Abusing Gras #2

Understanding Protease Natures "Protein Scissors" pre–Post Translation.

Hello Friends. In part #2 of Abusing GRAS, we are going to go heavy on definitions and functions because you need a basic understanding of nature’s “protein scissors” to understand how we are going to make large amounts of target metalloprotease post translational bacterial products. From the first article we remember that the reason we want to manufacture these products is as cleanup crew for all the polymerizing proteins in and around the gut and its axis components. We want to flood the zone as it has more electrostatic zinc fingers than the dimerized plaques can resist, and this breaks the pathogenic biofilms that have captured your tissues.

So, in that effort to optimize I’m having my Agent right this article from my notes to get to the point quickly. This is a technical document to help you understand the function or semi virulence and how to use esbl’s, mobile genetic elements, and promoters to force out unwanted semi pathogenic bacteria and viruses. By the end of this series, you will understand how to turn debilitating diseases like helicobacter pylori off with these products.

Of course, we can just have faith in Natures Order to promote health simply by chasing the good but in this case, we want to be specific. For example, what bacteria will specifically target alpha synuclein in the cytosol for detoxification of said dimers and promote the post translational modifications GRAS bacteria do to fold or unfold these plaques that cause Alzheimer’s and other dementias?

Me: What are Proteases and Their Biological Significance?

My Agent:

### Proteases: Nature's “Protein Scissors”

Proteases (also called peptidases, proteinases, or proteolytic enzymes) are enzymes that catalyze proteolysis, breaking down proteins into smaller polypeptides or single amino acids. They function by cleaving peptide bonds within proteins through hydrolysis, a reaction where water breaks chemical bonds. Proteases play crucial roles in numerous biological pathways, including:

- Digestion of ingested proteins

- Protein catabolism (breakdown of old proteins)

- Cell signaling pathways

- Immune system function

Without these enzymes, proteolysis would be extremely slow, potentially taking hundreds of years. Proteases exist in all forms of life, including viruses, and have independently evolved multiple times. Different classes of proteases can perform the same reaction through entirely different catalytic mechanisms.

## Hydrolysis: Breaking Bonds with Water

Hydrolysis (from Greek "hydro-" meaning water and "lysis" meaning to unbind) is a chemical reaction where a water molecule breaks one or more chemical bonds. In biological systems, hydrolysis is the cleavage of biomolecules where water is consumed to separate a larger molecule into component parts.

Hydrolysis reactions are often the reverse of condensation reactions:

- Hydrolysis adds water to break down molecules

- Condensation builds up molecules by removing water

In the context of carbohydrates, the hydrolytic breakdown into component sugar molecules is called saccharification (e.g., sucrose being broken down into glucose and fructose)

## Proteases in Bacteria

Bacteria secrete proteases to hydrolyze peptide bonds in proteins, breaking them down into their constituent amino acids. These bacterial and fungal proteases are vital to global carbon and nitrogen cycles by recycling proteins. Their activity is typically regulated by nutritional signals, with overall microbial community-level protease activity responding to carbon, nitrogen, or sulfur limitation.

Within bacteria, proteases also serve for general protein quality control by degrading unfolded or misfolded proteins. For example, the AAA+ proteasome controls protein quality within bacterial cells.

Some secreted bacterial proteases function as exotoxins and act as virulence factors in bacterial pathogenesis. The exfoliative toxin is one example—bacterial exotoxic proteases destroy extracellular structures, contributing to bacterial invasiveness and tissue damage.

## Proteasomes: Cellular Protein Degradation Machinery

Proteasomes are essential protein complexes responsible for degrading proteins through proteolysis. These complexes are found in all eukaryotes and archaea, as well as some bacteria. In eukaryotes, proteasomes exist in both the nucleus and cytoplasm.

The proteasomal degradation pathway is critical for many cellular processes:

- Cell cycle regulation

- Gene expression control

- Response to oxidative stress

The importance of this proteolytic pathway and ubiquitin's role was recognized by the 2004 Nobel Prize in Chemistry awarded to Aaron Ciechanover, Avram Hershko, and Irwin Rose.

### Structure and Function

The core 20S proteasome is a cylindrical protein complex with four stacked rings forming a central pore. Each ring consists of seven individual proteins:

- The inner two rings are made of seven β subunits containing protease active sites

- Access to these proteases is controlled by regulatory protein complexes

In eukaryotes, the ubiquitin-proteasome system (UPS) targets proteins for degradation by attaching ubiquitin molecules to them. Proteins destined for degradation require:

1. Attachment of ubiquitin (via E1, E2, and E3 enzymes)

2. An unstructured region of about 25 amino acids

The resulting polyubiquitin chain is recognized by the proteasome, allowing for ATP-dependent protein degradation. This process yields peptides about seven to eight amino acids long, which can be further broken down and used to synthesize new proteins.

### Bacterial Proteasomes

Actinobacteria have acquired their own proteasome degradation pathway, including:

- A 20S core particle

- A AAA protein motor called MPA (mycobacterial proteasome activator)

Unlike eukaryotic systems, MPA is a homohexameric motor complex with ATPase sites, an OB ring, and coiled coils. Protein targeting requires a prokaryotic ubiquitin-like protein (Pup) that functions similarly to ubiquitin but has an unrelated structure.

## Protein Denaturation: Unfolding Functional Structures

Denaturation is a process where proteins or nucleic acids lose their folded native structure due to external stresses or compounds, including:

- Strong acids or bases

- Concentrated inorganic salts

- Organic solvents (e.g., alcohol, chloroform)

- Agitation

- Radiation

- Heat

### Consequences of Denaturation

When proteins in living cells denature, cell activity is disrupted and may lead to cell death. Denatured proteins may exhibit:

- Conformational changes

- Loss of solubility

- Dissociation of cofactors

- Aggregation due to exposed hydrophobic groups

The loss of solubility resulting from denaturation is called coagulation. Most importantly, denatured proteins lose their three-dimensional structure and therefore their functionality.

### Protein Structure and Folding

Functional proteins have four levels of structural organization:

1. **Primary structure**: The linear sequence of amino acids

2. **Secondary structure**: Hydrogen bonds between peptide chains forming alpha helices or beta sheets

3. **Tertiary structure**: Three-dimensional folding of secondary structures

4. **Quaternary structure**: Assembly of multiple polypeptide subunits

Protein folding results from a balance between weak intramolecular interactions (hydrophobic, electrostatic, and van der Waals forces) and protein-solvent interactions. This process depends heavily on environmental conditions including temperature, salinity, pressure, and solvents.

When a protein denatures, its secondary and tertiary structures are altered while the primary structure remains intact. Since all structural levels determine function, denatured proteins can no longer perform their biological roles.

## Virulence Factors: Tools of Pathogenic Microbes

Virulence factors (also called pathogenicity factors or effectors) are structures, molecules, and regulatory systems that enable microbial pathogens to:

- Colonize niches in the host

- Evade or suppress the host's immune response

- Enter and exit host cells (for intracellular pathogens)

- Obtain nutrition from the host

### Sources and Transmission

Pathogens possess diverse virulence factors that may be:

- Chromosomally encoded and intrinsic (e.g., capsules, endotoxins)

- Acquired from mobile genetic elements like plasmids and bacteriophages (e.g., some exotoxins)

Factors encoded on mobile genetic elements can spread through horizontal gene transfer, potentially converting harmless bacteria into dangerous pathogens. For example, *Escherichia coli* O157:H7 gains most of its virulence from mobile genetic elements.

### Categories of Virulence Factors

#### Attachment, Immunoevasion, and Immunosuppression

- Adhesins, including lipoteichoic acid and trimeric autotransporter adhesins

- Capsules that inhibit phagocytosis

- Immunoglobulin proteases that break down host antibodies

#### Destructive Enzymes

- Hyaluronidase: breaks down connective tissue

- Proteases and lipases: degrade host proteins and lipids

- DNases: break down DNA

- Hemolysins: disrupt various host cells, including red blood cells

#### GTPase Regulators

Some virulence factors control host GTPase activation through:

- Acting as GEF (guanine nucleotide exchange factor) or GAP (GTPase-activating protein)

- Covalently modifying the GTPase itself

#### Toxins

Bacterial toxins fall into two main categories:

**Endotoxins**

- Components of gram-negative bacterial cell walls (lipopolysaccharide/LPS)

- The lipid A portion is toxic

- Trigger intense inflammation through cytokine release

- Can cause septic shock in severe cases

- Do not elicit an adaptive immune response

**Exotoxins**

- Secreted by bacteria

- Have diverse effects, including inhibiting host biochemical pathways

- Highly immunogenic, triggering antibody production

- Examples include tetanus toxin, botulinum toxin, and anthrax toxin

### Examples of Virulence Factors in Specific Pathogens

**Staphylococcus aureus**

- Hyaluronidase

- Protease

- Coagulase

- Lipases

- Deoxyribonucleases

- Enterotoxins

**Streptococcus pyogenes**

- M protein

- Lipoteichoic acid

- Hyaluronic acid capsule

- Destructive enzymes (streptokinase, streptodornase, hyaluronidase)

- Exotoxins (including streptolysin)

**Listeria monocytogenes**

- Internalin A and B

- Listeriolysin O

- ActA protein

**Yersinia pestis**

- Modified lipopolysaccharide

- Type three secretion system

- YopE and YopJ pathogenicity

**Candida albicans**

- Candidalysin (cytolytic peptide)

Other virulence factors include components required for biofilm formation (e.g., sortases) and integrins (e.g., beta-1 and beta-3).

So that basically sums up the very basics on how Protease function. If we go back in time to when these functions evolved there where only single and multi-cell lifeforms of microscopic size. our Microbiome is a conformation engine that requires the good in order to function in a healthy manner. as you read above it’s very easy to disrupt this balance by introducing toxins, mostly in your food, that then promote the bad bacteria and viruses. If you were ever looking for simple proof that you are what you eat” then the above description by my agent for you should explain that mystery.

In part #3 we will jump into Metalloproteinase and ADAM proteins and how the source of our existence is actually bacterial, and I will show several key functions that cannot be ignored if we are to fully comprehend the glories of the coming PROBIOTIC era of digitalmicrobiome cures and products. we sit on the edge of greatness.