9.5% of the large global population is plagued by Type 1 Diabetes (T1D). To view it from a statistical point of view, it is actually significant. Due to the reason that unlike Type 2 Diabetes, Type 1 Diabetes’ occurrence cannot be avoided simply, such as by controlling a healthy diet or exercising. Instead, it is a congenital disease, which means that humans can either inject insulin to alleviate the symptoms or operate genetic synthesis to step by step resolve it. We found out that among the proteins that influence Type 1 Diabetes, the Reg3 gamma protein is one that directly influences the beta cell. Since the overexpression of Reg3 gamma in pancreatic islets can trigger the activation of Janus kinase 2/signal. Thus far, the Reg3g protein has been implicated in positively driving beta cell regeneration, which are cells indispensable for insulin production. The dry lab team’s goal was to perform basic bioinformatic analyses to assess whether there may be additional proteins belonging in the same family that may contribute to treating T1D.

The Reg family protein consists of Reg1a, Reg3g, and Reg4. The Reg family includes a variety of protein types, with both distinct and shared functions appearing in different organisms. The Reg family is further divided into subfamilies and is numbered from one to four with each holding specific functions. These protein types and functions include lectins, acute phase reactants, antiapoptotic factors, or growth factors for pancreatic beta cells. The determining feature of REG family proteins is that they contain C-type-like lectin domain, which contributes to the ability to selectively bind a wide variety of ligands and carbohydrates. For instance, REG3A is able to recognize peptidoglycan through an EPN motif that supports calcium-dependent and calcium-independent carbohydrate binding (Lehotzky et al., 2010). Another commonality is that except for Reg4, all other Reg members have six exons and are located in the second chromosome in humans and clustered on 6C3 in mice (Hartupee et al., 2001).

C-type lectin fold is one of the most commonly found proteins. They are composed of 110 to 130 amino acids. Broadly speaking, they have regulatory roles in innate immune systems and have a dual role in the recognition and adhesion of pathogens. These domains facilitate binding with other lipids, proteins, lipids, or inorganic molecules. This characteristic may help Reg family proteins carry out their functions and explain why C-type-lectin domains are commonly found in these proteins. Additionally, C-type lectins accommodate plenty of sequence variations, which can be referred to as rigid scaffolds.

In addition, distinctive reg genes from different animals have a decent degree of sequence identity. In the Reg3 subfamily, REG3A(P05451) in humans is 61.85% similar to murine REG3A(O09037), 67.05% similar to murine Reg3g(O09049), and 69.54% similar to murine Reg3b(P35230). In addition, REG3G(Q6UW15) in humans is 65.52 %analogous to Reg3a(O09037), 68.79 % analogous to Reg3b(P35230), and 64.94 % similar to Reg3g(O09049).

Reg3g Family in Human
	REG3A	REG3G
Full Name and Alternative Names	Regenerating islet-derived protein 3-alpha Or Human hepatocarcinoma-intestine-pancreas (HIP) Or Human pancreatitis-associated protein (PAP)	Regenerating islet-derived protein 3-gamma Or Pancreatitis-associated protein 3 Or Regenerating islet-derived protein III-gamma (Reg III-gamma)
UniProt ID	P05451	Q6UW15
Length (Size)	166	175
Domain Sequence	36 to 164 (Total: 129 amino acids)	40 to 173 (Total: 134 amino acids)
Functions	Inhibitor: prevent the spontaneous precipitation of calcium carbonate Assist in neuronal sprouting in/with the brain and pancreas regeneration (tissues)	Mediates bacterial killing via binding to carbohydrate moieties of peptidoglycan on the surface Acts against Gram-positive bacteria Restricts bacterial colonization of the intestinal epithelial surface Restricts activation of adaptive immune responses by the microbiota
Express	Where? Abundant expression: in IL-17A-induced neonatal human epidermal keratinocytes (NHEKs) and in the lesional skin of psoriasis patients Low expression: in differentiated NHEKs Specialties Alzheimer disease and Down syndrome patients: Enhanced expression of PSP-related transcripts and intraneuronal accumulation of PSP-like proteins	Where? In the pancreas, where it is restricted to the exocrine pancreas. Specialties Moderate expression levels in the testis and weak in the heart, kidney, and placenta

Reg3 Family in Mouse
	Reg3A	Reg3B	Reg3G	Reg3D
Full Name	Regenerating islet-derived protein 3-alpha	Regenerating islet-derived protein 3-beta	Regenerating islet-derived protein 3-gamma	Regenerating islet-derived protein 3-delta
ID in UniProt	O09037	P35230	O09049	Q9QUS9
Length (Size)	175	175	174	175
Domain Sequence	40 to 173 (Total: 134 amino acids)	40 to 173 (Total: 134 amino acids)	40 to 172 (Total: 133 amino acids)	37 to 174 (Total: 138 amino acids)
Function	As hormone: in response to distinct stimuli like anti-inflammatory signals Via inhibition of inflammatory cytokines, it inhibits skin inflammation (PubMed:22727489)	Acts against intestinal Gram-positive and Gram-negative bacteria Builds protection against infection with S.enteritidis: via preventing the translocation from the gut lumen into intestinal tissues inhibits intestinal translocation of the Gram-negative Salmonella enteritidis bacteria upon oral infection (van Ampting et al., 2012)	Acts against Gram-positive bacteria Acts as an immunosuppressive promoter activates the JAK2/STAT3 signaling pathway triggers the generation of an immunosuppressive tumor microenvironment	Acts as a survival and growth factor Enables oligosaccharide binding activity, and signaling receptor activity Involves in responding to the antimicrobial humoral immune system that is mediated by antimicrobial peptide(Alliance of Genome Resources, Apr 2022)
Expressions	Where? in small intestine and pancreas and gut Additional Notes Secreted by activating its receptor EXTL3 and induce cell-specific signaling pathways Induced by IL17A in keratinocytes Regulated by injury, infection, inflammatory stimuli, and pro-cytokines via different signaling pathways	Where? in the small intestine in the colon and at an extremely low level in the healthy pancreas in murine intestinal epithelial cells Additional Notes associated with pancreatic tumor growth suppressed after chronic intragastric feeding of ethanol (Hartmann et al., 2013; Yan et al., 2011)	Where? in the small intestine, including Paneth cells Highly expressed in the lung epithelium while allergic airway inflammation (at protein level) in goblet and Clara-like cells epidermal expression increased by skin injury Additional Notes Hardly detectable in the colon (at protein level) Expressed by nociceptors	Where? Mainly in pancreatic islets and the digestive system Active in extracellular space

REG3G in Human

REG3G in Mouse

Both Reg3g in humans and mice contain the same kind of domain, the C-type-lectin domain, however, their respective sizes vary slightly. The domain length in H. sapiens Reg3g is 134 amino acids while mouse Reg3g has 133 amino acids. Regardless, protein prediction with Alphafold shows that Reg3 in humans or in mice has highly similar structures, and most of the parts are highly determined for the algorithm. The high degree of similarity suggests that the conclusions drawn from Xia et al. (2016) using mice, namely that Reg3g promotes beta cell regeneration, may be applied to human Reg3g. To get a better sense of degree of conservation of the Reg3g protein in animals, we performed BLAST searches in the NCBI database using both human and mammalian amino acid sequences as the query sequence. We found that human REG3G is also found in other primates, placentals, and carnivores (Figure 3). Since we are also curious what other proteins may hold similar if not redundant roles to REG3G, we performed a protein BLAST search among the H. sapiens proteome to identify similar proteins. Several secretory phospholipase A2 receptor isoform proteins show high sequence identity (Figure 4), suggesting that this protein family can be of future interest when studying beta cell regeneration. The mouse REG3G BLAST search turned up hits including primates as well, again supporting the notion that REG3G is highly conserved between mice and humans. Combining the findings above, we are encouraged by the high degree of conservation of REG3G and believe that the findings by Xia et al. (2016) will be transferable as we clone human REG3G and observe the effect of overexpression on beta cell proliferation.

In addition to performing basic bioinformatic analysis on our proteins of interest, our dry lab team planned on using experimental data to model how different uORF sequences would affect the final translational output of Reg3g. It is known that overexpression of a protein is not always beneficial. Therefore, we designed our construct with two versions of uORF sequences with different lengths to see which mode of overexpression can better promote beta cell regeneration. Our design is supported by knowledge that uORF affects translation efficiency by potentially stalling ribosomes and that a key parameter affecting translation is uORF length. However, due to timing constraints and logistical challenges on the wet lab side, we are unable to perform modeling using empirical data before the wiki freeze.

Renalase (hereafter RNLS) is a flavin-adenine dinucleotide (FAD domain-containing protein) (Xu et al., 2005). In our literature review, it is another protein we discovered that may be useful in treating Type 1 Diabetes besides Reg3g. RNLS functions as a cytokine, a signaling molecule, and interacts with an unidentified plasma membrane receptor to activate protein kinase B and mitogen-activated protein kinase pathway (Guo et al., 2014). By activating those pathways, it could save the beta cell from attacks and promote its regeneration. More importantly, in a CRISR screen for T1D targets, it was found that mutating RNLS leads to enhanced beta cell protection (Cai et al., 2020 ). In our project, we want to extend the findings of Cai et al (2020) further by creating specific mutations in the coding region of RNLS in order to evaluate whether current annotated domains are essential to RNLS function. To identify meaningful mutation sites, the goal of the dry lab team is to conduct bioinformatic analysis to evaluate both the function of renalase human and renalase mouse, identify similarities and differences, as well as highly similar protein and protein families.

Amino oxidase is the domain found in both human and mice RNLS and based on existing annotations, we believe it is the catalytic domain. Specifically, the type found in renalase is flavin-dependent amine oxidase (FAO). Generally speaking, this domain family has an annotated function of transferring a hydride to a FAD cofactor to oxidize an amine substrate. In this mechanism, the FAD cofactor is reduced, thereby oxidizing the imine bond in the amine.

Comparsion between Human and Mouse
Species	Homo Sapients (Human)	Mus Musculus (Mouse)
Size (AA)	342	342
Domain Sequence	99 ~ 297 (Total: 199 amino acids)	105 ~ 333 (Total: 229 amino acids)
Function	Metabolizes catecholamines and catecholamine-like substances Via superoxide-dependent mechanism using NADH as a cofactor (Desir & Peixoto, 2013)	Degrades circulating catecholamines and reduces myocardial necrosis Protect against renal ischemia-reperfusion injury (H. Thomas Lee et al., 2013)
Location of Expression	Glomeruli, tubule, mesangial cells, podocytes, renal tubule epithelial cells, and its cells supernatant The majority of the expression takes place in the kidney (Wang et al., 2012)	In terms of localization, the protein is annotated to be secreted to the extracellular space. We are unaware of empirical evidence on localization.

Through database and peer reviewed literature search, we found out that both human and mouse RNLS share similar annotated protein domains, which are the amino oxidase domain and the NAD_binding_8. Even though the two proteins in human and mouse are highly similar, its sequence has a little bit of difference. The amino oxidase domain in human RNLS is 199 amino acids long (Figure 6). The mouse RNLS has​​ 229 amino acids long (Figure 9). In addition to that, we used BLAST to align the amino oxidase domain in humans (Figure 7). The result shows that the same domain also exists in other isoforms of RNLS in humans. Furthermore, the function and the location of the expression are very similar. However, the renalase mouse seems to have an additional effect on myocardial disease. On top of that, we used BLAST with NCBI’s database to check the homology of both renalase human and renalase mice. For renalase humans, we discovered that it shares the same root with bats (Figure 8). For renalase mice, we found that they share a common root (Figure 10). Nevertheless, in order to have a better understanding of the protein structure of both renalase human and renalase mouse, we use AlphaFold to predict the 3D model of the protein (Figure 11). Since the wet lab team will be cloning a truncated version of RNLS without the amino oxidase domain, we noted the amino acid positions and noted down parts of the 3D structure that would be affected. Since the domain accounts for a large portion of the protein, the affected region encompasses both the interior pockets and the exterior (likely the hydrophilic region).

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