Proteasomes – The Human Garbage Can

Humans and all eukaryotic organisms (those with nucleated cells) have structures within the cells that are basically recycling bins.  This structure of multiple proteins is called a “Proteasome” and ironically is basically in the shape of a garbage can.  It is composed of a central cylinder shaped core (i.e., 20S, lighter gray sections of model below) created by two rings of a series of b1 to b7 sub-units.  The lid (i.e., 19S cap, dark gray parts of model below) of the proteasome is composed of a series of a1 to a7 sub-units that also creates a channel with a regulatory function.  The top of these a sub-units is composed of Rpt1 and Rpt2 followed by a subunits Rpt3,5,6,7,10, and 12.  The cellular process to degrade waste products starts with a three-step process of labeling them called “ubiquitization”.  The lid collects waste proteins labeled with one or more “ubiquitine” molecules (small black dots in model).  The label(s) are removed and the waste molecule is unfolded as it is fed into the proteasome cylinder.  Several proteolase (enzymatic protein degradation) functions are performed inside the core; b5 for chymotrophic, b2 for typsin-like, and b1 for caspase activities. Once inside the 20S section, b proteases break down the waste protein to simpler components (i.e., peptides).  These are expelled at the bottom cap and are now potentially reusable by the cell.  The net result would be a detoxification of the cell.  The system is named the “Ubiquitine Proteasome System”.  It plays a crucial role ensuring that the interior of whatever type cell it is in does not accumulate waste to levels that become toxic to the cell and interfere with the functions of that cell.

Representation of Yeast Proteasome¹

A recent study² published in 2010 investigated the role this system plays in cardiac muscles.  Dysfunction in the proteasome caused an accumulation of Akt kinase and the transcription factor p53, a mediator of apoptosis (cell self-destruction) resulting in hypertrophic cardiomyopathy and heart failure.  The dysfunction was attributed to the oxidized derivatives identified as Rpt3 and Rpt5, which are part of the lid (19S section).  The oxidative stress of these components was at least partially responsible for the proteasome dysfunction.  The degree of oxidation would adversely affect the proteasome’s ability to deliver the waste proteins to the 20S section.

The proteasome system in skin cells is involved in the clean up of UV damaged tissues and ends up regulating or inhibiting the rate of the repair process.  Similar dysfunctions in the lid section of these proteasomes may be a key to expediting cellular repairs from sun damage.  Skin care product developers may consider additives that would address minimizing this dysfunction and maximize the rate of repair.

The take-home lessons here are, “Make sure you tip your garbage man”.  They serve a very important function.  Also researchers in cellular repair of skin may use this information to discover similar mechanisms in skin metabolism and develop improved sun care products.

Another representation below³

¹McDonald,H.B, Byers,B., “A Proteasome Cap Subunit Required for Spindle Pole Body Duplication in Yeast”, Journal of Cell Biology, V.137;539-553;May 5, 1997.

²J.Predmore, S. Powell et. al., “Ubiquitin Proteasome Dysfunction in Human Hypertrophic and Dilated Cardiomyopathies”, The Journal of the American Heart Association,121;997-1004;2010.

³ AngieBioTech.com Image

Method Validation, Bonsai To Go and Improving GMP compliance

“The country does not matter, either the micro lab works or it doesn’t”

I have audited facilities in many different countries.  China (e.g., Shanghai, Jiangsu, Guangdong , Huizhou, Zhuhai), Taiwan, Thailand, Hong Kong, Korea,  Dubai, Israel, Italy, Canada, and the United Kingdom (i.e., Wales and England).

The regulatory compliance is subject to local regulatory agencies in the country involved, but the microbiology lab answers to a greater authority, the “Science of Life”.  Most labs have some degree of success in performing the testing.  However, I generally find that most labs can improve on the test methodology.   In two words- “Method Validation”.   The tools to measure growth success of different species on the actual test materials in question must be established in order to evaluate the real efficacy of the lab test program.  These tests are called “Microbial Recovery Validations”.   The lab should validate the method works to recover the general types of organisms expected, and especially the restricted organisms specified for the product.  For example, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella spp., Candida albicans, mold and any others specified as restricted for a given product or raw material.  

“100 things can go wrong in the microbiology lab that may give you false acceptable test results.”

I have audited over 60 manufacturer or microbiology lab operations (41 facilities or labs in 11 different countries).  In Asia I found they almost always had the same 17 items that needed correction or refinement before results could be verified.  There was a clear connection with all the failing labs.  The cosmetic industry in Asia, adopted clinical lab testing methods to meet the local government regulations for qualifying the microbiology lab.  A good start but other modifications and verifications are needed.  The methods have been validated to work for standard clinical test materials like blood, urine, and tissue.

However clinical methods are not always appropriate for organisms encountered in  manufactured products.  One reason is the products are at room temperature, not usually at human body temperatures.  A second reason was the clinical strategy of not risking test contaminations from positive controls. Thus the use of positive controls in the lab was considered not necessary.  As a result the labs did not have examples (i.e., stock cultures) of the organisms they were looking for.   Therefore in-line positive controls and media controls were not established to verify test results.   

Another concern is that cosmetic products are usually preserved, unlike clinical samples.  As a result, the test method may not neutralize the anti-microbial components in the test materials and result in false negative test results.

Even if this neutralization was performed as some companies did, the potential toxicity of the neutralizers were not evaluated.  Overuse of neutralizers could also result in no growth and false negative test results.   I can help you develop method validation procedures to verify the method was successful in recovering the microbes that could be present.

I have encounter inappropriate microbial test methods in US operations as well.  In the course of investigating a contamination of a raw material, it was found that the root cause of not detecting the microbes was over-neutralization in the test method .  This yielded false negative test results. We did resolve how to correct the situation by modifying the test procedure.

“Bonsai To Go Society?”

We live in a fast paced world.  Even traditionalists of different cultures have modified the traditional rules to conform to a faster pace.  Traditionalists respect the ancient values of original cultural societies.  However modern people have adopted quicker methods that may arrive at the same objective.  Some better than others and some with definite limitations.

                               Bonsai with 20 years training

The art is to create antiquity in young trees.  [The opposite of the cosmetic industry.]

Different methods are used to imply age.  Traditional methods of pruning over years of training can create a representative of a 100 year old tree with a long history of good and bad times.  

                                  Rapid Bonsai in two hours

 A rapid modern method would be to wire several saplings together and create what appears to be a single branching tree. In a short two hours a bonsai artist may transform saplings into an ancient miniature tree- Bonsai.      

                                  Details of rapid method 

Likewise there are newer rapid microbiological methods that need only be validated for the specific application that could be used for shorter release criteria to decrease manufacturing time.  The limitations need to be evaluated by method validation in order to substantiate quality, regulatory acceptability and practical cost of using a given technology.

Improving GMP compliance for manufacturing of cosmetic SPF products.

Cosmetic manufacturers that make SPF (Sun Protection Factor) products for Europe and non-American markets are not required to meet OTC (Over the Counter Drug) regulations that are enforced in the United States and Canada.  Consequently, they cannot legally manufacture these items for sale in the North American markets.  Compliance with US Federal Code of Regulations and Drug Product registrations are required to compete in this market.  I can audit your facility to determine what controls are lacking and provide a corrective action list to comply with the regulations.  The objective would be to assure the quality and safety of the product.  Consequently, the company should perform well in an official FDA audit.