Proteomics/Post-translational Modification/Proteolytic Processing

Chapter Edited and Updated by: Vrunda Sheth & Priyanka Yadlapati E-mail: vns4483@rit.edu, pxy8519@rit.edu

This Section:

Introduction
Proteolytic processing is a major form of post translational modification which occurs when a protease cleaves one or more bonds in a target protein to modify its activity. This processing may lead to activation, inhibition or destruction of the protein's activity. Many cellular processes are triggered by proteolytic processing. The attacking protease may remove a peptide segment from either end of the target protein, but it may also cleave internal bonds in the protein that lead to major changes in the structure and function of the protein.

Proprotein


Many proteins are synthesized as inactive precursors, which may be called proproteins or, in the case of enzymes, zymogens. These proproteins are synthesized in one tissue or organ, transported to another tissue or organ and activated there by proteolytic processing.

This figure shows an example of proteolytic cleavage which results in the activation of BID in the process of apoptosis, programmed cell death. BID is a pro-apoptotic protein which contains the BH3 domain, which is also known as the death domain. Capsase-8 is a protease that cleaves BID in the presence of an apoptotic signal to continue the process of apoptosis.

Proteolytic processing is a highly specific process. The mechanism of proteolytic processing varies according to the protein being processed, location of the protein, and protease. There is no general rule of the thumb for proteolytic processing. This section therefore provides different examples of proteolytic processing.

Proteolytic processing in secretory pathways
Proteolysis of precursor proteins regulates many cellular processes including gene expression, embryogenesis, the cell cycle, programmed cell death, intracellular protein targeting and endocrine/neural functions. In all of these processes, proteolytic cleavage of precursor proteins are necessary. The proteolysis is often done by serine proteases in the secretory pathways. These proteases are calcium dependent serine endoproteases and are related to yeast and subtilisin proteases and therefore called Subtilisin-like Proprotein Convertases (SPCs) or PCs. Seven members of this family have been identified & characterized and each have conserved signal peptides, pro-regions, catalytic and P-domains but differ in their C-terminal domains in mammals.

Autocatalytic cleavage of an N-terminal propeptide activates these proteases, which is required for folding and activity also release of  prodomain. It is known from previous studies that folding of the P domain forms an eight-stranded beta barrel to interact through a hydrophobic patch to a catalytic domain. Folding and activity needs a downstream domain of 150 amino acids. Concerning PCs their C-terminal regions play quite a role in subcellular routing. Furin, one of the seven members of the SPCs, serves as a model for other PCs. The intramolecular cleavage of prodomain allows furin to exit the endoplasmic reticulum (ER). Until the cleaved inactive proenzyme reaches the trans Golgi Network (TGN) where dissociation of prodomain is facilitated by calcium-enriched environment and acidic pH conditions, the prodomain remains attached noncovalently to it. For complete activation, further inhibitory reactions take place within the prodomain by a second cleavage. Proinsulin, Proglucagon and Proopiomelanocortin (POMC) are the well studied peptide hormone precursors.

PCs are responsible for the cleavage at specific sites in each of these precursors. The most frequent sites for cleavage are KR and RR. Proglucagon is processed by PC2 in the alpha cells and by PC1/PC3 in the intestinal L cells and releases active forms of glucagons-like peptides namely GLP-1 and GLP-2. The convertases PC2 and PC1/PC3 are majorly expressed in brain and neuroendocrine system to act on neuropeptide precursors in the Secretory pathway. Glucose, the second messengers in the neuroendocrine cells regulates the transcription and translation of PC2 and PC1/PC3. Other convertases like PC4 expressed in testis and an isoform of PC6 that lacks a TM domain belong to PC family. Recently discovered convertases are PC7, Furin, PACE4 and PC6B. These are expressed in tissues like liver, gut, brain, neuroendocrine system. There are still many unidentified enzymes that participate in cleavages at single basic residues and unusual sites, other than the subtilisin-like Proprotein Convertases.

Examples of proteolytic processing in various organs of our body
In concern with the proteolytic processing with respect to kidney the human prorenin is considered. The proteolytic processing of human prorenin in renal tissues is discussed here. The mouse proprotein convertase also known as PC1 accurately cleaves the prorenin and generates active renin. The human Proprotein Convertase also known as PC3 can also process the prorenin to generate renin but not as efficiently as mPC1 does. This is because of the differences in the sequences of the carboxy terminus from PC1 to hybrid hPC1/mPC1. The cleavage of prorenin bay PC1 in humans takes place at a pair of basic amino acids by removal of 43 amino acid prosegment from the amino terminus of the it along with identification of a functionally important site in the hPC1. Also PC1 generates active renin in other tissues like adrenal medulla and certain adrenal tumors. If human prorenin has either in Lys or Arg residues at the cleavage site then the hPC1 cannot generate active rennin.
 * Renal proteins


 * Blood protein

The Kell Blood Group Protein belongs to family of metalloendopeptidases. It’s a membrane Glycoprotein which shares a pentametric zinc-binding consensus sequence. It conserves all the amino acids required for the endopeptidase activity.Kell Blood Group proteins have a homology with M13 family in which a disulphide bond is covalently linked to a protein named XK, this spans the membrane 10 times, whereas kell proteins is linked to XK present is fifth extracellular loop. Activation of endothelins and processing of bioactive peptides can be done by Kell proteins.

Fodrin, Human Brain Alpha Spectrin plays an important role in the nervous system yet its mechanism is not completely understood. Its isoforms are present within the cells and in some neurotransmitter neurons. It is proteolytically processed by the calcium dependent proteases. This processing of fodrin is a central molecular mechanism in the development of long term memory. Foldrin has a proteolytically hypersensitive site in its centre for cleavage. Calcium-dependent binding of calmodulin takes place at this site.
 * Human Brain Spectrin

Autoprocessing
This is the process in which a protein modifies itself to convert a proprotein into an active protein. A classic example of this is the autoprocessing of furin family of endoproteases. Furin is the most widely used proteolytic processing enzyme and functions mainly for cleavage of proproteins needed to maintain cellular physiology.Furin family of protease is also called the PACE family.There are 7 identified members in this family and among them Furin is the most important protein.Most of these PACE family are active in the trans golgi network. Furin is transmembrane protein which is synthesized as a 100KDa protein. This protein undergoes catalytic cleavage in the Endoplasmic reticulum to become active furin which is 94 KDa. The proprotein however remains bound to the protein and acts as the furin inhibitor. The polypeptide release then occurs in the trans golgi network where furin becomes active.

An example of proteolytic processing by furin is processing of ProFibrillin.Fibrillin-1 is a glycoprotein which is a major component of the Extracellular matrix. It undergoes proteolytic cleavage in its C-terminal domain to form fibrillin-1 in the trans golgi network.

Proteolytic processing of the Human Immunodeficiency Virus type 1
Polyprotein precursors are processed to form the proteins that produce the retroviral particle. One of these precursors is the viral protease that processes the precursors itself. This viral protease cleaves the retroviral precursor proteins during the virus assembly. Fully infectious viral particles are produced by precise protease-mediated precursor processing and if the order of the cleavage changes, it results in the less infectious viral particle. To become active the proteases must form a dimer. The processing events are first carried out by the embedded PR, prior to the release from the GagPol precursor.The activated protease domain on one pair of GagPol molecules cleaves initial sites on those same molecules. Further substitutions at the first amino acid in the proteases, a proline, lifts this constraint and free the protease domain within GagPol to cleave additional native processing sites in the precursor not cleaved by the wild-type embedded protease.

Proteolytic processing- role in therapeutics
The fact that certain proteins can modify others by binding to specific site and alter its activity is used as a principle in the development of certain modern therapeutics. Here is an example of how the process of proteolytic processing has been used to find possible cure for diseases. Human coronavirus is the causative agent of the common respiratory disease in adults and children. There are no current cure for CoV as a result proteolytic processing is being exploited to find new drug targets. Biochemical analysis of HCoV –NL63 virus showed 2 viral papain like protease PLP1 and PLP2 which process viral replicase polyprotein. In this case they generated polyclonal antisera against 2 of replicase non structural proteins nsp3 and nsp4. It was found that PLP1 processes clevage site to release 1 to release nsp1 whereas PLP2 released nsp2 and nsp3. Also by using hexa ubiquitin substrate and ubiquitin vinylsulphonate inhibitor specific to deubiquitinization enzyme further show that CoV has deubiquititination activity. Understanding the concept of proteolytic processing and deubiquitinating of PLP’s can pave a way to the development of novel antisera regents directed against HCoV.

Lethal effects of proteolytic processing
Proteolytic processing not only helps in carrying out normal cellular processes but it also can cause processing of proteins to give lethal proteins. A classic example of proteolytic processing which develops lethal protein is development of Netherton syndrome. Netherton syndrome is a autosomal recessive congenital syndrome which causes ichthyosiform erythroderma caused by a mutation in the human SPINK5. Biochemical analysis of Netherton syndrome have shown an increase in the proteolytic processing of profilggrin into its constituent filaggrin monomers. Defects in filagrin processing cause congenital disorders like icthyosis. SPINX5 undergoes extreme proteolytic processing furin is assumed to be involved in the proteolytic processing of SPINX5 however the exact extent of processing is yet to be deteremined. In the absence of serine protease inhibitor SPHINX5 there is abnormal increase in the processing of profilagrin resulting in the increased production of filagrin monomers causing severely compromised epidermail barrier function.