Perfectionism on part of proteins in cargo delivery could save lives

August 2015

A minor fault in any member of the team of proteins carrying structural elements for melanin pigment maturation could deprive us of not just our colour, but could be fatal when combined with few other factors.

Trucks and lorries rule the world of cargo delivery. Any malfunction in them affects the timely delivery of the cargo at the destination and where and how they are used in the further processes. This chain of events is not very different at a cellular level. Our cells also have their own transport pathways responsible for the cargo delivery at the right destination at the right time. Any variations to that system shows up as symptoms to fatal diseases. Dr. Subba Rao and team from the Indian Institute of Science, Bangalore, unravel the nitty gritties of one such transport pathway in animal cells where failure to deliver the cargo, in this case melanin synthesizing enzymes, could result in fatalities.

Melanin pigments are responsible for the colour of our skin and also play an important role in protection against radiations and any other damage from light. Melanin pigment is produced in cellular organelles called melanosomes which need melanin synthesizing enzymes transported from other organelles. The enzymes transported into premature melanosomes facilitate the maturation into fully pigmented melanosomes. The transport pathway is completed with the help of four multi subunit protein complexes, BLOC 1, 2, 3 and Adaptor protein 3 complex.

BLOC 1 consists of 8 subunits, functioning in the upstream of the pathway while BLOC 2, a 3-subunit protein complex, functions towards the end of the pathway in directing the transfer of molecules towards maturing melanosomes for subsequent reactions. It does so by the specific method of “tethering” or by stabilizing the intermediate molecules that need to be transported.

Mutations in BLOC 1 or BLOC 2 proteins result in inefficient delivery of melanin synthesizing proteins to melanosomes and thus failure in full expression of the melanin pigment. This malfunction manifests in the form of albinism of skin, ear and eye, also referred to as oculocutaneous albinism. This is one of the primary symptoms in Hermansky-Pudlak Syndrome (HPS). The other symptoms are lung infections, which are mistaken as Tuberculosis in most cases in India. Both the lung pathology and albinism put together result in HPS but the confirmatory diagnosis is genetic sequencing of the patient and the parents. HPS generally shows up in children within the age group of 4-6. Out of the 16 possible genetic mutations that can result in HPS, only 9 are known so far. Three out of those nine subtypes are a result of mutations to the BLOC 2 protein.

Even though BLOC 1 and 2 play their respective roles in the overall transport pathway, their molecular functions are not yet clear. There are additional proteins that are responsible for membrane trafficking throughout the cell in most transport pathways. These proteins are called Soluble NSF (N-ethylmaleimide sensitive fusion proteins) Attachment Protein REceptor (SNARE). SNARE proteins, a family of about 60 proteins has been known for their role in membrane fusion during transfer of information. For the first time, the team from IISc has identified two members from the SNARE family that are involved in the transport pathway to melanosome. Immortal melanocyte cell lines from mice, both wild type and mutated, were used for the experiments. The expression of these cell lines were estimated by their absorbance at certain wavelengths and compared with levels of protein expressions found in healthy cells. The team concluded that not only do SNAREs play a vital role in the endosome and melanosome membrane trafficking but are also responsible for maintaining the melanosomal proteins in their stable states until delivered to the maturing melanosomes. Very strong interactions between the SNARE proteins and BLOC 1 has been reported in the initial steps of the transport pathway.

Dr. Subba Rao and team intends to further work on uncovering the details of the interactions between the SNAREs and BLOC 1 and 2 complexes. It is important to understand the specific roles that BLOC 2 plays in the cell and would help in filling the gaps in the transport pathway. How and what delivers the cargo at the destination is yet to be understood. Whether the membranes actually fuse for the transport of the proteins or only the proximity of molecules with the opposite membrane to the surface completes the transport? What are the guiding proteins? If the SNAREs go back to their respective states after the transfer is completed? These are few questions the team is looking forward to resolve in their future research.

 

Keeping DNA in good shape

December 1, 2014

A stitch in time saves nine, goes the old saying. And here a team of scientists at Indian Institute of Science study RAGs (recombination activating genes) to understand the stitching and unstitching of the DNA, which in certain ways leads to genomic instability and cancer.

The RAG complex consists of two genes, RAG1 and RAG2. These genes produce the RAG proteins – RAG1 and RAG2 — which are expressed in the B and T cells of our immune system. The B and T cells help in locating and dealing with foreign substances that enter our bodies like bacteria and other microbes. The cells can recognise foreign bodies using proteins on their surfaces. The RAG gene complex helps in the generation of these surface proteins.

The high number of surface proteins that need to be produced sometimes leads to genomic instability and “chromosomal translocations” – rearrangement of bits of the chromosome, which can lead to incorrect arrangement of genes. This can lead to diseases like lymphoma and leukemia.

In a previous study, Dr. Sathees C Raghavan and Rupa Kumari showed that RAG proteins cleave DNA when they spot a particular sequence of nucleotides. In this paper, they have focussed on studying the factors that can regulate DNA cleavage efficiency of the RAG proteins. This can improve our understanding of how the DNA cleaving activity of these genes is turned on and off.

They found that apart from the sequence of a particular DNA complex, the sequence of the regions surrounding it are important in determining where the RAG proteins bind and where they cleave. The presence of cytosine and thymine in a single stranded region of the DNA complex dictates the position of nicking. A minimum of two cytosines are required for the RAGs cleavage efficiency. The deletion of certain sequences could result in the loss of sequence specific nuclease activity of RAG but it retains its structure specific nuclease activity.

The further understanding of these factors which regulate the stability of the above mentioned DNA complex could help us decipher the mutations that act as the root causes leading to cancers like lymphoma and leukemia.

The paper has been published online on 14th November in The FEBS Journal. http://onlinelibrary.wiley.com/doi/10.1111/febs.13121/abstract

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Taking help from ageing cells to suppress tumours

December 8, 2014

As cells grow older, their DNA gets damaged. Depending on the extent of damage, the cell can repair the DNA and continue its life, or self destruct and die. A molecule called ATM kinase is involved in this decision making process.

Deepak Saini’s lab at IISc has delineated the role of ATM kinase in this important process. The extent of DNA damage either triggers activation of cancer causing genes, or deactivation of tumour suppressor genes. Both these processes can initiate uncontrollable multiplication of cells, leading to cancer. The other possible outcome of DNA damage, especially if very severe, is cell death. The decision of the cell’s fate lies in the hands of the genetic errors accumulated. If the errors cannot be repaired, or can be detrimental if left unrepaired, the cells enter cellular senescence, which is basically ageing. Cell function deteriorates and ageing of the organism is the inevitable result.

The senescent condition of the cells depend on their respective abilities to maintain a persistent DNA damage state without inducing death or repair. There are a number of molecules like ATM kinase and ROS (reactive oxygen species) that play a critical role in regulating cell fate after the genomic damage.

Cellular senescence can be divided into two distinct phases – initiation or early senescence and the maintenance of senescence. The present research delineates the roles of ATM kinase in the initiation of senescence and importance of ROS in maintaining senescence. ATM kinase is one of the key proteins which decides the fate of a cell; it also acts as a quantitative sensor for DNA damage. When DNA damage is not so severe, the cell repairs its DNA and continues growth; in severe damaged states, the cell dies.

In the intermediate stages of damage, the cell enters the senescent stage activated by ATM kinase. “Our studies show that senescence or aging is one of the cell fates in response to DNA damage and the decision is dependent on the dose of damage and ATM kinase protein. Aged cells generate free radicals which is critical in maintaining their status quo”, said Dr. Saini.

Since the other two alternatives after DNA damage – death and cancer – are obviously harmful, a possible way to push a cell toward senescence instead of the other options can have possible therapeutic value. Cell senescence can be induced in tumour and cancer cells by using a sub-lethal dose of stress, by agents like gamma rays, hydrogen peroxide etc. which triggers the DNA damage response leading to senescence. Further research on this could help us devise a very simple yet attractive tumour suppressing mechanism.

The paper will be published in the Journal of Cell Science and appeared online on 21st November. http://jcs.biologists.org/content/early/2014/11/20/jcs.159517.abstract

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A system to deliver drugs to individual cells

October 6, 2014

A system to package and deliver drugs to each cell of your body, depending on its needs, has been developed at IISc. “Nanocapsules” made from a special type of material can now deliver drugs right inside cancer affected cells in the body.

“Drug delivery systems” are mechanisms that can be programmed to release drug molecules at targeted cells in the body, using physiological cues present in the body itself. The major hurdle has been that these local cues are not consistent between cells; one needs systems that respond to multiple such cues. Prof. Ashok M Raichur and his team of scientists at the Indian Institute of Science, Bangalore, have demonstrated one of the very few systems that can respond to multiple cues.

There are three ideal characteristics that a drug delivery system should have: (1) the entire drug molecule should be encapsulated, which would prevent its premature release or degradation (2) it should carry the drug safely — and specifically — to the target site and (3) at the target site, it should release the drug molecules using the local physiological cues available.

Hollow nanocapsules were fabricated from special materials called biopolymers, which are materials that do not react with body tissues. These nanocapsules contain components that can respond to local cues integrated in the walls. To avoid premature release of the drug, the walls are crosslinked; this sort of architecture gives scope to load large amounts of drugs into the capsule. The wall structure also makes it possible for a small amount of local cues, like enzymes, to trigger the release of a large number of drug molecules.

The Food and Drug Administration(FDA) approved drug, polypeptide protamine (PRM), used to treat heparin induced toxicity, is one of the stimuli responsive components which is identified and actively cleaved into smaller fragments by trypsin like enzymes. The second component, chondroitin sulphate is susceptible to cleavage by enzyme hyaluronidase and has been used in the treatment of arthritis.

The Layer by Layer (LbL) assembly method used for fabrication of nanocapsules is carried under highly controlled mild conditions and thereby capable of incorporating the sensitive components (biopolymers) used here. It has the capacity to take up an array of materials ranging from small proteins to inorganic molecules. The nanocapsule surface was combined with a molecule used to identify cancer cells, folic acid (Vitamin B9, as we know it).

The drug delivery system was demonstrated using a population of cells in the lab – something called a “cell line”.

The paper appeared in the international journal RSC Advances on 17th September. http://pubs.rsc.org/en/content/articlelanding/2014/ra/c4ra07815b#!divAbs…

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Decoding transmembrane communication in living cells

April 24th, 2015

Living cells aren’t self-sufficient; they need to interact with their environment in order to survive. But these interactions are extensively controlled by the barrier called the cell membrane, a dynamic entity made up of lipids and proteins. Molecules are constantly passing in and out of the cell through the semi-permeable cell membrane, their movement often orchestrated by different forces and membrane components. This was the level of understanding of this barrier’s structure and function, posited by the ‘fluid mosaic’ model developed by Singer and Nicholson in 1972. Little was known then about minute details of the driving forces at the nano scale.

Until now, the nitty-gritty of how information traverses the membrane had been left to cell biologists’ countless hypotheses. Fast-forward to the 21st century, some of those assumptions have been put to rest by a recent study at NCBS. An interdisciplinary team has used living cells, synthetic lipid analogs and molecular dynamic simulations to understand transbilayer communication between molecules on either side of the bilayered cell membrane. The team consists of cell biologist Satyajit Mayor along with soft matter physicist Madan Rao and their teams at the National Centre for Biological Sciences, Bangalore, and synthetic chemists Ram Viswakarma (IIIM, Jammu) and Zhongwu Guo (Wayne State University, USA).

The team’s studies at the nanoscale have revealed that Phosphatidylserine (PS) is a key component that mediates the communication between the lipids on the inner leaflet, and actin and lipid-anchored proteins on the outer leaflet. PS gets the message across because of the presence of long chain-containing lipids such as those found in ‘solid fats’. That’s how the components on inner and outer leaflets communicate.  These studies show how integral PS is to signalling pathways of the cell, and therefore when absent results in irreparable damage.

“The uniqueness of the study lies in discovering a specific role for PS in nanocluster formation, a building block of ‘lipid rafts’ and how the chemistry of both the outer and inner leaflet facilitate this process. For this we have adopted an array of methods which combines biology, genetics, chemistry and physics to provide an explanation for the formation of nanoclusters” says Anupama Ambika Anilkumar, one of the first authors of the paper published online on 23 April, 2015, in the journal Cell.

Lipid rafts are microenvironments in the membrane made up of clusters of lipids and protein receptors, and are involved in molecule trafficking and assembly. Refuting early theories on the random combination of lipids to construct these lipid rafts, this study shows that the formation of these “nanoclusters” of lipids is an active process templated by the actin cytoskeleton on the inner leaflet. This understanding also points to further clues about the role of these clusters. They have been hypothesized to function as a ‘sorting station’ for components to be recruited for signalling events on the cell surface.

The discovery of PS as a vital component in the transmembrane communication would advance our understanding of the cell membrane’s microenvironment. It would also help scientists understand how these nanoclusters function and what proteins are involved in their assembly. Additional experiments in Mayor’s lab are underway to draw the complete picture of the cell’s communications and the anchors of the PS species. These entities also play an important role in various other cell functions and are hence important problems to pursue.

“There is no earlier evidence of how these clusters are formed by lipidic interactions. My colleague and co-first author, Riya Raghupathy established methods to assay the role of long-acyl chain lipid species, and along with Parvinder Pal Singh, developed the synthetic analogues used in this study. Anirban Polley, working with Madan, conducted molecular dynamic simulations; both of these are an integral part of this study and I am grateful for having such terrific collaborators,” said Anupama Anilkumar.

Decrypting this communication could help explain how signals are both read and interpreted by the cell, with implications for a number of diseases caused by alteration in lipid balance or composition. Understanding how these lipids, the “gatekeepers” of the cell, function might also help deter the progression of viral diseases, by potentially disrupting the interaction of membrane components with the viruses.

The paper can be accessed at:
http://dx.doi.org/10.1016/j.cell.2015.03.048

An “antioxidant-like” protein to fight free radical damage in the body

September 22, 2014

A protein found in high levels in some cancer cells can be used for treating diseases caused by oxygen free radicals in the body, a recent study has found.

Oxygen free radicals such as hydrogen peroxides and superoxides, called Reactive Oxygen Species (ROS), are found in the cells as byproducts of cellular metabolism. Uncontrolled levels of ROS in the cell can lead to oxidative stress. Diabetes, atherosclerosis and neurodegenerative diseases like Parkinson’s and Alzheimer’s disease find their roots at the damage caused by oxidative stress in the cells.

Patrick D’Silva’s group at the Indian Institute of Science have found that Magmas, a mitochondrial protein also regulates the level of ROS in cells, apart from its already known function.

Magmas is involved in protein transport in cells, and is found in elevated levels in certain cancer types. There is very little information already available about regulation of ROS in the body, and this paper brings forth a lot of missing links in this research area.

The team found that the levels of Magmas in the cell are dependent on the cellular ROS levels. Elevated levels of Magmas help in lowering the concentration of ROS and vice versa. It not only plays an important role in controlling the production of free radicals, but maintains the ROS homeostasis by efficient scavenging. This protects the cell viability and also increases cellular stress tolerance.

“By maintaining a free-radical balance in cell, this protein prevents stress mediated cellular damage to biomolecules such as DNA, proteins and lipids. Hence, overproduction of Magmas protein provides unique advantages to the cells against free radical stress”, said Prof D’Silva.

Higher levels of Magmas are typically found in metabolically active tissues, cancer cells and tissues at different developmental stages. In cancer cells, Magmas prevents cell death, and hence helps in the proliferation of cancer cells. Even in non-cancerous cells, Magmas shows controlled levels of ROS and much lesser oxidative stress.

Such molecules that regulate the number of free radicals can be used while designing possible therapies for oxidative stress related disorders. “The inhibitors or stimulators against Magmas can be used as a therapeutic intervention against cancer as well as multiple free-radical induced stress related diseases”, said Prof D’Silva.

Further research is required to elucidate the mechanism of ROS regulation by Magmas and to discover the other proteins involved in the regulatory circuit.

The paper appeared in the journal Cell Death and Disease on 28th August 2014.

Link: http://www.nature.com/cddis/journal/v5/n8/full/cddis2014355a.html

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