Lecture on HDAC Inhibitors

James Rusche, PhD, Sr. Vice President, Research and Development, Repligen Corporation, discusses the work his company has done to identify development candidate for Friedreich's Ataxia.

Ubiquitin signalling.



Proteins are targeted for degradation by the proteasome with covalent modification of a lysine residue that requires the coordinated reactions of three enzymes. In the first step, a ubiquitin-activating enzyme (known as E1) hydrolyzes ATP and adenylylates a ubiquitin molecule. This is then transferred to E1's active-site cysteine residue in concert with the adenylylation of a second ubiquitin. This adenylylated ubiquitin is then transferred to a cysteine of a second enzyme, ubiquitin-conjugating enzyme (E2). In the last step, a member of a highly diverse class of enzymes known as ubiquitin ligases (E3) recognizes the specific protein to be ubiquitinated and catalyzes the transfer of ubiquitin from E2 to this target protein. A target protein must be labeled with at least four ubiquitin monomers (in the form of a polyubiquitin chain) before it is recognized by the proteasome lid.[32] It is therefore the E3 that confers substrate specificity to this system.[33] The number of E1, E2, and E3 proteins expressed depends on the organism and cell type, but there are many different E3 enzymes present in humans, indicating that there is a huge number of targets for the ubiquitin proteasome system. The mechanism by which a polyubiquitinated protein is targeted to the proteasome is not fully understood. Ubiquitin-receptor proteins have an N-terminal ubiquitin-like (UBL) domain and one or more ubiquitin-associated (UBA) domains. The UBL domains are recognized by the 19S proteasome caps and the UBA domains bind ubiquitin via three-helix bundles. These receptor proteins may escort polyubiquitinated proteins to the proteasome, though the specifics of this interaction and its regulation are unclear.[34] The ubiquitin protein itself is 76 amino acids long and was named due to its ubiquitous nature, as it has a highly conserved sequence and is found in all known eukaryotic organisms. The genes encoding ubiquitin in eukaryotes are arranged in tandem repeats, possibly due to the heavy transcription demands on these genes to produce enough ubiquitin for the cell. It has been proposed that ubiquitin is the slowest-evolving protein identified to date.

SMRT DNA Sequencing

Pacific biosciences is developing a transformed DNA sequencing technology, which will revolutionize the field of genetic analysis by enabling researchers to answer questions important to human healthcare, It is called SMRT (Single Molecule Real-Time DNA Sequencing). A breakthrough technology based on the natural process that occurs every time living cells divides.

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Prior to division DNA is replicated by enzymes called DNA polymerase is which efficiently duplicating entire genomes in minutes, by reading the DNA and sequentially building a complementary strand with matching building blocks called nucleotides. Pacific biosciences SMRT sequencing harnesses the power of the polymerase as sequencing engine by eavesdropping on what works to replicate DNA, this approach is enabled by two proprietary technologies the first Phospholinked nucleotides. To visualize polymerase activity a different colored fluorescent label is attached to each of the four nucleotides ACG and T. In contrast to other sequencing approaches R phosholinked nucleotides carry their fluorescent label on the terminal phosphate rather than the base. Through this innovation enzyme cleaves away the fluorescent label as part of the incorporation process leaving behind a completely natural strand of DNA.This enables us to exploit the inherent properties of the DNA polymerase including high-speed long read length and high fidelity. The second key technology is a nano photonic visualization chamber called the Zero Mode Wave-guide or ZMW, It enables observation of the individual molecules against a required background of labeled nucleotides while maintaining a high signal-to-noise. This ZNW is a cylindrical metallic chamber approximately 70 nm wide it is illuminated/support creating an extremely small detection volume just 20 cL. Nucleotides defused in and out of the ZNW in microsecond. When the polymerase encounters the correct nucleotide it takes several milliseconds to incorporated during which time its florescent label is excited emitting light is captured by a sensitive detector. After incorporation the label is clipped off and diffuses away, The whole process repeats creating sequential bursts of light corresponding to the different nucleotides these are recorded thus building the DNA sequence.

Application

The Single Molecule Real Time sequencing will be applicable for a broad range of genomics research, namely:
  • De novo genome sequencing: The read length from the Single Molecule Real Time sequencing is currently comparable to that from the Sanger sequencing method based on dideoxynucleotide chain termination. The longer read length allows de novo genome sequencing and easier genome assemblies.
  • Individual whole genome sequencing: Individual genome sequencing may utilize the Single Molecule Real Time sequencing method for the personalized medicine.
  • Resequencing: A same DNA molecule can be resequenced independently by creating the circular DNA template and utilizing a strand displacing enzyme that separates the newly synthesized DNA strand from the template.

Activated T Cells attacking on cancer cells

Effux pumps


Active efflux is a mechanism responsible for extrusion of toxic substances and antibiotics outside the cell; this is considered to be a vital part of xenobiotic metabolism. This mechanism is important in medicine as it can contribute to bacterial antibiotic resistance.

Efflux systems function via an energy-dependent mechanism (Active transport) to pump out unwanted toxic substances through specific efflux pumps. Some efflux systems are drug-specific, whereas others may accommodate multiple drugs, and thus contribute to bacterial multidrug resistance (MDR).


Bacterial efflux pumps

Efflux pumps are proteinaceous transporters localized in the cytoplasmic membrane of all kinds of cells. They are active transporters, meaning that they require a source of chemical energy to perform their function. Some are primary active transporters utilizing Adenosine triphosphate hydrolysis as a source of energy, whereas others are secondary active transporters (uniporters, symporters, or antiporters) in which transport is coupled to an electrochemical potential difference created by pumping out hydrogen or sodium ions outside the cell.
Bacterial efflux transporters are classified into five major superfamilies, based on the amino acid sequence and the energy source used to export their substrates:

1. The major facilitator superfamily (MFS)
2. The ATP-binding cassette superfamily (ABC)
3. The small multidrug resistance family (SMR)
4. The resistance-nodulation-cell division superfamily (RND)
5. The Multi antimicrobial extrusion protein family (MATE).

Of these, only the ABC superfamily are primary transporters, the rest being secondary transporters utilizing proton or sodium gradient as a source of energy. Whereas MFS dominates in Gram positive bacteria , the RND family is unique to Gram-negatives.


Function

Although antibiotics are the most clinically important substrates of efflux systems, it is probable that most efflux pumps have other natural physiological functions. Examples include:

* The E. coli AcrAB efflux system, which has a physiologic role of pumping out bile acids and fatty acids to lower their toxicity.
* The MFS family Ptr pump in Streptomyces pristinaespiralis appears to be an autoimmunity pump for this organism when it turns on production of pristinamycins I and II.
* The AcrAB–TolC system in E.coli is suspected to have a role in the transport of the calcium-channel components in the E. coli membrane.
* The MtrCDE system plays a protective role by providing resistance to faecal lipids in rectal isolates of Neisseria gonorrhoeae.
* The AcrAB efflux system of Erwinia amylovora is important for this organism's virulence, plant (host) colonization, and resistance to plant toxins.
* The MexXY component of the MexXY-OprM multidrug efflux system of P. aeruginosa is inducible by antibiotics that target ribosomes via the PA5471 gene product[1].

The ability of efflux systems to recognize a large number of compounds other than their natural substrates is probably because substrate recognition is based on physicochemical properties, such as hydrophobicity, aromaticity and ionizable character rather than on defined chemical properties, as in classical enzyme-substrate or ligand-receptor recognition. Because most antibiotics are amphiphilic molecules - possessing both hydrophilic and hydrophobic characters - they are easily recognized by many efflux pumps.

Blastocyst Formation Video

The morula is a solid ball of about 16 undifferentiated, spherical cells. As cell division continues in the morula, the blastomeres change their shape and tightly align themselves against each other. This is called compaction and is likely mediated by cell surface adhesion glycoproteins.

CTLA-4 and Mechanism of action of anti-CTLA4

CTLA4 (Cytotoxic T-Lymphocyte Antigen 4) also known as CD152 (Cluster of differentiation 152) is a protein that plays an important regulatory role in the immune system. In humans, the CTLA4 protein is encoded by the CTLA4 gene.