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