Friday, June 7, 2019

Senescent Cells and Human Longevity


Although the average human lifespan has increased substantially over time due to the improvement in living conditions made possible by advances in public health, sanitation, medicine, etc., there is no selective advantage afforded by a longer life once the reproductive period has passed. Consequently, there is a normal and gradual deterioration of the tissues that is age-related.
 


Furthermore, there is a natural process referred to as cellular senescence – cells undergoing this change are no longer able to divide. Cellular senescence confers a reproductive advantage for the individual in that it helps block cancer cell proliferation; however, overtime it results in an increasing abundance of senescent cells (SNC) within the tissues. It seems that in animal studies, using the mouse model, in which the SNCs are selectively eliminated (senolysis),the median lifespan of individual test mice is extended, and the frequency of age-related diseases has been shown to be diminished. This result has encouraged the search for and development of drugs that selectively target SNCs.

In terms of this research, it is vitally important to discover the actual mechanisms that underly cellular senescence. In studies using cells grown in culture, it has been shown that SNCs are in a state of permanent cell cycle arrest. This state is apparently initiated and maintained by the p53-p21 retinoblastoma (RB) and p16-RB tumor suppressor pathways. The factors that can trigger this process are –
  • Oxidative stress
  • Shortening to telomeres – repetitive DNA sequences at the ends of chromosomes that afford protection
  • Prolonged mitotic activity
  • DNA errors during replication
  • Mitochondrial impairment.
SNCs produce a so-called, “Senescence associated secretory phenotype” (SASP). SASP has been shown to negatively impact normal tissue architecture through a variety of processes including the onset of fibrosis and the inhibition of stem cell functionality. Although SASP has a protective function in regard to the development of cellular neoplasm, in later life it seems to provide a protective function against the onset of cancer. This raised the possibility that the selective elimination of SNCs from older patients might exert an anti-cancer effect.

Given this data, it would seem that therapies that can effectively eliminate SNCs might produce a two-fold health advantage by increasing longevity and by decreasing the onset of cancer in later life. Encouraging results from animal model studies have shown that drugs that target those pathways that block apoptosis – programmed cell death – promote senolysis and afford an anti-cancer potential. In regard to future research, this may provide a very fruitful line of enquiry.

Tuesday, February 26, 2019

Microbial Carcinogens in the Human Large Intestine

The microbial micro-environment in the human large intestine is intricate and complex. In fact, there are many hundreds of small molecules and metabolites produced by this diverse population of microflora that may profoundly influence human health. Many of these substances are produced by enzyme-directed pathways that have been shown to be programmed by so-called, “bacterial biosynthetic gene clusters.” 

One class of these molecules, colibactins, has been shown to be produced from a gene cluster called the, polyketide synthase island (PKS). PKS occurs in certain strains of Escherichia coli (E-coli) that seem to be prevalent in the microbiota of colorectal cancer (CRC) patients. Up until this time, despite many years of painstaking research, little has been discovered regarding the structure and the mode of action of colibactins.

Dr. Matthew Wilson and his colleagues at Vertex Pharmaceuticals have recently published a paper in the journal Science in which they describe the mode of action of colibactins. According to the author, “colibactin alkylates DNA in cultured cells and in vivo, forming covalent modifications known as DNA adducts. These colibactin-DNA adducts are chemical evidence of DNA damage and represent a detectable signature of exposure to colibactin. Misrepaired DNA adducts may generate mutations that contribute to colorectal tumorigenesis.”

In their research Wilson’s group identified the colibactin-DNA adducts as involving the cyclopropane ring and that the site of alkylation involves the nucleotide adenine within the DNA backbone (see diagrams below). Furthermore, it is believed that these adducts could lead to mutations in the oncogenes or tumor suppressor genes that drive CRC-related tumorigenesis.



Although, this model of colibactin involvement in DNA modification is significant, many questions remain unanswered in regard to how tumorigenesis is subsequently initiated in CRC. However, it is line of research that offers some promise in elucidating cancer-causing mechanisms.

Thursday, January 24, 2019

Evolution of an Enzyme from Short Peptide Pieces

Enzymes are complex protein molecules that are responsible for accelerating chemical reactions in the living cell that essentially make life possible. Each unique enzyme is responsible for a particular chemical transformation. The sum total of all of these reactions is what is referred to as cellular metabolism. The primary structure of enzymes – the sequence of amino acids embedded with the protein structure – is encoded within the particular gene responsible for the production of each unique enzyme.

It is of great interest to understand how such complex protein structures evolved from simpler structures that are known to have been available early in the evolution of life on planet earth – amino acids and peptide. In regard to proteins, the class of molecules that represent short pieces of protein is referred to as peptides. Peptides are short strands of amino acids tied together through peptide bonds. 



Enzymes generally require complex folding in their structures to foster their catalytic activity. Peptides are generally too short for this folding to occur. Recent research into the structure of metalloenzymes – enzymes that employ metal ions in their structures – suggest that metal ions may have helped induce folding in precursor peptides. Metalloenzymes are ubiquitous in nature and play fundamental roles in cellular biology and chemistry.

Sabine Studer from the Dana-Farber Cancer Institute, Boston and her colleagues have attempted to more fully understand the processes through which enzymes have evolved. They have done so by devising techniques for facilitating the transformation of a peptide capable of binding zinc into a functional enzyme with a complex globular structure.

According to the authors, “Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (k cat/K M ∼ 10 6 M -1 s -1). The simultaneous optimization of structure and function in a na├»ve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.”

The techniques they successfully employed to accomplish this include (see diagram below)
Computational redesign
Cassette Mutagenesis – An in-vitro technique for altering genetic structure i.e. mutations.
DNA shuffling
Random mutagenesis
Alanine scan – Alanine is one of the amino acids that plays a critical role in protein structure.


Note: that the majority of the techniques employed involve manipulating the genetic information.

The work cited above is of seminal importance in the overall search for elucidating the molecular mechanisms that may account for the evolutionary development of life on planet earth from its elemental beginnings

Sunday, December 16, 2018

The Impact of Climate Change on the Alaskan Permafrost

The process by which the earth transfers heat has led to an acceleration of warming in the northern climes caused by the unabated increase in greenhouse gases in the atmosphere. This effect has been particularly felt in Alaska. As a result, the heretofore permanent nature of the Alaskan permafrost is changing. This is particularly troublesome in that locked within this permafrost is organic material that is ,by its nature, rich in carbon and greenhouse gases. This process by which melting permafrost leads to increased release of greenhouse gases that subsequently leads to increased melting of permafrost represents a disturbing feedback loop mechanism with inherently dangerous consequences.


A Report from the New York Times helps elucidate the real gravity of this change.

Saturday, October 6, 2018

RNA Modifications Regulate Gene Expression During Development

RNA plays a critical role in gene expression within the cellular environment. Both messenger RNA (mRNA) and transfer RNA (tRNA) are involved in translating the instructions encoded in the DNA sequence of expressed genes into the manufacture of protein products at the site of the ribosomes. This process is, of course, happening continually throughout the life cycle of the cell. In the development of the human embryo in utero, the timing and orchestration of gene expression is vitally important; the precision of these processes is an absolute requirement for the successful creation of a viable individual at birth. The timed expression of development-related genes is determined by gene expression programs.

There is current evidence that there are modifications of RNA that function as post-transcriptional regulators of these gene expression programs. These regulators apparently impact a wide variety of eukaryotic biological processes. Michaela Frye from the Department of Genetics at the University of Cambridge, UK and her scientific collaborators stated in a recent article in the publication, Science, that “N6-methyladenosine affects the translation and stability of the modified transcripts, thus providing a mechanism to coordinate the regulation of groups of transcripts during cell state maintenance and transition. Similarly, some modifications in transfer RNAs are essential for RNA structure and function. Others are deposited in response to external cues and adapt global protein synthesis and gene-specific translation accordingly and thereby facilitate proper development.”


N6-methyladenosine

Gene expression in multicellular organisms is determined by a complex set of interacting and dynamic processes that require the coordination of mRNA metabolism and protein synthesis. There has been considerable investigation into the mechanism of transcriptional networks related to tissue-specific stem cell differentiation. However, the regulation of gene expression programs during development is especially crucial and must be unerringly precise. The recent evidence points to specific modifications of RNA as crucial to the regulation of cellular transcriptomes and proteomes during development.

At present there are over 170 modifications in RNA reported, but it is only following the relatively recent development of precise analytical tools that these modifications can be identified and quantified with precision. In addition to the N6 methyladenosine modification of mRNA as a crucial regulator of gene expression, other modifications including 5-methylcytosine and N1-methyladenosine are involved in the modification of both noncoding RNA and mRNA.

A detailed examination of some of these changes has been reported in the Science publication authored by Michaela Frye as noted above. These findings help to elucidate the molecular and cellular mechanisms that are involved in the intricate process of development.

Sunday, July 29, 2018

An Elegant and Monumental Experiment


The year is 1952 – two years prior to the discovery of the three-dimensional structure of DNA.  At that time there were essentially two schools of thought regarding the class of organic compounds responsible for heredity, namely, proteins or nucleic acids.  Two researchers Alfred Hershey (1908 – 1997) and his assistant Martha Chase (1923 – 2003) collaborated on a research project that they felt could unambiguously provide the answer to this fundamental question.  The experiment described below is remarkably simple, precise and elegant; the ramifications of their results speaks for itself in regards to the discoveries that would follow including Watson and Crick’s elucidation of the structure DNA and ultimately the complete sequence of the human genome (2003).

Hershey and Chase focused their attention upon the bacteriophage – a type of virus that preferentially attacks bacterial cells.  Like all viruses, the phage is made up of two distinct classes of compounds – a protein coat that surrounds a DNA core (there is also a class of viruses that use RNA as the infecting agent, the AIDS virus, for example).



Their experimental approach involved two precise steps outlined below
  •          They labeled the phage (T2) with radioactive Phosphorus (P32) prior to introducing T2 to bacterial host.  Phosphorus is predominantly found in DNA where it is a major constituent and found in protein in insignificant amounts.  After infection, they found that P32 was no longer in the phage, but was found in the host indicating the phage DNA was transferred to the host. 
  •          In the second experiment, they preferentially labeled the phage protein with radioactive Sulfur (S35).  Sulfur is a significant part of the composition of proteins but does not exist in nucleic acids (DNA).  In their analysis subsequent to infection, they found that S35 remained with the phage but was not found in the bacterium.



The results from these experiments, clearly demonstrated that the infectious agent was DNA and not protein.  This conclusion was so significant at the time that James Watson stated that, “the Hershey-Chase experiment had a much broader impact than most confirmatory announcements and made me ever more certain that finding the three-dimensional structure of DNA was biology's next important objective.

Note, that Hershey won the Nobel Prize for his work in 1969 along with Max Delbruck and Salvador Luria.  This kind of experimental approach also demonstrates the roles that imagination, dedication, persistence and creativity play in conducting scientific research.