Morphoproteomics

Morphoproteomics is a novel medical discipline that combines histopathology, molecular and cellular biology and protein biochemistry to define the protein circuitry in diseased cells. In this way it should be possible to identify specific target for customized therapeutic intervention. One preclinical study on prostate cancer overlaps data from phosphorylation analysis with phospho-specific probes and morphological evaluation of cellular compartmentalization. The identification of peculiar localization of certain phospho-analytes demonstrated the efficacy of this kind of personalized studies and their clinical utility. Another study demonstrated the feasibility of this approach in neck and head squamous carcinoma, that often has poor prognosis and high mortality rate. In this case, mTOR pathway was analyzed in terms of potential clinical significance of its activated components. Rapamycin is an inhibitor of this pathway. The inhibitory effects on cellular growth were directly correlated with mTOR activation, and a paint of the block was obtained by immuno-hystochemistry. On conclusion, morphoproteomics gives details about the signal transduction by using the combination of several techniques. This approach has been positively evaluated in certain disease, like cancer where an alteration in protein circuitry often causes the disease. Other pathological model would be available soon to validate this approach also in other disease.

Protein macroarray

Protein macroarrays are chips of gold or silver where proteins are bound and
analyzed. Several methods have been developed to attach the proteins on chip surface without interfering with their folding or functionality. In some cases, it has been useful to use particular tags at the N or C terminal of proteins of interest to facilitate the binding. The SNAP tag is one of common tag used for this purpose. This tag is based on the fusion of your protein to a small mammalian protein, the O6-alkyl-guanine- DNA- alkyl-tranferase (AGT). AGT can covalently bind a benzyl group from its substrate, resulting in a self labeling process with extremely low background. In macroarray chips, the SNAP tag can contribute to stabilize the binding through a covalent interaction. Macroarrays will be really important tools to understand the mechanism of protein- protein interaction, ligand receptor interaction as well as drug to target interaction. After the proteome project, it’s crucial that also technology will be updated to obtain quantitative results on protein functionality. The main fault of protein macroarray is the high expense to produce chips; thus it will be necessary to find out new solutions to reduce costs, and the use SNAP tag may be one of those.

Novel technique to diffract molecules

Ankylography is a novel technique that is setting up to determine the three-dimensional structure of proteins or in general macromolecules. This technique is based on the opportunity to measure the diffraction pattern through a spherical detector.
A coherent x-ray beam is launched over a single particle and the diffraction records of scattered waves is registered on a curved surface. In this way, the three- structure of an object is encoded by the 2D spherical pattern and is solved in a single shot.
Ankylography is currently at the beginning, but several applications have been already imagined: for instance, it will be possible to study the enzymatic conformational changes in the real time of the reaction, thus making realistic the opportunity to develop drugs and inhibitors for each enzymatic state. Indeed, the biggest difference between ankylography and traditional x- ray diffraction is the capability in the first case to determine the structure of a single molecule and not an average of different conformations.
To do this, additional studies and developments are needed, from a new generation of powerful x- ray generators to algorithms to elaborate the huge amount of data derived from similar experiments. Fortunately, US and Europe governments are interested in ankylography and seem to invest money in it.

Role of natural antioxidant elements in cardiovascular disease prevention

Natural antioxidant elements consist of carotenoids, vitamins, mineral salt contained in food. These components are important to remove free oxygen derived compounds that are highly reactive and generate lipid peroxidation and DNA damage. Lipid peroxidation is one cause of lipoprotein modifications. In particular low density lipoproteins (LDLs) are intensively modified by free radicals and several forms of altered LDL have been identified.
The overall effect is an increasing likelihood to produce atherosclerotic lesions, followed by an increased risk to have cardiovascular problems. In vitro some evidences about the ability of natural antioxidants to remove free radicals have been observed, but it’s not totally clear what is the significance in human, because clinical studies still are controversial. Even if some studies demonstrated a protective role of natural antioxidants against cardiovascular diseases, it’s quite difficult to evaluate the exact concentration of these components in the serum, and few dose- response studies are available up to date. Vitamin E seems particularly involved in cardiovascular disease protection because low levels of this compound have been measured in patients who had stroke or other cardiovascular problems. Unfortunately, prospective study is a limited tool to evaluate the efficacy of vitamin supplement as a tool to prevent heart failure. Further analyses must be done to ascertain the role of antioxidants in disease prevention; at this time we can still eat a lot of fruit and vegetables for our pleasure.

Novel applications of nanobodies

Camelid derived single domain peptides are a novel form of antibody which maintain the same antigen binding properties but have greater stability and smaller size than traditional antibody. These molecules can be conjugate with several chromophores, for instance with green fluorescent protein for cellular imaging applications.
A group from the University of Munich (German) applied its deepen knowledge about GFP modifications to nanobodies, this is the name of camelid peptides. The result was an improvement in GFP brightness modulation. By performing a phage display screening, they found out seven different molecules able recognize GFP and enhance or minimize its fluorescent signal. To validate the system, they produced a GFP- tagged oestrogen receptor and a nuclear binding enhancer nanobody, in presence of hormone the receptor moved into the nucleus and GFP signal increased five- fold its brightness. Several applications can be thought for this tool. For instance, nanobodies for each cellular compartment can be useful to determine how the protein of interest tagged with GFP change their position inside the cells in response to specific stimuli. Alternatively, protein- protein interaction can be studied because nanobody can bind one protein and after interaction with the second protein fluorescent signal can be modulated, as well as it should be possible to evaluate the duration of interaction self. A novel and flexible tool is now available for biologists.

The value of negative results

Negative results are similarly important than positive results for science advances. Indeed, a large number if hypothesis could be excluded if we considered negative results from other studies. Unfortunately, negative results are not so easily accepted and published in peer reviewed journal, thus are not available for science community.
Scientists from the German Research Centre for Environmental Health and from the University of Munich spent their time to collect negative results from protein-protein interaction studies and built a database, called “negatome”. They used data from literature search and from structural information retrieved into Protein Data Bank and identified almost two thousand of not interacting pairs. The novelty is that the not-interaction is experimentally demonstrated and published, while in previous work not- interaction was determined from not co-localization: if two proteins are differentially located, they will not interact. This sentence could be true, but is not predictive of interaction, just co-localization. The Negatome can contain some false negative pairs, but in general it can be considered a useful tool to compare and confirm results from immunoprecipitation or two hybrid system or other techniques that usually generate some false positive. We hope that this example should be followed by other databases for negative results.

Monoclonal antibodies

Monoclonal antibodies are important tool in molecular biology, diagnostics and clinical studies. These protein are produced by a single cells isolated from immunized animals. Current protocol requires an immunization of an animal host, for instance rabbit or mouse; then spleen cells are selected in vitro and B cells are isolated. B cells from spleen are fused with tumoral mouse cells of mieloma in order to stabilize and make possible the B lymphocyte culture. Indeed, B cell culture is difficult to set up and maintain.
The hybridoma technology allows overcoming these difficulties because of genetic transformation of mieloma cells. Finally, hybridomas are serially diluted and the antibodies are obtained from cell hybridomas cultures derived from a single cell. Which are the advantages of monoclonal antibody in respect with polyclonal ones? Monoclonal antibodies are codified by the same gene and none point mutations are present to generate some difference into antibody population. Thus, the whole population is identical and specifically recognizes one antigen. Cross reactivity is reduced with monoclonal antibodies and the interaction between antigen and antibody is usually more stable. Furthermore, this technique is also really flexible because it’s virtually possible to create antibody versus each antigen, when it’s possible to immunize the host animal. Which are the applications for monoclonal antibody? Monoclonal antibodies are currently used in molecular biology and biochemistry laboratories for imaging, western blotting, immunoprecipitation and so on. A lot of protocols are based on antibody use. In diagnostics, monoclonal antibodies are used in ELISA dosage or in flow cytometric analyses, as well as infection detection or pregnancy diagnosis.

Clinical applications of monoclonal antibody are prevalently in oncology. In 1997, the first monoclonal antibody was approved for non- Hodgkin lymphoma treatment. Since this year, several antibodies have been optimized against breast cancer, leukaemia, colon cancer and recently lung cancer. Each antibody recognizes a tumoral antigen and specifically kills only the cells (tumoral) that present that molecule. Thus, adverse effects associated with the use of monoclonal antibodies are reduced if compared with traditional drugs. Based on their specificity, antibodies can be used to carry other useful drugs to cells. For instance, an antibody can be conjugated to radioactive compounds to be addressed to cancer cells. Furthermore, other drugs can be carried into the brain, giving the capability of monoclonal antibody to overcome the blood- brain barrier. Parkinson’s disease can be treated with this approach. Improvement of biochemical characteristics of monoclonal antibodies is one challenge for scientists for the next future. Indeed, it’s important to improve the delivery of monoclonal antibody into all districts of human body. The specificity will be a must if clinical or diagnostic applications are planned for the monoclonal antibody. Furthermore, cheaper technology must be optimized to allow large scale production. Research development in this field is really promising.

Tetracycline derivative can modulate SMN expression

Survival of motor neuron centromeric (SMN) genes are involved in spinal muscular atrophy when mutated. This disease is an autosomal recessive disorder that progressively determines the loss of spinal alpha motor neurons, causing death during the childhood.

Deletion in SMN1 gene generates a truncated protein without functionality. In a similar situation another protein of the same family SMN2 is produced in larger amount, but this spliced form is unable to overcome the loss of SMN1 and restore its normal activity. SMN2 lacks an exon (7) important for protein function, this is the reason for which SMN2 cannot completely restore SMN1 activity. Researchers observed that tetracycline derivatives could interfere with splicing mechanism and promote the inclusion of exon 7 into SMN2 mRNA. They identified the less toxic ones in order to modulate SMN2 expression without collateral effects. Rather than the toxicity, the second problem that researchers had to solve was the blood brain barrier crossing because tetracycline derivatives cannot cross this barrier. Scientists proposed firstly an injection into brain, then the use of osmotic pump to internalize these useful drug in SMA patients. Giving the lethality of this disease, it’s important that all routes are checked and considered in order to find the right one.

Novel function for IHD1 in glioma

Genetic mutations are usually identified in cancer. These alterations can cause loss of function, when one mutation interferes with protein functionality, or gain of function when a mutated protein over-works or is over-expressed in a wrong cellular district. In this case, a protein maintains the same function as a normal one. In some cases, mutation can generate novel functions in a protein.
This is the case of IDH1, the isocitrate dehydrogenase 1 that usually converts the isocitrate into alpha- ketoglutarate. When this protein is mutated, an overproduction of 2 hydroxyglutarate (2HG) is abundantly recovered into cells. 2HG is toxic for brain and its presence is correlated with cancer. X-ray diffraction demonstrated that the mutation at arginine 132 results in the formation of a distinct active site, different from those that catalyzes the normal reaction. So, a novel mutation has been identified and a novel function has been recognized. IDH1 could be considered a new target for therapeutic intervention. A selective and specific small molecule inhibitor could interact only with the mutated form, without interfering with the wild type protein and the glucose metabolism. This discover could hopefully help scientists to identify a treatment for glioma.

Novel mouse model for BRCA1 breast cancer

BRCA1 is one important marker for breast cancer; in particular BRCA1 is mutated in the invasive ductal cancer and in general is associated with lack of oestrogen receptor, progesterone receptor and Her/ ERB2, making useless current therapies for breast cancer. Giving the lethality of double mutant Brca1, none animal models were available up to date to study the role of this gene in tumorigenesis.

Everyone knows the importance to study cancer from its molecular basis. Scientists from the Netherlands Cancer Institute of Amsterdam proposed a conditional mutant in which tissue specific inactivation of Brca1 was performed by Cre Recombinase. This system allows to dispose on one new model that mimics several characteristics of human mammary tumour. Furthermore, numerous new inhibitors or useful molecules could be tested on this model in order to find out some drugs to care BRCA1 cancers. The work, published on the British Journal of Cancer, open new perspectives in breast cancer research. Indeed, the next generation of mouse models will regard resistance mechanisms and double mutant animals will be likely generated in order to highlight the correlation between lack of BRCA1 and the role of other proteins often mutated in breast cancer, such as TP53.