produced by the ribosomes therefore mistakenly end up in the ER as well. The researchers have shown that this is particularly true for proteins intended for the mitochondria. The misdirection of mitochondrial proteins in particular, attracted the researchers' attention as previous studies had already suggested that NAC was somehow involved in protein transport. "Our study has now shown that NAC prevents the erroneous transport of non-authentic proteins into the ER, thus indirectly promoting the transport of these proteins into the mitochondria," says Dr. Gamerdinger highlighting his group's findings. They have shown that animals with low NAC levels only live half as long as animals with normal NAC levels. This therefore revealed that ER and mitochondrial stress are the result of erroneous ribosome transport to the ER, as NAC is no longer there to inhibit the transport of non-authentic proteins into the ER.

The combination of fluorescence microscopy and bioinformatics is successful

Different techniques had to be applied in order to demonstrate the function of NAC. "The application of different methods was the only way that we could obtain a comprehensive picture and make clear statements about the processes," said Prof. Deuerling. The researchers used high-resolution confocal fluorescence microscopy to identify proteins that ended up at the wrong place, i.e. the ER lumen rather than in the mitochondria. Bioformatic approaches were used to analyse system-wide data. "Using these two approaches helped us to find out that NAC mainly prevented cytosolic ribosomes (which translate proteins without an ER signalling sequence) from adhering to the ER membrane, while ribosomes that synthesise ER-specific proteins arrived at their intended ER destination largely unaffected by NAC, and helped only by the SRP," said Dr. Gamerdinger. 

Basic research for the treatment of Alzheimer’s

The study focuses on aspects of basic research and aims to find out why the NAC is essential and which cellular processes are affected by the complex. However, the study has also come up with many interesting application-related aspects. "We have seen that animals with low NAC levels only live half as long as those with normal NAC levels. Other researchers have also observed that Alzheimer's patients have lower than normal NAC levels. However, whether NAC has a direct effect on the pathogenesis of Alzheimer's is not yet known and needs to be explored further," said Prof. Deuerling.

The next step is therefore to carry out a structural analysis in order to study how the interplay of NAC and SRP affects the sorting of proteins on the mechanical level. "We want to find out how NAC sits on the ribosome and prevents proteins from binding to the ER translocation pore," says Dr. Gamerdinger, referring to future research. "We will also explore whether the function of NAC decreases with age and under protein stress, which, amongst other things leads to Alzheimer's and other diseases," concluded Prof. Deuerling.

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